CBDclos fused to phytochelatin (PC) EC20 with LacI promoter
This part has a constitutive promoter, so our PBAN(Spodoptera litura) can constitutive express.
IPTG-induced and T7-driven expression of TEV protease
This protein generator produces TEV protease when induced with IPTG in a DE3 strain.
We created K1319004 in RFC[25] to enable potential
The part is combined from other BioBricks and a codon-optimized TEV protease CDS (K1319004).
RhlI Acyl-homoserine-lactone synthase from P.aeruginosa SQ6
This sequece encodes RhlI, an acyl-homoserine-lactone synthase required for the synthesis of BHL (N-butanoyl-L-homoserine lactone), and HHL (N-hexanoyl-L-homoserine lactone) autoinducer molecules which bind to RhlR.
It includes both the complete coding sequence of RhlI and part of noncoding sequence between RhlR and RhlI gene.
It comes from the genomic sequence of P.aeruginosa SQ6.
Human ferritins do loads more iron atoms then bacterial ferritins. Furthermore they are forming metall cores that are less contaminated with phosphates then e.g. bacterial ferritins. Due to this fact, this should result in a purer iron crystal with an improved magnetic moment.
This design was provided by iGEM Calgary and the HuFerritin as a single protein coding biobrick was constructed by iGEM Berlin as this biobrick was not accessible in this version in the registry.
iGEM Calgary 2013 from a synthesized codon optimized construct
SigD-dependent promoter of the LytABC locus (PlytA) of Bacillus subtilis
PlytA promoter of the ''B. subtilis'' LytABC operon, under the control of SigD (major), the transcripts being predominants at the exponential growth phase, and SigA (minor).
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. PlytA_ENX_fwd: TGCAGAATTCGCGGCCGCTTCTAGAG ctcatcctttgcacctcg PlytA_SNP_rev: AGCCTGCAGCGGCCGCTACTAGTA tcattatattttatcgtcaacctatt
This part consists of histidine tag and RFP. His-tag is useful for purifying RFP.
RhlR regulatory protein from P. aeruginosa SQ6
Coding a transcription regulator RhlR. RhlR is necessary for transcriptional activation of the rhlAB genes encoding the rhamnosyltransferase. It also functions as a transcriptional activator of elastase structural gene (lasB). RhlR binds to autoinducer molecules BHL (N-butanoyl-L-homoserine lactone), and HHL (N-hexanoyl-L-homoserine lactone). This part includes the noncoding sequence of RhlR.
It includes both the complete coding sequence and part of noncoding sequence of RhlR.
It comes from the genomic sequence of Pseudomonas aeruginosa SQ6.
Tal Naor, Ricarina Rabinovitz, Ittai Rubinstein, Yair Maimon
The promoter Plux is induced by the binding of an AHL-luxR dimer. This bio-brick leads to the synthesis of the enzyme Green Renilla Luciferase, which catalyses a luminescent reaction. lacI can be used to repress the promoter Plac.
BBa_R0062: Plux, BBa_B0030: Strong RBS, BBa_B0030: Strong RBS, BBa_B0010:T1 (terminator) from E. coli.
Cyan Fluorescent Protein (CFP) "Cindy Lou" coding region, intellectual property-free
We had to make sure that we could do the transformation properly.
Neomycin resistance gene. This gene can be used for selection of cells with an efficient transformation. This gene codes neomycin phosphotransferase which breaks neomycin, kanamycin, and gentamicin molecules. If you use these gene as selectivity gene in bacteria, and your laboratory lacks neomycin, you can replace it for kanamycin by adding 35 mg/ml per liter of LB media. The best concentration of neomycin to use is 80 mg/ml per liter of LB media. This gene can be also used as a selective gene for mammalian cells using gentamicin.
This gene was obtained from pCDNA 3.1 myc His(A).
Encodes for an enzyme, that is capable of cleaving A- and B-antigens in the human ABO-Bloodgroup system. As the name suggests, the galactose and connected saccharide residues are cleaved from the antigen, leaving behind the the Oh-Antigen which is characteristic of the Bombay-blood type. Part in RFC25.
Part in RFC25, in order to ease fusion with other parts, for example immobilisation tags.
The protein encoded by this sequence naturally occurs in Clostridium perfringens. This sequence was optimized for codon usage in E. coli K12 and synthesized.
Supernova is the monomeric version of KillerRed (BB_K1184000). Codon optimized Supernova with WTLac promoter allows for induction of Supernova expression with IPTG.
RBS + SspGyrB C-intein RFC[105] assembly construct (with His6)
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena B??scher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Sch??fer, Carolin Schmelas, Silvan Schmitz, Max Waldha
RBS + SspGyrB N-intein RFC[105] assembly construct (with His6)
This part has a constitutive promoter, so our PBAN(Bombyx) can constitutive express.
Dye-decolourising peroxidase from Bacillus subtilis
BsDyP gene (1251bp) from Bacillus subtilis that has been found to be effective in degrading lignin and azo dyes, in an oxidative process.
From genomic sequence of Bacillus subtilis; BsDyP gene inserted into a pET-21a (+) expression vector, provided to us by XXX Research Group.
RBS+NpuDnaE C-intein RFC[105] assembly construct (with His6)
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Buescher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schaefer, Carolin Schmelas, Silvan Schmitz, Max Waldhauer
This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting those intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the Nostoc punctiforme DnaE N-terminal split intein. The corresponding C-terminal construct is BBa_K1362101 . Upon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond. This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our [http://2014.igem.org/Team:Heidelberg split sfGFP experiment] and the respective parts in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on! ===Nostoc punctiforme DnaE split intein (NpuDnaE)=== Inteins can be found in all kingdoms of life, however their use for the organism remains yet unknown. In the past years many intein sequences were found in the genomes of cyanobacteria. NpuDnaE is a naturally split intein from the alpha subunit of Dna Polymerase III in Nostoc punctiforme bacteria which shows [[#References|[2]]] extraordinarly high splicing activity and therefore is on of the gold-standards for inteins since many years. Amino Acid Sequence: N-Intein: AEY | CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN C-Intein: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN | CFN
The part is assembled from different sources. The intein sequences come addgene or were kindly provided by different working groups. BBa_J04450 was used as mRFP selection marker. The scars and overhangs are part of the RFC[???] intein cloning strategy and are further explained on our [http://2014.igem.org/Team:Heidelberg/parts/rfc wiki] and in RFC[???] [[#Reference|[1]]]. Please see the corresponding basic parts for the individual detailed information.
Phytochelatin (PC) EC20 fused to linker-dCBD with LacI
Encodes for an enzyme, that is capable of cleaving off N-Acetlygalactosamine from A-group blood antigens, thereby turning them into H-antigens. Can be used to convert erythrocytes from blood type A to O. Part in RFC25 standard.
The protein encoded by this sequence naturally occurs in Elizabethkingia meningoseptica( also called Chryseobacterium meningosepticum or Flavobacterium meningosepticum). This sequence was optimized for codon usage in E. coli K12 and synthesized.
We add an araBAD promoter up to this part to regulate the PIII; expression wtih ara and temperature.
To regulate PIII; expression with temperature on the translation level used RNAT.
The RNAT(PrfA) comes from bacteria genome and PIII; comes from M13 phage genome.
NiBP fused to dCBD with linker in RFC 25
promoter with CIλ and LacI operator sites
AHL Generator that can be used in E.coli and L.lactis
bphR2 gene, from Pseudomonas pseudoalcaligenes KF707, encodes for a regulatory protein (bphR2) which can detect polychlorinated biphenyls (PCBs). This gene regulates negatively bphR1 promoter involved in the transcription of biphenyl-catabolic genes (bph), degrading PCBs. In absence of PCB, bphR2 is bound to the bphR1 promoter. When compound diffuses into the cell, it binds to protein allowing a conformational change. BphR2 is release from the promoter that its allows the transcription of bph genes and therefore the degration of PCBs. Our aim was to finish and characterize the part of 2013 Paris Saclay's team. We used bphR2 in our biosensor project of PCBs. Instead of the genes of degradation, we have RFP (BBa_E1010) to detect 2 hydroxy-3',4' biphenyl.
Moreover, the sequence contained a pstI site from 142 to 148bp after ATG site so we have synthetized it by mutating the site, keeping the same amino acid.
The gene was synthetized from the sequence of bphR2 (BBa_K1155009)because there isn't the sample in the registry.
This part has a constitutive promoter, so our PBAN(Adoxophyes sp.) can constitutive express.
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha
"This part was generated in a modified version of RFC25, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGATGGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Stop codons are underlined."
The LMU Bacillus RBS collection is based on a wobble-library with the sequence ARRRRRRGATA and the theoretical translation initiation rates of the RBS in this library, calculated by the salis lab's RBS calculator (https://salis.psu.edu/software/). The 7 RBS submitted as BioBricks are the ones that offer the best coverage of translation initiation rate space, 1 being the optimal Bacillus RBS and 7 being the weakest.
Alexia Satouf, Clara Bouyx, Aimeric Agaoua, Lambert Antoni, Vincent Castel
SH3pep-LZA is a link wich mediates proteins interactions.
Amplified from pWW2181,Whitaker et al. PNAS 2012
Codes for the sfGFP protein, an optimized fluorescent reporter. Was originally optimized for Bacillus subtilis, but then showed high fluorescence in Lactococcus lactis.
Used primers: forward - 5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGATGTCAAAAGGAGAAGAGCT-3' reverse - 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTACTTATAAAGCTCAT-3' to PCR sfGFP(Bs) from the plasmid and add the BioBrick prefix and suffix.
PCR on plasmid MG_sfgfp(Bs), used in the study 'Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging', see references. Plasmid kindly provided by the Molecular Genetics group of the Rijksuniversiteit Groningen.
Bastian Wagner, Christian Mende, Andreas Schmidt, Max Dombrowsky
This part is the full renilla luciferase. The split protein has also been submitted as biobricks: BBa_K1486021.
sfGFP fused to dCBD with linker in RFC 25
Red imported fire ant (Solenopsis invicta) Spread:The red imported fire ant, a eusocial species, are far more aggressive than most ant species. Animals, including humans, often encounter them by inadvertently stepping on one of their mounds, which causes the ants to swarm up the legs, attacking en masse. The ants respond to pheromones released by the first ant that attacks, thereafter stinging in concert. Characteristics: Fire ants are red and black in coloration and, like all insects, they are protected by a hard exoskeleton and have six legs. Worker ants have round heads with mandibles, an armored thorax midsection and an abdomen, made up of the pedicle and the gaster. The head is typically copper brown in color. In addition to their mandibles, fire ant workers also possess an abdominal stinger. Damage: They are considered to be a pest, not only because of the physical pain they can inflict, but also because their mound-building activity can damage plant roots, lead to loss of crops Control:(2) Hot water Pouring hot water on the mounds is effective and environmentally friendly, but may require 3 or 4 applications to kill the colony. Water should be at least scalding hot, but does not need to be boiling. This works best when you use 3 to 4 gallons of water in each application. WARNING: Hot water kills grass and shrubbery and may cause severe burns if spilled. (2)Liquid nitrogen: it could be the most effective and most environmental friendly method to eradicate the species.
The aresnic inducible banana odor generator converts isoamyl alcohol to isoamyl acetate in the presence of arsenic.
We digested the Biobricks with CIP to assure that the plasmids did not re-ligate to themselves.
The arsenic promoter with the arsR repressor gene is naturally found in E. coli. The ribosome binding site, the ATF1 gene, and the double terminator combined sequence was obtained from the Biobrick BBa_J45199.
Encodes for an enzyme, that is capable of cleaving off galactose from B-group blood antigens, thereby turning them into H-antigens. Can be used to convert erythrocytes from blood type A to O. Part in RFC25 standard.
The protein encoded by this sequence naturally occurs in Bacteroides fragilis. This sequence was optimized for codon usage in E. coli K12 and synthesized.
This is a regulated promoter with a eforRed protein
S-adenosyl methionine-dependent methyltransferase relate to Met-DMSP byosynthesis pathway(SAMmt)
This part is S-adenosyl methionine-dependent methyltransferase gene of Fragilariopsis cylindrus. The gene code S-adenosyl methionine-dependent methyltransferase relate to Met-DMSP byosynthesis pathway from Fragilariopsis cylindrus. which catalyze third step of Met-DMSP biosynthesis pathway.
This part was excluded intron. And there was PstⅠ recognition site so I made single base substitution in recognition site.
This part has a constitutive promoter, so our PBAN(Lymantria dispar) can constitutive express.
Sascha Hein, Rene Sahm, Malte Blumenroth, Tobias Gabriel, Sebastian Palluk
Chondroitin 4 sulfotransferase-derived peptide binding to NADH Oxidase of S. pneumoniae
Short peptide of 13 amino acids corresponding to amino acids 279 to 290 of the human protein Chondroitin 4 sulfotransferase (UniProt [http://www.uniprot.org/uniprot/Q9NRB3 Q9NRB3]). It has been shown to inhibit NADH oxidase (NOX) mediated binding of ''Streptococcus pneumoniae'' to human A549 cells (Muchnik ''et. al.'', 2013). Displayed on the surface of ''Bacillus subtilis'', it should enable ''B. subtilis'' to bind specifically to ''S. pneumoniae''. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
For generation of the part's nucleotide sequence, the amino acid sequence of the peptide (Muchnik et. al., 2013) was reverse transcribed with regard to the codon usage of Bacillus subtilis. Oligos were designed as precut by EcoRI and PstI to shorten the synthesized fragments and to simplify the cloning into pSB1C3.
The part was generated by annealing of artificially synthesized oligonucletides: CSP_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCGAACCACACATCACTTCCGGCGTCAGCGAGAGAA ACCGGTtaatACTAGTagcggccgCTGCA CSP_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT TTCTCTCGCTGACGCCGGAAGTGATGTGTGGTTCGC CATGCCGGCttcctccttacTCTAGAagcggccgcG
Prefix homologous recombination for Synechocystis ("Left flank")
This is a prefix region of DNA for homologous recombination into a neutral site within the genome of Synechocystis sp. PCC 6803. Kunert, A., Hagemann, M. & Erdmann, N. (2000) Construction of promoter probe vectors of Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB. J. Microbiol. Methods. 41, 185-194 Angermayr, S. A., Paszota, M. & Hellingwerf, K. J. (2012) Engineering a cyanobacterial cell factory for production of lactic acid. Appl. Environ. Microbiol. 78, 7098-7106
This part comes from genomic sequence of Synechocystis sp. PCC 6803
Synechocystis prefix homologous integration region
MerP, mercuric transport protein periplasmic component
merP is in charge of periplasmic transportation of mercury. This protein has two conserved cysteine residues for specific binding with the mercury ion, Hg(II).
Source organism: Serratia marcescens. Plasmid pDU1358 kindly donated by Anne O. Summers.
TEV protease with anti-self cleavage mutation S219V, codon optimized for E. coli
This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.
The part does not contain start or stop codons, because it was designed for RFC25 compability
The part was codon optimized using the genomic amino acid sequence, designed for RFC25 and mutated S219V for anti-self cleavage properties.
M13 bacteriophage g5p with a FLAG tag on C-terminal
This part is coding the M13 bacteriophage g5p with a FLAG tag on C-terminal, which could act as an single strand DNA binding protein and RNA binding protein at the same time. These properties make g5p protein an important regulatory protein for M13 phage. In the life cycle of M13 phage, g5p protein would bind to newly synthesized viral genome ssDNA, resulting in the correct direction to the phage packaging site. Also, g5p protein would repress the expression of g2p protein by binding to the leader sequence of viral mRNA.
Between the g5p and FLAG coding sequencing, there are 9 nucleotides linker sequence coding a 3 amino acid peptide. It is not certain whether this peptide would affect the regular function of g5p protein.
It is derived from the commercial M13KE vector(New England Biolabs) used in phage display system by PCR.
GFP (mut3b) with SsrA-DAS+2 degradation tag
GFP (mut3b) (see part BBa_E0040) with added engineered NYADAS-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The NYADAS tag encodes peptide sequence AANDENYNYDAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] The two additional residues ???NY??? extends tag between SspB and ClpX binding site, thus preventing clash when both these protein are bound to tag.[3] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [4]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] McGinness, K. E., Baker, T. a & Sauer, R. T. Engineering controllable protein degradation. Mol. Cell 22, 701???7 (2006). [4] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (DAS in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0051.
sfGFP with MLRR tag under arabinose promoter
Przemysław Nuc, Melania Nowicka, Weronika Krzynowek, Jakub Bartoszewicz, Katarzyna Rżosińska
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
This part was generated in a modified version of RFC25, and has the following prefix and suffix: {||prefix with EcoRI, NotI, XbaI, SD and NgoMIV:|GAATTCGCGGCCGCTTCTAGATGGCCGGC|-|suffix with AgeI, SpeI, NotI and PstI:|ACCGGTTAATACTAGTAGCGGCCGCTGCAG|}Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Stop codons are underlined.
Coding for the trans-acting regulator SmtB
In 1993, Huckle and co-workers reported the isolation and analysis of the smt locus from Synechococcus sp.. Gene smtB, one of the components of the locus, encodes a transcriptional repressor of gene smtA, with which is required for normal tolerance to Zn2+ and Cd2+. The product of smtB functions by binding to the smt operator-promoter(smtO-P) and dissociating in the presence of Zn2+ due to the classical helix-turn-helix motif similar to many DNA-binding proteins. It can also be genomically rearranged when induced by Cd2+, leading to a deletion within smtB because of a highly iterated palindrome (HIP1), a highly represented octanucleotides(5′-GCGATC-GC-3′) which traverse both borders of the excised element. These two mechanisms both cause the elevated expression of smtA.
According to the mechanisms of smt locus represented above, gene smtB should function with the accompany of smt operator-promoter. Previous cases selected smtA whose product functions by encoding MT-like to sequester metal ions, while our case intended to take advantage of the trigger lies in this locus. By imitating former researches, in which a promoter less lacZ gene was fused upstream of smtA, we respectively splice two selected reporter genes and sequence from smt operator-promoter to smtB, equivalent to replacing smtA with reporter genes, to visualize the effects of metal ions on the locus.
Metallothioneins(MTs), discovered in horse kidney in 1957 by Margoshes and Vallee are identified as low-molecular weight and sulphydryl rich proteins which bind metal ions in metal-thiolate clusters and whose synthesis increases in response to elevated concentrations of certain metals. In 1988, Olafson and co-workers reported the amino acid sequence of a metal-associated protein isolated from cyanobacterium Synechococcus sp.. With the function of binding metal ions remaining, it only has less than 20% homology in amino acid sequence with known MTs in eukaryotes and thus we call it class II MT or on some occasions MT-like. Owing to the works of Robinson and Huckle, the corresponding locus of Synechococcus PCC7942, smt locus was isolated and analyzed. It includes smtA which encodes the class II MT and a divergently transcribed gene smtB which encodes a trans-acting, autoregulatory repressor that exerts metal ion-inducible negative control over smtA transcription. Between them lies a 100-bp operator-promoter region(smt O-P, BBa_K1509001), which is responsible for the divergent orientation of smtA and smtB.
Sortase A non-circularization construct (with His6)
Phytochelatin (PC) EC20 fused to CBDclos with T7 promoter
Germacradienol/germacrene D synthase CDS.
Supernova is the monomeric version of KillerRed (BBa_K1184000). It has been codon optimized for E. coli.
The original KillerRed is BBa_K11840000.
This part is a combination of a pBAD promoter(BBa_I13453), RNA thermometer(BBa_K115002）
The source of the part will be its components.
Sortase A non-circularization construct (with Smt3 and His6)
IIA(Glc) - E.coli glucose-specific phosphotransferase enzyme IIA
Fahmi Dwilaksono, Indah Nurulita, Rian Adha Ardinata, Mochammad Isro Alfajri, Arief Budi Witarto
Phosphotransferase system (PTS) is a major carbohydrate active -transport system, which is executed by a cascade of protein rally. For glucose uptake, IIA(Glc) is the only enzyme specific to glucose transport. We measure the concentration of glucose in the medium using catabolite repression mechanism toward lacI promoter activity upstream of a reporter gene, mRFP. We expect by over-expressing IIA(Glc) in E. coli, we will obtain different range of glucose standard curve in our measurement system.
The gene coding for IIA(Glc) from E.coli - crr - was isolated by colony PCR using forward primer which contains with restriction sites of EcoRI and XbaI and reverse primers which contains restriction site for SpeI and a double stop codon.
GFP (mut3b) with AAV-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added AAV-ssrA degradation tag (part BBa_I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (AAV in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E0040, tag sequence was obtained from part I11012, but same tag was also used in paper by Andersen et al., (1998).[2]
Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. The bactericidal activity of lantibiotics is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores. This BioBrick differs in its overhangs from BBa_K1351012.
The tRNA synthetase/tRNA needed for incorporating o-(2-nitrobenzyl)-L-tyrosine (ONBY) at a UAG codon
The part includes the gene coding for the Methanocaldococcus jannaschii synthetase mutated to charge the orthogonal tRNA (also in the part) with the non-canonical amino acid o-nitrobenzyl tyrosine (ONBY) along with the proper promoters and terminator . This part encodes for the incorporation of ONBY during translation at AUG amber stop codons in a cell with Release Factor 1 (RF-1) knocked out such as "Amberless" E. coli or "RF-0".
Evolved from the Methanocaldococcus janaschii tyrosyl synthetase and tRNA by the Schulz group. Specific mutations sourced from Deiters, A., Groff, D., Ryu, Y., Xie, J. and Schultz, P. G. (2006), A Genetically Encoded Photocaged Tyrosine. Angew. Chem. Int. Ed., 45: 2728???2731.
RFC[25]-compatible dark quencher based on K1319000 (E0030)
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP). Three mutations were introduced that eliminated fluorescence: * L90I * Y145W * H148R
Point mutations were introduced, starting from K1319000 (RFC25-YFP) as the template.
Nikolas Layer, Philipp H. O. Mayer and Philip Roessler
N-Intein part of the ssp GyrB split intein. Can be used in conjunction with the 6 amino acid long C-Intein to N-terminally immobilize a peptide onto a matrix. For immobilisation, parts need to be fused to the C-terminus of the N-intein. The N-terminus of the C-Intein is fused the matrix. The split intein recombines and splices itself out, thereby specificially immobilising the N-terminus of the peptide to the matrix. The sequence of the corresponding C-Intein is: H2N-GVPVHN-COOH (was aquired by peptide synthesis and is thus not part of the registry) This part can also be used in order to create fusion proteins, by attaching the C-Intein to a proein instead of the matrix. Part in RFC25 standard.
Part was designed in RFC25, since it is specifically used for fusion of peptides
Intein of DNA grase subunit B of the cyanobacterium Synechocystis species, strain PCC6804. Sequence optimized for codon usage in E. coli K12 and synthesised.
Translational unit of mRFP-galectin3-his
This part is a translational unit of a mRFP-galectin-3-his (B0032.E1010.K1319003.K1319016.B0015)
The stop of K1319003 was removed and substituted by a His6-tag.
The part is a combination of other BioBrick parts.
RBS + NpuDnaE N-intein RFC[105] assembly construct (with His6)
Deinococcus radiodurans UV DNA damage endonuclease - uvsE
TODO - mention the different starting points
Deinococcus radiodurans R1 genomic DNA from Chromosome 1
This part is an inverting regulator sensitive to LacI (BBa_C0010, BBa_C0012, etc.) The T7-lac promoter is a hybrid regulatory region consisting of the T7 promoter of T7 phage next to LacO. The hybrid design allows for strong promotion that can nevertheless be:  repressed by LacI  induced by lac or IPTG, showing a high expression level.  CAP protein will not influence the expression. Strong promoter. Only E.Coli BL21(DE3) can use this part.
expression PⅢ, which is from M13 phage genome, with the regulation of araBAD promoter.
pRhamnose regulating csgB and csgA curli proteins
This part is meant to express the csgB and csgA curli genes, which respectively code for the production of the curli protein that is anchored to the cell membrane (CsgB); and for the major curli protein (CsgA), which forms long fibrils with other CsgA monomers in a self-assembly process. These CsgA long fibrils bind to the anchored CsgB protein forming the amyloid-like curli structure. The expression of these two genes will, then, occur at the same time and under the same conditions (in the presence of Rhamnose in this case) This construct was designed as a negative control for BBa_K1316013 and BBa_K1316014, to see if expressing CsgA constitutively has a positive impact on the speed of curli formation
This construct was designed as a negative control for BBa_K1316013 and BBa_K1316014, to see if expressing CsgA constitutively has a positive impact on the speed of curli formation
csgA and csgB were PCRed out of the K540000 Lyon 2011 iGEM part.
GFP (mut3b) with LVA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LVA-ssrA degradation tag. The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part E1010, the tag sequence was obtained from paper by Andersen et al., (1998).[2]
The RBS is from pmsp3 promoter, the strongest promoter isolated from Magnetospirillum bacteria. mntH was functionally expressed in Azospirillum sp. B510. It encodes a divalent metal cation transporter. The MntH protein and mCerulean protein are linked by a fusion protein linker. The purpose of the linkage is to improve the expression/folding of MntH and for better visualization of successful transformation. The original use is to express MntH in Magnetospirillum magneticum strain AMB-1.
We researched the optimum linker sequence for fusion protein to facilitate successful fusion. The mCerulean protein is expected to improve the folding of MntH in AMB-1.
RBS is predicted from pmsp3 promoter from Magnetospirillum magneticum strain AMB-1 genome. MntH is from part BBa_K1505000. mCerulean is from part BBa_K133066.
This part is a combination of a pBAD promoter(BBa_I13453), RNA thermometer(BBa_K115003), and a RFP gene.
Escherichia coli CRP protein is a cAMP-activated global transcription factor. CRP protein binds cAMP and leads to the activation of transcription of more than one hundred promoters. This protein enhances the possibility of RNA polymerase holoenzyme binding and initiating transcription.
Introduced a mutation from ACTTCA to ATAATT.
RBS + NpuDnaE C-intein RFC[105] non-slicing control construct (with His6)
This part is a combination of a pBAD promoter(BBa_I13453), RNA thermometer(BBa_K115001)
This construct encodes a truncated version of E. coli W3110 SerA ( D-3-phosphoglycerate dehydrogenase), missing the last 75 aa of the full sequence, and without stop codon. SerA is requiered for serine biosynthesis. Following the work of Peters-Wendisch et al. 2005, this mutated version of the enzyme is expected to be no-longer inhibited by Serine, and to allow the production of a high serine concentration.
The part was obtained by PCR from E.coli strain W3110 and SLIC assembly. The construction removed the EcoR1 restriction sites in the gene by the silent mutations of a GAA codon to a GAG codon.
Amplified by PCR from E.coli W3110 genomic DNA.
Response regulator of the cusCFBA-cusRS divergent operons fused with a leucine zipper.
Amplified from pWW2021,Whitaker et al. PNAS 2012
Black cutworm (Agrotis ipsilon) Spread: This Caterpillar can be found, as various species, throughout North America. Characteristics: Cutworms common in Georgia fields are black (Agrotis ipsilon (Ashmed)), granulate (Agrotis subterranea (Fabricius)) and variegated cutworms (Peridroma saucia(Hubner)). These are moths in the family Noctuidae. Full-grown cutworm larvae are 1.5 to 2 inches long. Coloration will vary among species, but all tend to be stout-bodied caterpillars with four sets of prolegs. They have the tendency to curl into a ball when disturbed. Damage: Almost any plant can be attacked in the seedling stage. Cotton and certain vegetables sometimes have stand reductions. Climbing cutworms, such as the granulate cutworm, can be serious foliage feeders on some crops such as peanuts. Control: Bacillus thuringiensis, a widely available caterpillar-killing bacterium,is a very effective control for climbing cutworms as well as for the surface feeders.
Signal peptide of B. subtilis minor autolysin LytE
Signal peptide (amino acids 1 to 25) of ''Bacillus subtilis'' minor autolysin LytE (probable peptidoglycan endopeptidase, Uniprot [http://www.uniprot.org/uniprot/P54421 P54421]). Targets proteins for secretion via the Sec pathway (Tjalsma ''et. al.'', 2000). This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
This part was generated by amplification from BioBrick [http://parts.igem.org/Part:BBa_K1351007 BBa_K1351007] with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytE_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC ATGAAAAAGCAAATCATTACAGCTACGACAGC LyteSP_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TGCAGATGCCGCTCCTGCAAATAAC
RBS + SspDnaX C-intein RFC[105] assembly construct (with His6)
SdpI with RBS: Immunity against the cannibalsim toxin sdpC of B. subtilis
This gene codes for the immunity against a cannibalism toxin of Bacillus subtilis namely the sdpC. The sdpI gene is regulated by a separate promoter (PsdpIR) which is responsible for the operon sdpIR. Cannibalsim toxins are mainly produced by b. subtilis in mid to late stationary phase when nutrients are limited.
No changes were preformed regarding the sequence referring to the bio brick standard.
The sequence comes from a PCR with B. subtilis gDNA template.
This part is a combination of a pBAD promoter(BBa_I13453), RNA thermometer(BBa_K115002), and VIII gene from M13 phage(c).
This reporter part is a combination of a constitutive promoter, AraC gene, a pBad promoter (inducible with arabinose), and a RFP gene. The purpose of this part is to provide the user with a simple, but complete, inducible RFP genetic device. Adding arabinose with any concentration higher than 0.01 mM seems to cause a significant expression of the RFP gene. This is useful if the user plans to build a larger plasmid construct in a short time or characterize a specific part. This part can be inserted in a cell line that does not contain any AraC in its genome, which is important if the user is not using Top 10 cells.
The RBS, RFP, and Terminator were taken from BBa_J61002 using SpeI and PstI restriction enzymes.
The part is obtained from ligating several biobricks.
RBS + NpuDnaE N-intein RFC[105] non-splicing control construct (with His6)
Ag43+FLAG+MCS under the control of pBAD promoter
Antigen 43 (sometimes called Ag43 or fluffing protein) is a phase-variable outer membrane protein encoded by flu gene. It is native to E.Coli K12 strain and is usually expressed at about 50, 000 copies/cell. Ag34 precursor is 1039 amino acids long and subsequently becomes cleaved into alpha and beta chains (499 and 488 amino acids long respectively). The beta subunit forms a β-barrel pore via which alpha-subunit translocates to the cell surface, and with which it remains non-covalently joined. The surface alpha chain can be released by a brief heat treatment at approx. 60oC. Ag43 is an autotransporter protein, therefore it possesses all information necessary for translocation to the cell surface in its coding sequence. Ag43 mediates autoaggregation, via a velcro-like mechanism (Heras et. al., 2014), and plays a role in E.coli biofilm formation. Interestingly, the alpha subunit is able to express foreign peptide sequences on E.coli cell surface if inserted just in front of codon 148 (Kj??rgaard et. al., 2002). BioBrick Bba_K1352001 is a modified version of the Bba_K1352000 BioBrick. It is composed of an Ag43 coding sequence with an in-frame FLAG epitope tag flanked by BglII and HindIII multiple cloning sites inserted within the alpha-subunit in front of codon 148 of Ag43. Expression of Ag43 is under the control of the pBAD promoter; it includes a ribosome binding site as well as two transcriptional terminators. Structure of the BioBrick was designed to allow easy insertion of foreign protein sequences of choice at codon 148 with a simple restriction digest with BglII and HindIII followed by ligation. Potential uses of this BioBrick include surface display of foreign peptide sequences, E.coli aggregation tests as well as synthetic vaccines production.
FLAG tag epitope flanked by multiple cloning sites was designed to be in-frame with Ag43 ORF; it is optimized for E.coli codon bias.
This BioBrick was PCR amplified from Bba_K1352000; in-frame FLAG tag epitope flanked by multiple cloning sites was inserted via In-Fusion (Clonetech).
This part can be used for IPTG-induced expression of K660004 (iLOV).
The part was assembled by Gibson assembly starting from K731520. The open reading frame was exchanged.
The part was combined from previously existing BioBricks.
Kis Antitoxin (Kis-Kid Toxin-Antitoxin System)
Kid antitoxin regulated by lacI promoter
signal sequence from USP45 fused to aiiA
- one nucleotide in the signal sequence of USP45 had to be changed because it contained the illegal restriction site PstI
-aiiA was isolated out of the biobrick BBa_C0060. -ssUSP45 was isolated from the synthetic gene ssUSP45DspB made by IDT.
tsPurple Reporter (RBS + Coding + Terminator)
Reporter to add onto parts to see if they are functional
Nitric oxide reductase (norC) from Pseudomonas aeruginosa PAO1
This gene codes for the nitrite reductase (nirS) that converts nitrite (NO2-) into nitric oxide (NO). It is cloned into the pSB1C3
RBS + SspDnaB C-intein RFC[105] assembly construct (with His6)
Choi hiu Ying, LEE Cheuk Lam, Tse Yui Shing, Yeung Sin Yu
This sequence encoded for the delta15 desaturase. The gene is from Synechocystis sp. and optimised for the expression in E. coli. During the fatty acid conversion process, delta 15 desaturase catalyses the conversion of linolenic acid (18:2) into alpha-Linolenic acid, ALA (18:3) by adding a cis-doube bond at carbon 15.
The enzyme cut site must be designed according to iGEM prefix and suffix requirements.
The amino acid sequence of delta 15 desaturase is derived from Synechocystis sp. but codon optimised for the expression in E. coli.
This part is derived from Streptococcus mutans UA159, a gram positive bacterium. The main function of this promoter is to sense a quorum sensing chemical, competence-stimulating peptide (CSP), and trigger down string gene.
Since there is no accurate description of actual position of nlmC promoter in previous study, to ensure the function of this promoter, we amplified the sequence from two comE binding site( used for detecting CSP) to the last nucleotide before gene nlmC. As a result, the gene sequence consists of two comE binding site, nlmC promoter and RBS.
Oriental Leafworm Moth (Spodoptera litura) Spread:It is found in the Indo-Australian tropics. It is also established on most Polynesian islands where it occurs in a variety of island forms. Characteristics: Adult moths measure between 15-20 mm (0.59-0.79 inches) in length and have a wingspan of 30-38 mm (1.18-1.5 inches). Forewings are gray to reddish-brown, with a complex pattern of creamy streaks and paler lines along the veins. Hind wings are grayish-white with grayish-brown margins. Males have a blue-grey band from the upper corner (apex) to the inner margin of each forewing. Larvae have bright yellow stripes along the back and the sides. Larval color varies from pale green to dark green, Damage: Oriental Leafworm Moth Spodoptera litura is a Noctuid moth which is considered as an agricultural pest. It is also known as the Cluster caterpillar, Cotton leafworm, Tobacco cutworm, and Tropical armyworm. It has a very wide host range of over 120 plant species, including: lettuce, cabbage, beetroot, peanuts, geranium, cotton, banana, fuchsias, acacia, African oil palm, amaranth, alfalfa, strawberry, sorghum, sugarcane, tomatoes, asparagus, apple, eggplant, beet, beans, broccoli, elephants ear, horsetail she oak, corn, flax, lantana, papaya, orange, mango, leek, among many others. Control: The use of Bacillus thuringiensis (BT) may effectively control this pest. Other forms of biological, horticultural, and cultural control that have been studied include: planting near derris and garlic plants, breeding resistant plants from wild plants for example groundnuts from wild groundnuts, breeding resistant plants using bacterium Bacillus thuringiensis genes, using a Baculovirus, using the nematode Steinernema carpocapsae, and using the fly Exorista japonica.
RBS + SspDnaB N-intein RFC[105] assembly construct (with His6)
CBD2 is EnLinker-CBH2-EnLinker-CBH1 from T.reesei. TBC
superfolder GFP with N-terminal pdu-microcompartment tag
We have used superfolded GFP with the N-terminal pdu-microcompartment tag in order to get fluorescence images that would show localised spots of higher GFP-density, indicating that the microcompartment tag successfully inserts the attached protein into the microcompartments
We have designed Primers to amplify sfGFP from a plasmid, provided by Dr. Ciaran Kelly, in order to attach the microcompartment tag upstream of the sfGFP-coding region.
We received a sample of sfGFP in a holding vector, provided by Dr. Ciaran Kelly.
PelB leading sequence as RFC[105] N-terminal insert
Ag43+FLAG+MCS(-)beta-hairpins under pBAD promoter
Antigen 43 (sometimes called Ag43 or fluffing protein) is a phase-variable outer membrane protein encoded by flu gene. It is native to E.Coli K12 strain and is usually expressed at about 50, 000 copies/cell. Ag34 precursor is 1039 amino acids long and subsequently becomes cleaved into alpha and beta chains (499 and 488 amino acids long respectively). The beta subunit forms a β-barrel pore via which alpha-subunit translocates to the cell surface, and with which it remains non-covalently joined. The surface alpha chain can be released by a brief heat treatment at approx. 60oC. Ag43 is an autotransporter protein, therefore it possesses all information necessary for translocation to the cell surface in its coding sequence. Ag43 mediates autoaggregation, via a velcro-like mechanism (Heras et. al., 2014), and plays a role in E.coli biofilm formation. Interestingly, the alpha subunit is able to express foreign peptide sequences on E.coli cell surface if inserted just in front of codon 148 (Kj??rgaard et. al., 2002). The shape of α-subunit of Ag43 resembles letter L. It consists of a 'β-helix domain [which form] the stem of the letter L, followed by three rungs flanked by four β-hairpin motifs that bend the protein by about 110?? and a C-terminal (...) parallel β-helix domain [which forms] the bottom of the letter L' (Heras et. al., 2014). It has been indicated that this unique shape plays a crucial role in cell-to-cell aggregation via velcro-like mechanism, in which α-subunits form a dimer by coling around each other. This interaction is strengthened by Van der Waals interactions, hydrogen bonds and salt bridges facilitated by the L-shape (Heras et. al., 2014). Recent research demonstrates that disruption of the bend and straightening of the shape by removal of two β-hairpin sequences eliminates self-association of Ag43 proteins (Heras et. al., 2014). Removal of β-hairpins does not interfere with protein translocation to the cell surface membrane. This BioBrick is a modified version of the Bba_K1352001 BioBrick. It is composed of an Ag43 coding sequence with an in-frame FLAG epitope tag flanked by BglII and HindIII multiple cloning sites inserted within the alpha-subunit in front of codon 148 of Ag43. Two beta hairpin sequences within Ag43 were removed via In-Fusion (Clonetech). Expression of Ag43 is under the control of the pBAD promoter; it includes a ribosome binding site as well as two transcriptional terminators. Structure of the BioBrick was designed to allow easy insertion of foreign protein sequences of choice at codon 148 with a simple restriction digest with BglII and HindIII followed by ligation. Potential uses of this BioBrick include surface display of foreign peptide sequences when aggregation is undesirable, as well as synthetic vaccines production.
PCR amplified from Bba_K1352001; two hairpin sequences (H1 268 AATVTGTNRLGAFSVVA 284; H2 341 GAAVSGTRSDGKAFSIG 357) were removed via In-Fusion (Clonetech).
Yellow fever mosquito (Aedes aegypyi) Spread:The yellow fever mosquito, Aedes aegypti, is a mosquito that can spread the dengue fever, chikungunya, and yellow fever viruses, and other diseases. The mosquito can be recognized by white markings on its legs and a marking in the form of a lyre on the thorax. The mosquito originated in Africa, but is now found in tropical and subtropical regions throughout the world. Characteristics: The mosquito can be recognized by white markings on its legs and a marking in the form of a lyre on the thorax. Damage: The yellow fever mosquito, Aedes aegypti, is a mosquito that can spread the dengue fever, chikungunya, and yellow fever viruses, and other diseases. Control:(1) Empty water from containers such as flower pots, birdbaths, pet water dishes, cans, gutters, tires and buckets regularly to disrupt the mosquito breeding cycle. (2)Consider using an insect repellent, be sure to follow the label directions for applying the repellent. For help selecting a mosquito repellent, try our Insect Repellent Locator.
E. coli IIA(Glc) gene (BBa_K1508002) followed by double terminator (BBa_B0015)
Construct was assembled by suffix insertion, namely BBa_B0015 fragment was inserted into BBa_K1508002 backbone.
IIA(Glc) gene from Escherichia coli; double terminator from iGEM Registry
ModA is a subunit of ModABC(ATP-binding cassette) trabsporter protein which can bind molybdenum. Molybdenum is an essential component of a large number of enzymes, where it is present in the form of two types of cofactor: the iron???molybdenum cofactor, in nitrogenase, and the molybdopterin cofactors in all the other known molybdoenzymes. Molybdenum is a relatively abundant element in the earth???s crust, in two main chemical forms, MoS2,and MoO2-. Only Mo(VI)in the form of molybdate is readily soluble and stable in aqueous solutions, and it is thus the only relevant source of molybdenum for biological systems. We want to use ModA to bind molubdenum then carry it to the plants.
We change several sites of the gene to evaluate the binding efficiency.
CWB domain of B. subtilis minor autolysin LytE
Cell wall binding domain and signal peptide (amino acids 1 to 207) of ''Bacillus subtilis'' minor autolysin LytE (probable peptidoglycan endopeptidase, Uniprot [http://www.uniprot.org/uniprot/P54421 P54421]). This domain has been used for displaying beta-lactamase on the cell surface of ''B. subtilis'' (Chen ''et. al.'', 2008). Used in the [http://2014.igem.org/Team:LMU-Munich/Project BaKillus project] to display pathogen-specific peptides to mediate adhesion of ''B. subtilis'' to pathogens. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
Due to the N-terminal signal peptide, this part allows only C-terminal fusions of proteins to be displayed on the cell surface. The CWB domain of LytE consists of three N‐terminal LysM cell wall‐binding motifs, ranging from amino acid 28 to 193. To minimize possible effects of a translational fusion to the CWB domain, the part includes the downstream unstructered region until amino acid 207. An additional linker can be inserted to allow more structural flexibilty.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytE_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC ATGAAAAAGCAAATCATTACAGCTACGACAGC LytE_207_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT CGATGAAGATGAAGCAGGTTTTGAGC A SpeI site was removed by changing T to C at position 399 without altering the protein sequence by fusion PCR with the following mutagenesis primers: LytE_SpeI399mut_fwd: GTCCTGAAACTGAAAGGTTCAACcAGTTCAAGCAGCTCCAGCTCATCAAAAG LytE_SpeI399mut_rev: CTTTTGATGAGCTGGAGCTGCTTGAACTgGTTGAACCTTTCAGTTTCAGGAC
Sortase A non-circularization construct (with Smt3 and His6; without RBS)
GoldenGate compatible AmilCP, Blue chromoprotein
A variation of the part BBa_K592009 that has been made compatible with the GoldenGate MoClo Assembly Standard as it is free from BsaI and BpiI recognition sequences. The chromoprotein naturally exhibits strong blue color when expressed. Codon-optimised for expression in dicotyledenous plants.
Domesticated for GoldenGate by removal of internal BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants.
promoter with CIλ and Tet operator sites
Pseudomonas AutoInducer-1 response unit (luxR promoter)
All the inserts were digested with XbaI and PstI, and all the vectors were each time cut with SpeI and PstI.
All the parts have come from existing iGEM distribution kits.
mOrange with 6x His tag(BBa_K1491006) and a strong RBS (BBa_B0034). It can be used as a reporter.
RBS is BBa_B0034 and mOrange is BBa_E2050
This protein prevents the production of oxygen radicals. Our cells will produce this enzyme to block the production of oxygen radicals within the cells to prevent harmful effects.
We ordered this part to be synthesized. Sequence originally belongs to Bos taurus (bovine)
The wss operon was extracted from P.fluorescens, an organism that produces cellulose acetate.
Ag43 with PstI sites removed under the control of pBad/araC promoter
BBa_K1352000 is the modularized version of BBa_K759001, which is compatible with iGEM Assemly Standard RCF10.
We found that BBa_K759001 contained 6 additional PstI sites within the coding region of Ag43. We therefore removed the additional PstI sites, to meet the requirements iGEM Assemly Standard RCF10.
Rescued from BioBrick BBa_K1352000 and subjected to PCR-based site directed mutagenisis
This is a translational unit that include RBS and amilCP.
Cotton bollworm Helicoverpa armigera （Hubner） Spread:The pink bollworm has spread to cotton-growing regions throughout the world. Characteristics: The larva is green, khaki, grey-brown or brown with dark spots. The topside is darker than the bottom side and a yellow or light brown stripe goes round the middle portion by the spots. Damage: The cotton bollworm is a moth, the larvae of which feed on a wide range of plants, including many important cultivated crops. It is a major pest in cotton and one of the most polyphagous and cosmopolitan pest species. It should not be confused with the similarly named, related species Helicoverpa zea.The cotton bollworm is a highly polyphagous species.The most important crop hosts are tomato, cotton, pigeon pea, chickpea, sorghum and cowpea. Other hosts include groundnut, okra, peas, field beans, soybeans, lucerne, Phaseolus spp., other Leguminosae, tobacco, potatoes, maize, flax, Dianthus, Rosa, Pelargonium, Chrysanthemum, a number of fruit trees, forest trees and a range of vegetable crops. Control: Cultural controls, with the exception of the use of Bt cotton and the use of mating disruption and sprays of the Entrust formulation of spinosad are acceptable to use on organically grown cotton.
Sortase A circularization construct (with Smt3 and His6)
A right promoter which can sense formaldehyde while there is a left promoter which can erpress the HxlR protein theoretically.The HxlR protein can enhance the function of the P-formaldehyde.
The start codon of the HxlR protein is UUG.
We get this part from Bacillus subtilis 168.
Double CBD (dCBD) with N-terminal linker
The BCAM0228 Response Regulator from Burkholderia
This is the coding sequence of the BCAM0228 two-component regulator of the BDSF sensory BCAM0227-BCAM0228 system. This system is found in Burkholderia spp.
The sequence had 1 EcoRI site that was mutated out by site-directed mutagenesis.
The source of this part is Burkholderia cenocepacia J2315 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Coding region for cellulose binding domain. When fused to a protein, protein attaches to cellulose
Sequence was optimized for Escherichia coli and Arabidopsis thaliana codon usage.
Domain derived from cellulose binding protein from Chlostridium cellulovorans(WP_013291799).
merA, mercuric reductase from Serratia marcescens
Mercuric reductase catalyzes the reduction of Hg to Hg2+.
Mutagenesis of adenine to guanine at position 1026 to remove EcoRI site.
A araBAD promoter with araC transcripted from the negative direction.
We generate this regulatory part to regulate our reporter gene expression in our project.
The two parts are all from previous parts.
Inclusion of this part on a plasmid will facilitate the uptake of the plasmid as a single-stranded phagemid into the M13 phage. Note that this part does not code for phage coat proteins so phage containing this M13 ori and your part will not multiply. Therefore, the M13K07 or similar helper phagemid is required for phage production. This part is packaged preferentially over M13K07, reducing the risk of uncontrolled phage multiplication.
Orientation in relation to ori may affect packaging ability. Also, 3??? end contains the promoter for the M13 gene II. This is a strong promoter and may cause unwanted expression of down stream genes. We recommend placing coding regions upstream of part.
This promoter can sense the whether there is formaldehyde or not.It can be activitated by formaldehyde with the help of the protein hxlR.This promoter may have some leakage in the strain E.coli DH5a .
There may be different expression levels when you use it in different stains of E.coli.
This part comes from the Bacillus subtilis genomic sequence.
Combination of T7 promoter with the lac operator. For the promoter to be active both T7 RNA polymerase and the lac operon inducer (lactose or an analogue such as IPTG) must be present. This double regulatory system reduces the promoter leakage MtrCAB is an electron transport complex of the bacteria Shewanella oneidensis
In order to eliminate the illegal restriction sites, the entire construct was PCRed in different parts and reassembled via the Golden Gate Assembly method
Caroline M. Ajo-Franklin laboratory (Goldbeck et al. ACS Synth. Biol. 2013, 2, 150−159)
Phytochelatin (PC) EC20 fused to CBDcex with LacI promoter
n our project, we will biologically synthesize PBAN with our E.coli. We store the PBAN inside a trapping device (check this out at our Device page). In the device, there will be appropriate lighting and nutrient sources that will attract insects.Once an insect is attracted into our device and ingests the nutrient sources we provide, it will also inevitably come in contact with our PBAN, which is evenly mixed with the nutrient sources. As the PBAN works its magic and activates the pheromone synthesis of the attracted insect, more of this species of insect???s counterparts will be attracted and later captured.Owing to the first feature mentioned above, PBANs are species-specific, which means that it doesn't matter if other kind of insect fly into our device and eat PBANs, because the insects we don't want to catch will not be stimulated by PBANs to produce pheromone; our PBANs are only for what we want to catch, we are sure that our method won't affect other kinds of insects.
Artificial synthesis from known amino acid sequence.
Gypsy Moth (Lymantria dispar) Spread: It has a range which covers Europe, Africa, and North America. Characteristics:Gypsy moth caterpillars change appearance as they grow. Young caterpillars are black or brown and about ?? inch (.6 cm) in length. As they grow, bumps develop along their backs along with coarse, black hairs. Each of the 11 sections of a developed caterpillar will have two coloured spots, the first five pairs, blue, and the last six, red. Mature caterpillars can be as long as 2 ?? inches (6.35 cm). Damage: It is classified as a pest, and its larvae consume the leaves of over 500 species of trees, shrubs and plants. The gypsy moth is one of the most destructive pests of hardwood trees in the eastern United States.he gypsy moth was considered a nuisance just ten years after their release. It included an account of all the trees being defoliated, caterpillars covering houses and sidewalks and that the caterpillars would rain down upon residents. The first outbreak occurred in 1889. An eradication program was begun in 1890. Control: Tanglefoot Pest Barrier or Sticky Tree Bands can be placed around tree trunks to help curtail the caterpillars movement into and out of the tree canopy. Apply Bacillus thuringiensis, var. kurstaki or Monterey Garden Insect Spray (Spinosad) to the leaves of trees to kill gypsy moth caterpillars.
RBS + SspDnaX N-intein RFC[105] assembly construct (with His6)
The glmS riboswitch has a self-cleavage activity through which it regulates gene expression.
anti Pseudomonas aeruginosa secreting system
Lo Hiu Fung, Lau Tsz Kit, Chu Lok Ting, Wu Chun Ting
We have cloned a modified thioesterase gene, ???tesA, which codes for an E. coli thioesterase protein that lacks the N-terminal 26-amino acid signal peptide sequence, and is named here as ???leaderless TesA??? or ???TesA. The ???TesA protein is expected to be expressed exclusively in the cytosol of E. coli to catalyze the conversion of fatty acyl-ACP (acyl carrier protein) or fatty acyl-CoA to free fatty acids (Figure 1). ???TesA is predicted to enhance free fatty acid production in the genetically-modified E. coli strain.
The genome sequence of Escherichia coli K-12 substr. W3110 is available in GenBank at http://www.ncbi.nlm.nih.gov/gene/12930860.
Galactose-induced gene 4 DNA binding domain (Gal4DBD)
This yeast protein is a positive transcriptional regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which are important for galactose import and conversion to glucose []. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. GAL4 is used to investigate gene expressions in several organisms (bacteria, plants, fruit flyes) []. The protein is controlled by a strong promotor. Binding to UAS-G leads to the expression of the corresponding gene. ==References== BRAND, AH., PERRIMON, N.: Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401-15. TRAVEN, A., JELICIC, B., SOPTA, M.: Yeast Gal4: a transcriptional paradigm revisited. EMBO Rep. May 2006; 7(5): 496???499.
We received it from the AG Weber at the BIOSS Center, Freiburg, Germany.
CBDcenA with linker fused to NiBP in Freiburg format (RFC 25)
Lactose/IPTG inducible promoter with RBS, RFP reporter tagged with LVA-ssrA degradation tag and terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. This part can be considered as improved version of BBa_J04451 as it does not contain StuI restriction site coding for additional 'RP' residues between RFP and LVA tag.
Part was assembled using BrickClip assembly (BBF RFC 104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1]
This part has a constitutive promoter, so our PBAN( Aedes aegypti) can constitutive express.
MDFA is an antiporter which can make E-coli have a ability to tolerate high alkiline environment up to pH 10.
We use pcr to get the target genes from E-coli and invert them into pet28a which can multicopy.
The kanamycin resistance gene tn903 aph from e.coli for use with Synechocystis sp. PCC 6803. The kanamycin gene to allow transformation in Synechocystis sp. PCC 6803 (Poteete et al, 2006).
genomic DNA of kanamycin containing strain of Synechocystis sp. PCC 6803
Endo-1,4-beta-glucanase (cellulase gene)
Endo-1,4-&-beta-glucanase (EG 1) is an enzyme that breaks down the 1,4-beta-D-glycosidic linkages in cellulose to produce glucose. The coding sequence of this gene was from Neisseria Sicca bacterium that uses cellulose acetate as its sole carbon source. This enzyme is specific for 1,4-beta linkages in cellulose.
Since the gene is usually secreted by the original bacterium (Neisseria Sicca), it contains a signal sequence for extra-cellular secretion. We removed this signal sequence and replaced it with a secretion tag specific for E.coli, so that the enzyme can be secreted extracellularly. In order to allow protein purification and assays, we added a His-tag at the C' terminus of the gene.
The gene encoding trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) was isolated from Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene.
Pseudomonas fluorescens (strain: PC20, biotype: F) NCBI Gene ID: 7871527
Sortase A non-circularization construct (with His6; without RBS)
Ptac-NiCoTB, improves nickel and cobalt internalization
This part comprises a strong E. coli promoter with...
SigD-dependent flagellin promoter (Phag) of Bacillus subtilis
Phag promoter of ''B. subtilis'' flagellin under the control of SigD.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. Phag_ENX_fwd: TGCAGAATTCGCGGCCGCTTCTAGAG ttcacaggaaagaaatttacttgacc Phag_SNP_rev: AGCCTGCAGCGGCCGCTACTAGTA gaatatgttgttaaggcacgtcc
Laminin-derived peptide binding to NADH Oxidase of S. pneumoniae
Short peptide of 9 amino acids corresponding to amino acids 661 to 668 of the human protein Laminin (subunit alpha-5, UniProt [http://www.uniprot.org/uniprot/O15230 O15230]). It has been shown to bind to NADH oxidase (NOX) of ''Streptococcus pneumoniae'' and inhibit bacterial binding to human A549 cells (Muchnik ''et. al.'', 2013). Displayed on the surface of ''Bacillus subtilis'', it should enable ''B. subtilis'' to bind specifically to ''S. pneumoniae''. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
The part was generated by annealing of artificially synthesized oligonucletides: L5P_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG TGTCAAGAATGTTCACCGGGATTT ACCGGTtaatACTAGTagcggccgCTGCA L5P_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT AAATCCCGGTGAACATTCTTGACA CATGCCGGCttcctccttacTCTAGAagcggccgcG
This is a functional coding sequence for the protein NixA, which is a nickel transporter from H. pylori. This part will result in nickel intolerance from higher levels of intracellular nickel.
The Anderson promoter provides strong constitutive expression of NixA in E. coli.
Anderson promoter is that of part BBa_J23102. Terminator is that of part BBa_B1006. NixA gene is naturally found in H. pylori. This part was synthesized by GenScript in the vector pUC57. Part was then transferred into pSB1C3 using standard cloning procedures.
Synthetic Phytochelatin EC(20) rfc25 fusion format
Sequence is not natural. Basis for the sequence was from the following reference: Bae W, Chen W, Mulchandani A, Mehra RK (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol Bioeng. 70(5):518-24.
To regulate PIII expression with temperature on the translation level used RNAT.
The RNAT(Rose42) comes from bacteria genome and PIII; comes from M13 phage genome.
mRFP with 6x His tag (BBa_K1491008) and a strong RBS (BBa_B0034). It can be used as a reporter.
RBS + Circular Xylanase with His6 linker
ranscriptionfactor，the ipgC???mxiE complex activates transcriptionfrom the pipaH* promoter
gene clusters encoding type III secretion systems
Split sfGFP reconstitution N-terminal splicing half
This part is derived from Streptococcus mutans UA159. The main function of this promoter is to trigger downstream gene continually.
In order to maintain the complete function of this promoter, RBS is retained when amplifying the sequence with PCR. Consequently, the part consists of one constitutive promoter and RBS.
merB, Organomercurial Lyase from Serratia marcescens
Amplified from pDU1358, kindly donated by Anne O. Summers.
Bacterioferritin (BFR) M52H heme-deletion
The M52H Mutagensis leads to an deletion of the BFR heme residue. As a result, BFR will have a reduced iron release rate and keep it's iron content inside of the protein scaffold.
SsrA degradation tag as RFC[105] C-terminal insert
Luna Barroco de Lacerda, Maria Clara Tavares Astolfi
The biorremediator device is composed by our ???essential biobrick??? (BBa_K1355001) + a MerA gene (BBa_K1355000). In the presence of mercury, The MerR protein will bind to it and MerT, MerP and MerA protein will be expressed and bioremediation will start! The MerP and MerT proteins are responsible for the transport of mercury from the periplasm to the cytoplasm, leading him to the mercury ion reductase (MerA)! The beautiful MerA be responsible for the reduction from Hg2+ in Hg0, which is volatile at room temperature and able to passively leave out the bacteria.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the MerA Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for merA Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of merA gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + merA Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +merA Biobrick ;
The cblD promoter from Burkholderia
This is a promoter that is activated by the OmpR-like two-component response regulator BCAM0228 (Bba_K1315007) in the BCAM0227-BCAM0228 sensor system. The Burkholderia diffusible signal (BDSF) is being picked up by the BCAM0227 sensor kinase that is hypothesised to eventually lead in involvement of BCAM0228. BCAM0228 is then activating cblD.
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector
Lactose/IPTG inducible promoter with RBS, RFP reporter tagged with LAA-ssrA degradation tag and terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity.
Nickel Binding Protein (NiBP) Freiburg format (RFC[25])
Nickel Binding Protein in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
OEPCR primers were designed to allow for the replacement of the standard BioBrick prefix and suffix with the Freiburg prefix and suffix.
This part is based on the existing BioBrick BBa_K1151001 by the 2013 Salente Lecce iGEM team.
NADPH-dependent flavinoid reductase relate to Met-DMSP byosynthesis pathway(REDOX)
This part is NADPH-dependent flavinoid reductase gene of Fragilariopsis cylindrus. The gene code NADPH-dependent flavinoid reductase relate to Met-DMSP byosynthesis pathway from Fragilariopsis cylindrus. which catalyze second step of Met-DMSP biosynthesis pathway.
Fuse lambda cI with LGF2 mutated from Asp to Ala at site 78. As one of the components in Bacterial Two-hybrid System, it causes a less interaction with GAL11P compared to the nomalized WT.
From Agilent Technologies' BacterioMatch ⅡTwo-Hybrid System Vector Kit.
Mer operon, biological system found to detoxify organic and inorganic forms of mercury
converts organomercuric and cationic mercury to the less toxic volatile mercury.
The original plasmid obtained from A. O. Summer's lab contained a the EcoRI restriction site within the operon. Mutagenesis by the UMN iGEM team removed this internal restriction site.
This part was acquired courtesy of A. O. Summer's lab, in the pDU1358 plasmid.
quorum sensing machinery that activates GFP expression
This part is the combination of two parts, BBa_I13202 and BBa_T9002. The first part (BBa_I13202) produces a luxI enzyme under the control of a lac repressible promoter. The second part (BBa_T9002) produces luxR, which combines with the luxI to activate the promoter that controls expression of GFP.
Chitin-binding wasp protein 2 (PdomMRNAr1.2-08705.1)
CBDcenA with linker fused to NiBP driven by LacI
Split sfGFP reconstitution N-terminal non-splicing control half
Signal peptide of B. subtilis endonuclease YhcR
Signal peptide (amino acids 1 to 46) of ''Bacillus subtilis'' endonuclease YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]. YhcR is secreted and covalently anchored to the petidoglycan wall by the sortase YhcS. Despite its signal protein of RR-type, which is typical for proteins exported via the Tat pathway, export of YhcR is not Tat-dependent and seems to function via the Sec pathway (Tjalsma ''et. al.'', 2004). A YhcR-based surface display system has been shown to covalently anchor heterologous proteins to the ''B. subtilis'' cell wall (Liew ''et. al.'', 2012). It was used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides and mediate adhesion of ''B. subtlis'' to pathogens. It consists of four components: the sortase substrate with a signal peptide (this part), a linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) and a cell wall-anchoring domain ([http://parts.igem.org/Part:BBa_K1351010 BBa_K1351010]) as well as the sortase YhcS itself ([http://parts.igem.org/Part:BBa_K1351011 BBa_K1351011]), whose overexpression increases the efficiency of the surface display.
The native signal peptide of YhcR was used instead of known Sec-dependent signal peptides as Liew ''et. al.'' did 2012.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. SP_YhcR_ ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC ATGCTGTCTGTCGAAATGATAAGC SP_YhcR_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT AGCTTCGAACGTGTACATTACATTTAAG
RFP from Discosoma striata (coral) with LAA-ssrA degradation tag (wt)
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012)
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0050, but same tag was also used in paper by Andersen et al., (1998).[2]
CBDcex fused to Phytochelatin (PC) EC20 with T7 promoter
RBS + TerDnaE3 C-intein RFC[105] assembly construct (with His6)
cell division topological specificity factor MinE
The clp gene from Xanthomonas campestris
c-di-GMP Repressed DNA Binding Protein (Clp) Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads. http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris
The sequence had 1 PstI site that was mutated out by site-directed mutagenesis.
The source of this part is Xanthomonas campestris pv. campestris 8004 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Pseudomonas AutoInducer 1 (PAI-1) Biosensor
This is a composite part that consists of 4 biobricks. In the front it has ptet(Bba_R0040) which is constitutively on. This produces LasR all the time with lasR(Bba_C0179) next in the sequence. Only in the presence of the Pseudomonas Autoinducer 1 (PA1), LasR forms an complex with PA1 and it can then activate the plasB(Bba_R0079) promoter that leads to the production of GFP(Bba_E0040).
The pqsA promoter from Pseudomonas
The Pseudomonas quinolone signal (PQS) is imperative in modulating mechanisms that enable Pseudomonas aeruginosa to survive at times of stress. P. aeruginosa is the main pathogen responsible for severe deterioration in the Cystic Fibrosis lungs. The production of this signal relies on the pqsABCDE operon. The transcriptional factor PqsR (BBa_K1315000) positively regulates this operon. http://www.ncbi.nlm.nih.gov/pubmed/25225275 http://www.ncbi.nlm.nih.gov/pubmed/16735731 http://www.ncbi.nlm.nih.gov/pubmed/21539583 http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs http://www.ncbi.nlm.nih.gov/pubmed/12393198
The source of this part is the genome of Pseudomonas aeruginosa PAO1 provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Monomeric Red Fluorescent Protein from Discosoma striata
Mutant of the Biobrick BBa_E1010, in order to make it compatible with the Freiburg-Standard, two AgeI-Restriction sites were deleted monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
The Biobrick has the Following Prefix and Suffix: Gatcgaattccgcggccgcttctagataaggaggagccggc Gatcctgcagcggccgctactagtattaaccggt
Codon optimized KillerRed with WTLac promoter allows for induction of KillerRed expression with IPTG.
CBDclos fused to Phytochelatin (PC) EC20
CWB domain of B. subtilis major autolysin LytC
Cell wall binding domain and signal peptide (amino acids 1 to 333) of ''Bacillus subtilis'' major autolysin LytC (Uniprot [http://www.uniprot.org/uniprot/Q02114 Q02114]). This domain has been used for displaying heterologous proteins on the cell surface of ''B. subtilis'' (Tsuchiya ''et. al.'', 1999). Used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides by C-terminal fusions to mediate adhesion of ''B. subtlis'' to pathogens.
Due to the N-terminal signal peptide, this part allows only C-terminal fusions of proteins to be displayed on the cell surface. The CWB domain of LytC consists of three N‐terminal type II cell wall‐binding motifs, ranging from position 30 to 318. To minimize possible effects of a translational fusion to the CWB domain, the part includes the downstream unstructered region until position 333. An additional linker can be inserted to allow more structural flexibilty.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytC_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC TTGCGTTCTTATATAAAAGTCCTAACAATGTG LytC_333_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TGAATCTTGATCACCGTGACCC
Split sfGFP reconstitution positive control
cI repressor protein regulated by L-rhamnose promoter
Ptac-NiCoTB-rcn-csgBAEFG, turns E. coli into a functional Ni and Co biofilter
This part, when expressed in a rcnA E. coli strain, allows the formation of a functional Ni2+ and Co2+ biofilter capturing up to 3 mg of metal per g of bacterial dry weight when exposed to 20 ??M Ni2+ or Co2+, in only 10 mn. This is one of the best performances and the fastest reported until now.
[lien] and [lien] were cloned by 3A assembly.
CBDcex fused to phytochelatin (PC) EC20 with LacI promoter
Methanococcus jannaschii tyrosyl-tRNA synthetase
Pyrrolysyl-tRNA Synthetase introduces the Non Natural Amino acid p-Azido-L-Phenylalanine at the TAG codon.
SuperYFP2 with 6x His tag(BBa_K1491004) and a strong RBS (BBa_B0034). It can be used as a reporter.
RBS is BBa_B0034 and SuperYFP2 is BBa_K864100
Sortase A circularization construct (with His6; without RBS)
Chaperone,the SicA???InvF complex activates transcription from the psicA promoter
Deinococcus radiodurans uracil-DNA glycosylase 2
Split sfGFP reconstitution C-terminal non-splicing control half
Contactin-4-derived peptide binding to NADH Oxidase of S. pneumoniae
Short peptide of 13 amino acids corresponding to amino acids of the human protein Contactin-4 (isoform 3, UniProt Q8IWV2-3). It has been shown to bind to NADH oxidase (NOX) of Streptococcus pneumoniae and inhibit bacterial binding to human A549 cells (Muchnik et. al., 2013). Displayed on the surface of Bacillus subtilis, it should enable B. subtilis to bind specifically to S. pneumoniae. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
The part was generated by annealing of artificially synthesized oligonucletides: C4P_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG TGGGAATGTAAAGCGAACGGAAGACCGAAACCGACATAT ACCGGTtaatACTAGTagcggccgCTGCA C4P_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT ATATGTCGGTTTCGGTCTTCCGTTCGCTTTACATTCCCACATGCCGGCttcctccttacTCTAGAagcggccgcG
DsRed Expression Cassette under a Constitutive Promoter
This part has a constitutive promoter, so our PBAN(Solenopsis invicta) can constitutive express.
Pseudomonas AutoInducer-1 response unit (lasB promoter)
Transcription factor involved in pyoverdine synethesis gene transcription
outer membrane porin protein C from Escherichia coli BL21(DE3)
One OmpC molecule consists of 16 transmembrane, antiparallel β-strands, which produce a β-barrel structure surrounding a large channel and are connected by seven internal loops and eight external loops. Three OmpC molecules form a pore structure on the outer membrane of an E.coli cell, which allows small hydrophilic molecules to pass through.
In general, the amino acid sequences of the external loops are less conservative, and thus these loops may be relatively tolerant to insertion and deletion. We used the external loop as the point of insertion for foreign peptides (copper binding polypeptide or polyhistidine) for cell surface display.
This part is from genome of Escherichia coli BL21(DE3).
RBS + Gp41-1 N-intein RFC[105] assembly construct (with His6)
RBS + TerDnaE3 N-intein RFC[105] assembly construct (with His6)
RBS + SspDnaB N-intein RFC[105] non-splicing control construct (with His6)
Juliette Paillet, Alexandre Duprey, Emiel van der Kouwe
This part is a combination of a pBAD promoter(BBa_I13453), RNA thermometer(BBa_K115001), and a RFP gene.
All this parts come from the distribution.
Cellulose binding domains with streptavidin domain generator
Different sequences for cellulose-binding domains to prevent homologous recombination. Removed cut sites for RFC-10 compatibility.
Cellulose-binding(clostridium cellulovorans)
Cell wall-anchoring domain of B. subtilis endonuclease YhcR
Cell wall-anchoring domain (amino acids 1086 to 1217) of ''Bacillus subtilis'' endonuclease YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]). YhcR is secreted and covalently anchored to the petidoglycan wall by the sortase YhcS. A YhcR-based surface display system has been shown to covalently anchor heterologous proteins to the ''B. subtilis'' cell wall (Liew ''et. al.'', 2012). It was used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides and mediate adhesion of ''B. subtlis'' to pathogens. It consists of four components: the sortase substrate with a signal peptide ([http://parts.igem.org/Part:BBa_K1351008 BBa_K1351008]), a linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) and a cell wall-anchoring domain (this part) as well as the sortase YhcS itself ([http://parts.igem.org/Part:BBa_K1351011 BBa_K1351011]), whose overexpression increases the efficiency of the surface display. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. CWAD_YhcR_ ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCTGAAGCCAGTGATATTGAAGACCC CWAD_YhcR_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TCACGTTCTGGAGGCGCTC
This is a reporter biobrick that can be regulated by C4-HSL and reduced by C12-HSL, also,there need another two coding sequences, RhlR and LasR.
Modified rhlA coding sequence from Pseudomonas aeruginosa SQ6
RhlA together with RhlB encodes Rhamnosyltransferase I for mono-rahmnolipids biosynthesis in Pseudomonas aeruginosa.
A mutation has been done to remove the PstI restriction site contained in the coding sequence.
Peptide 1018 is a synthetic cationic peptides, derived from natural peptides such as the human cathelicidin LL-37 and the bovine peptide indolicidin. This part work as biofilm inhibitory compounds. Anti-biofilm peptides are similar to cationic antimicrobial peptides (which are active against planktonic bacteria), comprising both cationic and hydrophobic amino acids.
This part will have broad spectrum effect on gram-negative and gram-positive bacterias
RFP from Discosoma striata (coral) with LVA-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LVA-ssrA degradation tag. The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. This tag is commonly attached to repressor proteins for use in various gene networks (e.g., oscillators). The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; protein stability; Km of binding to the protease; temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction sites. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from paper by Andersen et al., (1998).[2]
This is the composite T7 RNA Polymerase and DNA Polymerase
We used the biobrick (BBa_E0840) available in the database of the iGEM since 2004 to be connected to our ???essential biobrick??? (BBa_K1355001) and - together - form a system capable of biodetect mercury in bacteria. In the presence of mercury, The MerR protein will bind to it and MerT, MerP and the Green Fluorescent Protein (GFP) protein will be expressed and the biodetection will begin! The MerP and MerT protein are responsible for the transport of mercury from the periplasm to the cytoplasm and the GFP will act as rep??rter to alerting the presence of mercury in the middle and the higher concentration of mercury is, higher will be the GFP expression. And If there is no mercury in the middle, the synthesis of MerT, MerP and GFP protein will be repressed by the MerR repressor protein.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the GFP Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for GFP Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of GFP gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + GFP Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +GFP Biobrick ;
Smt3-Murine DNMT1(731-1602) fusion protein (with His6)
* Smt3: ''Saccharomyces cerevisiae'' * DNMT1: ''Mus musculus''
aadD - Kanamycin Resistance Gene Coding Sequence
To test the RdRP against a control, we used a deliberately mutated version of itself. To mutate it, we used the restriction enzyme BgLII (code: agatct). This was not thought through but arbitrarily chosen, and resulted in a shift of frame and the introduction of a stop codon (tga).
Principally, our goal with this part was to create a non-functioning version of our RdRP which we could test it against, to ensure it was the RdRP proper that was having the desired effect of replicating our RNA.
The source from which this mutant is derived is our RdRP proper, part BBa_K1442100.
The M13ori-pCas9 composite part consists of the M13 origin of replication (M13ori) and the pCas9 part (BBa_K1218011) containing a tracrRNA sequence, the cas9 gene, and a minimal CRISPR array. M13ori (BBa_K1445000) is a 500bp noncoding sequence that is recognized by the gene II of the M13 phage, which results in the uptake of the plasmid containing the ori into an assembled phage coat. This part does not contain any complete phage genes so phage production requires a Helper Phagemid such as M13K07. There will be some packaging of Helper Phagemid but their packaging signal weakened so parts containing the M13ori are preferentially packaged. pCas9 contains the native trcrRNA and promoter region upstream of the Cas9 protein. The type II Cas9 protein is the native, active version from streptococcus pyogenes with the exceptrion of a SNP to erase an illegal EcoRI cleavage site. Downstream of the Cas9 protein is a minimal CRISPR array. This minimal array only includes two CRISPR repeats flanking a spacer region. It is this spacer region that determines which DNA sequence is targeted by the Cas9 endonuclease. Spacer sequence can be easily replaced with the protocol found here.
We have experienced some toxic effects by the pCas9 part so we suggest growing part-containing cells in lower concentrations of antibiotic, even if plasmid contains a high copy origin of replication.
The M13 origin of replication from Litmus28i and part BBa_K1218011 from the registry.
Fuse lambda cI with LGF2 mutated from Glu to Ala at site 75 (the Glu which locates after three continuous Ala behind lambda repressor is at site 68). As one of the components in Bacterial Two-hybrid System, it causes little interaction with GAL11P compared to the normalized WT.
The LGF2 is mutated from GAA to GCA at site 75.
It is from Agilent Technologies' BacterioMatch ⅡTwo-Hybrid System Vector Kit and modified by Site-Directed Mutagenesis(SDM).
RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter
his part serves as a functional metallothionein for heavy metal binding and conferred resistance to heavy metals when combined with an appropriate promoter. CRS5 gene is the metallothionein that binds to heavy metals and it is fused to GST for stability.
The part contains a KpnI cut site between the biobrick prefix and the RBS that is a result of removing the T7 promoter from part BBa_K1460001.
This part is BBa_K1460001 with the T7 promoter removed.
RFP from Discosoma striata (coral) with AAV-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part E1010) with added AAV-ssrA degradation tag (part I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: 1) ClpXP and ClpAP protease and SspB mediator concentrations; 2) Protein stability; 3) Km of binding to the protease; 4) Temperature [3]. [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3]Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part E1010, tag sequence was obtained from part I11012, but same tag was also usead in paper by Andersen et al., (1998).[2]
Luna Barroco de Lacerda, Maria Clara Astolfi
The Bioaccumulative biobrick is composed of our ???essential biobrick??? (BBa_K1355001) + a Metallothionein (BBa_K346004) available in the database of the iGEM since 2010.In the presence of mercury, The MerR protein will bind to it and MerT, MerP and Metal Binding Protein (MBP) proteins are synthesized and bioaccumulation will start! The MerP and MerT protein are responsible for the transport of mercury from the periplasm to the cytoplasm, leading him to MBP! The MBP binds to the mercury in the cytoplasm, rendering it inactive.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the MBP Biobrick (BBa_K346004); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for MBP Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Purification of the Fragment of interest from the gel; Ligation the gel purification of MBP gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + MBP Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +MBP Biobrick ;
This is a functional coding sequence for the mercury transport proteins merT/merP from P. aeruginosa. This part will result in mercury intolerance due to greater mercury transport into engineered cells.
The Anderson promoter provides strong constitutive expression of merT/merP in E. coli
Anderson promoter is that of part BBa_J23102. Terminator is that of part BBa_B1006. merT/merP genes are naturally found in Pseudomonas aeruginosa. This part was synthesized by GenScript in the vector pUC57. The part was then transferred into pSB1C3 using standard cloning procedures.
Red Fluorescent Protein (RFP) "Rudolph" coding region, intellectual property-free
An intellectual property free protein coding region which may be used by any scientist to create a biological machine.
This part has a constitutive promoter, so our PBAN(Mamestra brassicae) can constitutive express.
Uncharacterized wasp protein 1 (PdomMRNAr1.2-02758.1)
sfGFP with R4-tag under arabinose promoter
sfGFP under arabinose promoter with integrase R4
Modified rhlB coding sequence from Pseudomonas aeruginosa SQ6
RhlB together with RhlA encodes Rhamnosyltransferase I for mono-rahmnolipids biosynthesis in Pseudomonas aeruginosa. This part includes bp upstream noncoding gene and bp downstreanm gene of RhlB. Two mutations have been done to remove the PstI restriction sites contained in the coding sequence, which won't change the amino acid sequence.
Two mutations have been done to remove the PstI restriction sites contained in the coding sequence, which won't change the amino acid sequence.
Leafrollers (Adoxophyes sp.) Spread: The obliquebanded leafroller (OBLR) is native to and widely distributed throughout temperate North America. Characteristics: hatched larvae have a yellowish green body and a black head and thoracic shield. Mature larvae are 20 to 25 mm in length and the head and thoracic shield may be either black or various shades of brown. Damage: Leafrollers, the larvae of certain tortricid moths, often feed and pupate within the protection of rolled-up leaves. Several species can cause problems on fruit and ornamental trees in California. The fruittree leafroller, Archips argyrospila, is the most common leafroller pest in landscapes throughout the state. It occurs on many ornamental trees???including ash, birch, California buckeye, box elder, elm, locust, maple, poplar, rose, and willow???and is particularly damaging to deciduous and live oaks. It also attacks numerous fruit and nut trees including almond, apple, apricot, caneberries, cherry, citrus, pear, plum, prune, quince, and walnut. Control(1) Several parasites attack OBLR larvae but do not adequately control the pest.(2) Apply sprays during June to kill the first summer brood adults and newly hatching larvae.
GFP (mut3b) with DAS-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added DAS-ssrA degradation tag (see part BBa_M0052). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. SsrA tags encode peptide sequence that is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag encodes peptide sequence AANDENYADAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
GFP comes from part BBa_E0040, the tag sequence was obtained from part BBa_M0052.
CBDclos fused to Phytochelatin (PC) EC20 with T7 promoter
GFP (mut3b) with LAA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LAA-ssrA degradation tag (see part BBa_M0050). The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes E.coli wild type ssrA peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. ===References=== [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additional residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LAA in this case) are recognized by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part BBa_E1010, the tag sequence was obtained from part BBa_M0050.
This is a regulated promoter with a yellow chromoprotein
Spinach2.1 flanked by tRNALys3 with terminator BBa_B0052
Spc - Spectinomycin Resistance Gene Coding Sequence
The promoter Plux in induced by the dimer AHL-luxR. As a result, amilCP, a blue chromophoire protein, is synthesized. TetR is a repressor of the promoter Ptet.
BBa_R0062: Plux, BBa_B0030: Strong RBS, BBa_K592009: amilCP, BBa_B0010:T1 (terminator) from E. coli.
This part is the codon optimized version of KillerRed (BBa_K1184000). It has been optimized for E. coli by eliminating the rare codons.
Increase the functionality of KillerRed through codon optimization.
This part was codon optimized from the original KillerRed (BBa_K1184000)
Converts L-methionine to mathanethiol, ammonium and 2-oxobutanoate
RBS + Antifungal GAFP-1 + Double terminator
The nadE gene encodes Naphthyl Acetamide synthetase which can boot up the NAD+(H) level in the cells facilitating the electron transferring rate in MFCs (Microbial Fuel Cells).
The sequence is a full length ORF containing the initiation codon and the termination codon.
This part is aquried from genome the E.coli K-12 substrain MG1655.
Xylose Inducible Promoter for Staphylococcus aureus
Signal peptide of B. subtilis major autolysin LytC
Signal peptide (amino acids 1 to 25) of ''Bacillus subtilis'' major autolysin LytC (N-acetylmuramoyl-L-alanine amidase, Uniprot [http://www.uniprot.org/uniprot/Q02114 Q02114]). Targets proteins for secretion via the Sec pathway (Tjalsma ''et. al.'', 2000).
This part was generated by amplification from BioBrick [http://parts.igem.org/Part:BBa_K1351006 BBa_K1351006] with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. LytC_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC TTGCGTTCTTATATAAAAGTCCTAACAATGTG LytcSP_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT ATCGGCCAAAGCTGTTGGC
RBS + Circular λ-Lysozyme with rigid linker
Phytochelatin (PC) EC20 fused with CBDclos with LacI promoter
OmpA surface protein as RFC[105] N-terminal insert
Lactose/IPTG inducible promoter with RFP reporter tagged with DAS-ssrA degradation tag followed by terminator. The tagged RFP is degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. Presence of SspB is crucial for effective protein degradation.
Part was assembled using BrickClip assembly (BBF RFC104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1]
Isobutanol pathway with adhA from Lactococcus lactis under Ptac control
This is a T7 RNA polymerase under an arabinose promoter
This regulatory part is made up of the Arabinose-Inducible promoter, pBAD, and its transcriptional inhibitor/activator, AraC. AraC inhibits gene expression in the absence of Arabinose by binding to the operator units present within the AraC and pBAD promoter sequences, and producing a DNA loop that prevents the RNA polymerase and CAP from binding and initiating transcription. When Arabinose is supplied, this binds to the AraC dimer and releases it from the operator site found in the pBAD promoter, at the same time it enhances transcription by allowing binding to the enhancer sites upstream.
This part was cloned out of existing templates (such as BBa_K325218, BBa_325219, all of which contain sfGFP downstream of the pBAD promoter)
RFP from Discosoma striata (coral) with DAS-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added DAS-ssrA degradation tag (part BBa_M0052). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYADAS and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0052, but same tag was also used in paper by Andersen et al., (1998).[2]
The manA promoter from Xanthomonas campestris
This enzyme converts hydrogen peroxide into water Our cells will produce this enzyme to degrade oxygen radicals within the cells to prevent harmful effects.
We clonned this part from human hepatocellular carcinoma cell genome cDNA
This part is a combination of a constitutive promoter(BBa_J23119), RNA thermometer(BBa_K115002), and a RFP gene(BBa_E1010).
Sascha Hein, Daniel Sachs, Christian Sator
Outer membrane protein A (OmpA) is a common outer membrane protein found in bacteria that serves various purposes. For our purposes, the beta-barrel transmembrane domain can be utilized to anchor otherwise soluble proteins in the outer cell membrane. Lpp serves to direct the fusion protein, while a unstructured linker (GGGSGGGS) serves to separate OmpA from its cargo in order to avoid interactions between the two proteins. The expressed protein is structurally identical to BBa_K103006. This version of the biobrick has been moved into the new backbone, pSB1C3, which contains the gene for chloramphenicol resistance. This resistance gene contains an internal SacI site, making the SacI site at the end of the gene almost unusable for RE cloning. BBa_K1489003 mutagenizes this SacI site into a KasI in order to avoid this issue.
SacI site is kept in this version of Lpp-OmpA-Linker, is removed in BBa_K1489003.
Part sequence is from BBa_K103006, which originally comes from the E. coli genome.
J23101+B0034+VHb (Vitreoscilla haemoglobin)
Intein protease with arabinose inducible regulatory promoter/ repressor araC-Pbad-T7RBS-His-SspDnaBC
The rpfG gene from Xanthomonas campestris
This is the coding sequence for the RpfG(regulation of pathogenicity factor G) protein. It is the regulator in the two-component signal transduction system of Xanthomonas campestris pv. campestris that is activated by the sensor kinase RpfC(Bba_K1315002) in the presence of DSF(diffusible signal factor).
The source of this part is Xanthomonas campestris pv. campestris(Xcc) genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
The OprF is a major outer membrane protein of Pseudomonas aeruginosa. This protein functions as a nonspecific porin to allow the passage of small hydrophilic molecules, plays a structural role in maintaining cell shape and outer membrane integrity, and is required for growth under low osmolality. The structure of OprF has been proposed to consist of three domains, the N-terminal forming h-barrel structure, a loop or hinge region, and the C-terminal associated with peptidoglycan. Based on the predicted secondary structure and information found in the literature, we chose Ala196 as potential fusion sites for displaying CBP. CBP is the abbreviation of copper binding peptide made up of seven amino acid.
Based on the predicted secondary structure and information found in the literature, we chose Val(188) as potential fusion sites for displaying CBP. CBP is the abbreviation of copper binding peptide made up of seven amino acid.
The OprF gene is from Pseudomonas aeruginosa.
E.coli optimized green renilla luciferase under LuxR-AHL regulated promoter
To assamble this part, and others like it, we used a specialized Gibson Barcode System to allow for a modular construction of gates with various components using the same Gibson reaction conditions, allowing for the assembly of a large number of constructs in parallel to each other
p70-CsgA-His*2, double His-tagged curli generator
This is a MoClo formatted C0080_CI araC gene. This is a Level 0 MoClo part with flanking sites C on the 5' side and site D on the 3' side of the part.
The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA; I = TCTA.
dCas9: Expression cassette under a xylose inducible promoter
Chitin-binding wasp protein 1 (PdomMRNAr1.2-03231.1)
NpuDnaE intein RFC[105] circularization construct
Deinococcus radiodurans uracil-DNA glycosylase 1
Expa4 plant secretion signal, localizes to extracellular space
Coding region for first 20 aminoacids of plant protein Exansin4. Secretion signal for plants (derived from A. thaliana). When fused to a protein, directs the protein to extracellular space.
Derived from Expansin4 from Arabidopsis thaliana(NP_181500).
NisA codes for the nisin precursor. Together with the genes NisB, NisT, NisC, NisP, NisR and NisK it is responsible for producing the lantibiotic nisin in Lactococcus lactis. Can be used in the Lactococcus lactis NZ9800, a strain with a deletion in the original NisA gene, to let it produce nisin.
Obtained by PCR on the genome of L. lactis NZ9700, a nisin producing strain.
Myristoilation signal sequence as RFC[105] N-terminal insert
This tandem promoter was created using the MoClo assembly method. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. The promoter is induced by atc and arabinose. The characterization data will be updated before the jamboree.
We looked through the literature and decided to attach the two promoters without any extra sequence between them
This was formed using BBa_I13453 and BBa_R0040
HRE is a enhancer and regulate gene transcription depend on hypoxia.
This part will help our cells to sense hypoxic conditions in order to activate our other vital parts that allow oxygen re-support.
This is a modification of the standard plac operon with everything before the -35 site removed, the Or2 site from the lambda promoter inserted at the beginning, and a 17-bp spacer added between the two.
Pcons+B0034+PBAN(Helicoverpa armigera Hubner )
''Bacillus subtilis'' sortase YhcS (UniProt [http://www.uniprot.org/uniprot/P54603 P54603]), responsible for anchoring its substrae YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]) covalently to the peptidoglycan wall of ''B. subtilis''. A YhcR-based surface display system has been shown to covalently anchor heterologous proteins to the ''B. subtilis'' cell wall (Liew ''et. al.'', 2012). It was used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides and mediate adhesion of ''B. subtlis'' to pathogens. It consists of four components: the sortase substrate with a signal peptide (this part), a linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) and a cell wall-anchoring domain ([http://parts.igem.org/Part:BBa_K1351010 BBa_K1351010]) as well as the sortase YhcS itself ([http://parts.igem.org/Part:BBa_K1351011 BBa_K1351011]), whose overexpression increases the efficiency of the surface display. This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix: {| |prefix with EcoRI, NotI, XbaI, SD and NgoMIV: |GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC |- |suffix with AgeI, SpeI, NotI and PstI: |ACCGGTTAATACTAGTAGCGGCCGCCTGCAG |} Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3. YhcS_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC GTGAAAAAAGTTATTCCACTATTCATCATTGC YhcS_SNP_rev: gatcCTGCAGcggccgctACTAGTa TTAAGTCACTCGTTTTCCATATATAATATAGCG An EcoRI site was removed by changing T to C at position 399 without altering the protein sequence by fusion PCR with the following mutagenesis primers: YhcS_EcoRI435mut_fwd: CGTCTTCTCCTGTCATATGGAGAATTtACATATGAAATTGTAAAAACAAAAATTGTCG YhcS_EcoRI435mut_rev: CGACAATTTTTGTTTTTACAATTTCATATGTaAATTCTCCATATGACAGGAGAAGACG
Phytochelatin (PC) EC20 fused to linker-dCBD with T7 promoter
The promoter Plux in induced by the dimer AHL-luxR. As a result, amilCP, a blue chromophoire protein, is synthesized. Lambda phage CI is a repressor of the promoter PCI.
This is a composite part that consists of 4 biobricks. This is an alternative PA1 Activated System from Bba_K1315009 that instead uses pluxR (Bba_R0062). In the front it has ptet(Bba_R0040) which is constitutively on. This produces LasR all the time with lasR(Bba_C0179) next in the sequence. Only in the presence of the Pseudomonas Autoinducer 1 (PA1), LasR forms an complex with PA1 and it can then activate the pluxR(Bba_R0062) promoter that leads to the production of GFP(Bba_E0040).
M13 bacteriophage g8p with a FLAG tag on C-terminal
beta-Neurexin-derived peptide binding to S. aureus adhesin SdrC
Short peptide of 11 amino acids corresponding to the N-terminus of the human protein neurexin beta-1. It includes the binding site (HHIHHFH) for the Staphylococcus aureus adhesin SdrC. Displayed on the surface of Bacillus subtilis, it shoud enable B. subtilis to bind specifically to S. aureus.
Oligos were designed as precut by EcoRI and PstI to shorten the synthesized fragments and to simplify the cloning into pSB1C3.
The part was generated by annealing of artificially synthesized oligonucletides: Nrx1b_ENX_SD_Ngo_precutEP_fwd: AATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCGCACCACATTCACCACTTTCACGGATCA ACCGGTtaatACTAGTagcggccgCTGCA Nrx1b_SNP_Age_precutEP_rev: GcggccgctACTAGTattaACCGGT TGATCCGTGAAAGTGGTGAATGTGGTGCGC CATGCCGGCttcctccttacTCTAGAagcggccgcG
This part is a combination of a pBAD promoter(BBa_I13453), RNA thermometer(BBa_K115003)
sfGFP in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
Lactose/IPTG inducible promoter wuth RFP reporter tagged with AAV-ssrA degradation tag followed by terminator. The tagged RFP is actively degraded within cell, thus provides better temporal resolution of red fluorescence and promoter activity. Part was assembled using BrickClip assembly (BBF RFC 104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1] Reference [1] Trubitsyna, M., Michlewski, G., Cai, Y., Elfick, A. & French, C. E. PaperClip: rapid multi-part DNA assembly from existing libraries. Nucleic Acids Res. 1???8 (2014). doi:10.1093/nar/gku829
GoldenGate compatible Green Flourescent Protein (GFP)
A variation of the part BBa_E0040 that has been made compatible for Golden Gate by removal of internal BsaI and BpiI recognition sequences. Fluoresces under UV light when the gene is expressed.
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences.
This part is sensitive to long chain acyl-CoA ,with a FadR binding site. In the absence of long chain acyl-CoA, FadR represses the transcription. In the presence of long chain acyl-CoA, FadR dissociates from the binding site so that the transcription begins. E.coli uses this promoter to regulate fatty acid metabolism.
The promoter Plux is induced by the binding of an AHL-luxR dimer. This bio-brick leads to the synthesis of the enzyme Green Renilla Luciferase, which catalyses a luminescent reaction. TetR can be used to repress the promoter Ptet.
This is a T7 DNA polymerase under an arabinose promoter
kerUS keratinase from Brevibacillus brevis
Coding region of kerUS keratinase from Brevibacillus brevis with signal for extracellular secretion, which is cleaved following secretion Codes for a 45.9kDa (predicted)pre-processed protein, cleaved to 29kDa active protein
codon optimisation for E. coli, ensuring no BioBrick restriction enzyme sites were present
is the codon optimised (for E. coli) kerUS gene from: Jaouadi, N. Z., Rekik, H., Badis, A., Trabelsi, S., Belhoul, M., Yahiaoui, A. B., ??? Jaouadi, B. (2013). Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities. PloS one, 8(10), e76722. doi:10.1371/journal.pone.0076722 Genbank Number: KC152966
spaS Subtilin (antimicrobial peptide, Freiburg standard)
Subtilin Lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. The bactericidal activity of lantibiotics is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores.
SmtA metallothionein fused to linker-dCBD
This part has a constitutive promoter, so our PBAN(Agrotis ipsilon) can constitutive express.
merT is found in the Serratia marcescens plasmid pDU1358 on the other side of the bidirectional promoter opposite of merR. It is 351 base pairs long and is hypothesized that it codes for a mercuric transport protein.
Source organism: Serratia marcescens. Amplified from plasmid pDU1358, kindly donated by Anne O. Summers.
cell division inhibitor of the MinC-MinD-MinE and DicB-MinC systems that regulate septum placement
p70-CsgA-His, His-tagged curli generator
ComE is part of a two-component regulatory system (TCS) that controls competence development in Streptococcus pneumoniae. ComE is the response regulator to histidine kinase ComD. Upon binding and detection of the quorum-sensing peptide CSP, transmembrane receptor ComD autophosphorylates using ATP. Subsequently, cytosolic protein ComE is transphosphorylated by ComD. ComE~P activates the comCDE and comAB operons for expression of late competence genes.
The gene was functionally expressed in Azospirillum sp. B510. It encodes a divalent metal cation transporter. The protein can be used to increase transportation level of divalent metal ions into the cells. Its potential applications include heavy metal bioremediation (by increasing uptake efficiency) and protein expression in cell surface (by fusion with mntH).