Expression Plasmid coding for His-tag, also containing a thrombine cleavage site between coding region and Histag, so that His-tag can be cleaved off. Vecotr opening with PstI/XmaI, clone part into it with PstI/NgoMIV. Ampicillin resistance.
very strong RBS 6xHis-Tag Thromin-cleavage-site NgoMIV-compatible restriction site for in-frame insertion
Halorhodopsin gene allow chloride ion to be pumped in by the level of light intensity. Halorhodopsin gene is under regulation by constitute T7 promoter to allow protein coding to be maximized, and the bidirectional terminator is added for high efficiency termination of both forward and backward transcription
The double terminator wass tested to stop effectively on the transcription of both forward and backward transcription to make our protein generator be a controlled system
Halorhodopsin comes from part BBa_K559000, which is come from genomic sequence of N.pharaonis (genebank ID:J05199.1)
Strong T7 promoter and RBS including start codon for protein expression in E. coli or in-vitro. Part is provided in RFC 25 assembly format.
* sequence is taken from the commonly used pET3a expression vector * deleted XbaI site between prom. and RBS * T7 promoter sequence (1-19) equals the one in BBa_I712074 * Conservative choice of part boundaries and modifications -- the part's size could probably be reduced.
This part contains the N-terminal 180 amino acids of OmpA fused to SH3 domain in human Grb2 protein. The N-terminal of OmpA allows the moiety fused to it to be presented on to the outer membrane of E. Coli. SH3 domain allows for binding to multi-proline peptide in human cells, which functions as a crucial adaptor module in signaling. In our part, this is specially for binding to the proteins for transportation by E. Coli. All the coding sequence is under T7 promoter and lac repressor binding site. The parts is most efficiently presented onto the membrane when induced at 0.1mM IPTG at 18 centigrade in BL21 E. Coli strain.
High expression efficiency as well as inducible expression is most desirable when considering a coding sequence.
OmpA is derived from the genomic DNA of BL21 (DE3) E. Coli strain while SH3 is from human Grb2.
T7 terminator for protein expression in E. coli or in vitro. Part is provided in R.F.C. 25 assembly format.
* taken from pET3a expression plasmid * added two STOP to 5' * conservative choice of part boundary, could probably be shortened
Halorhodopsin under constitute T7 promoter to regulate the gene transcription to be maximized and no restrict to other regulatory factor
The coding sequence of N.pharaonis J05199.1 is selected from 61nt to 700nt and the T7 promotor placed in front by 3A assembly
Halorhodopsin come from N.pharaonis, gene bank J05199.1
This is a mutant T7 promoter that includes a lacO sequence. This is repressible by the LacI protein and is induced in the presence of IPTG. The promoter sits in the 5' region of the gene of interest and initiates transcription when the cell expresses T7 RNA polymerase. Be sure you are using a cell strain that includes this gene (DE3).
There are three portions to the T7 promoter: the recognition site, the melting box (or TATA box) and the initation site. Mutations in these sequences caused differential behavior from the wild type.
The part consists of a sequences that resemble a T7 promoter and the lacO sequence found in E. coli.
beta-lactamase (ampicillin resistance marker)
T7 promoter-driven BMC structural genes with a His6-tagged component for purification
T7 promoter diving the expression of His6-tagged EutS, with subsequent Plac-driven EutM, EutN, EutL, EutK
Clone BBa_K311004 into pET28a via NheI and NdeI
Added HIV protease site between OmpA and SH3 for easier release.
The HIV protease site is embedded in the protein so that a proteolytic cleavage can release the binding cassette.
OmpA is from BL21(DE3) E. Coli genomic DNA. SH3 is from human Grb2 protein. HIV protease site is from a former part.
It is a repeat-spacer-repeat region designed according to E.coli's CRISPR/Cas system. It also includes a T7 promoter and T7 terminator. THe spacer is designed to target on a segment of non-coding-sequence in E.coli's genome. This biobrick can be transformed into E.coli cells to hopefully construct a suicide system with E.coli's CRISPR/Cas system.
The repeat-spacer-repeat region design should be consistent with the operating principles of E.coli's CRISPR/cas system.
The 'repeat' sequence in CRISPR/cas system of E.coli strain K12.
T7 promoter for expression of functional RNA
OmpA is used to present HIV aspartyl protease outside the E. Coli membrane so that it can cut other proteins with the specific sequence outside in the media.
Since HIV protease is a dimer, dimerization is a great concern to its function. Function of this protease is tested in BL21.
HIV protease sequence is from former part BBa_I712667. OmpA sequence is from BL21(DE3) e. Coli strain.