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.
This is a fusion between ompA and the DNA binding protein zif268 under the control of pLAC. The protein should be present on the outer surface of cells and bind the zif268 recognition sequence (GATGCTGCA). This will also allow cells to be anchored to a glass slide with the appropriate DNA attached. This can serve as a way to position cells in any desired pattern.
NOTE- the sequence as listed here is wrong. Because part K165006 is supplied on a Silver lab vector (Bba_J63010) with a modified biobrick prefix, the scar between ompA and zif268 is non-standard. The G at position 700 is not there. This has been verified by sequencing. Deleting the G keeps zif268 in frame with ompA. Part length is 1091bp. The sequence was generated automatically and I couldn't figure out how to edit it.
parts come from iGEM distribution and from iGEM HQ
Ralstonia solanacearum prhA gene, encoding an outer membrane protein that senses a signal on plant cell walls and transduces it through the bacterial cell envelop to stimulate transcription from several operons
Not yet edited, other than changing stop codon to TAATAA
Ralstonia solanacearum GMI1000 plasmid pGMI1000MP, complete sequence NCBI Reference Sequence: NC_003296.1
prhA standard biobrick double stop codon
Extracellular domain of T-cell surface glycoprotein CD4.
Obtained from biobrick part BBa_I712084.
Export of recombinant proteins in Escherichia coli using ABC transporter of Erwinia chrysanthemi
Jung Hoon Ahn from Korea Advanced Institute of Science and Technology
EGF-TliA, EGF-LARD1 fusion proteins were secreted when ABC transporter was present. E. coli harboring the PrtDEF of E. chrysanthemi secreted more fusion proteins than without ABC transporter. In fact, in enzyme activation assay(with Hitachi Spectrophotometer) we observed that although our protein -EGF- has disulfide bonds, it was excreted succesfully. In addition, when compearing with lack of ABC domain, we saw that protein excretion into medium (supernatant) is 10-fold more with ABC transporter-PrtDEF-. PrtDEF functioned well at 37??C, the optimum growth temperature for E. coli
We used whole plasmid in order to excrete a fusion protein with one of two signal tags which are TliA and LARD1
Sequence and Features PrtDEF: ABC transporter of Erwinia chrysanthemi for PrtA EGF: Human Epidermal Growth Factor
This is a fusion between ompA and the DNA binding protein zif268 under the control of pBAD. The protein should be present on the outer surface of cells and bind the zif268 recognition sequence (GATGCTGCA). This will also allow cells to be anchored to a glass slide with the appropriate DNA attached. This can serve as a way to position cells in any desired pattern.
NOTE- the sequence as listed here is wrong. Because part K165006 is supplied on a Silver lab vector (Bba_J63010) with a modified biobrick prefix, the scar between ompA and zif268 is non-standard. The G at position 840 is not there. This has been verified by sequencing. Deleting the G keeps zif268 in frame with ompA. Part length is 1364bp. The sequence was generated automatically and I couldn't figure out how to edit it.
EstA : inactive fusion protein for bacterial surface expression
Marie Burghard, Henrik Cordes, Jascha Diemer, Adrian Eilingsfeld, Sven Jager, Philipp Rottmann, Rene Sahm, Arne Wehling
This Esterase A is a inactivated membrane bound esterase from Pseudomonas putida. Its active site is mutated so it shows no more hydrolytic activity. It can be used for cell surface display like its shown in the following paper: Taek Ho Yang, Jae Gu Pan, Yeon Soo Seo, Joon Shick Rhee Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli Appl Environ Microbiol. 2004 December; 70(12): 6968???6976. doi: 10.1128/AEM.70.12.6968-6976.2004
This certain variant of EstA should be used for cell surface expression and displaying of almost every kind of enzyme. There is no ATG at its 5' because its an C-terminal fusion protein. If you want to use you need to design it in the following way: N - Peri plasmatic signal sequence - Protein of interest - EstA - C We did not use any kind of BioBrick Assembly due to the generation of stop codons. If you would like to fuse EstA to any kind of protein, please use SOE PCR or an alternative kind of assembly strategy.
Our parts derives from an established inactive EstA mutant on pEST100 vector, which was used in the following paper: Becker, S., Theile, S., Heppeler, N., Michalczyk, A., Wentzel, A., Wilhelm, S., Jaeger, K.-E., et al. (2005). A generic system for the Escherichia coli cell-surface display of lipolytic enzymes. FEBS letters, 579(5), 1177???82. doi:10.1016/j.febslet.2004.12.087
Marie Burghard, Henrik Cordes, Jascha Diemer, Sven Jager, Philipp Rottmann, Rene Sahm, Arne Wehling
RBS-PhoA-His6tag-pNBEst13-Myctag-EstA is a long name for a long protein construct. It is a fusion protein which is secreted into the peri plasma in order to be integrated into the outer membran. When the phoA signal sequence is cut off, the N-terminus is formed by our pNB-Est13. This esterase shows actitivty towards PET and is anchored onto the cell by its inactive EstA membrane anchor.
We got it synthesized by GENEART but added the RBS via PCR.
This is no natural or even native construct. We got it synthesized by GENEART. but added the RBS via PCR.
Juliette Paillet, Alexandre Duprey, Emiel van der Kouwe
GFP is presented outside E. Coli as a positive control for protein presentation
His tag is inserted between OmpA and GFP for easier detection in Western Blotting.
OmpA is cloned from BL21(DE3) E. Coli strain. GFP is from Clontech plasmid pGFP-N1
p70-CsgA-His, His-tagged curli generator
p70-CsgA-His*2, double His-tagged curli generator
This is a fusion between ompA and the DNA binding protein Gli-1 under the control of pLAC. The protein should be present on the outer surface of cells and bind the Gli-1 recognition sequence (GACCACCCAAGACGA). This will also allow cells to be anchored to a glass slide with the appropriate DNA attached. This can serve as a way to position cells in any desired pattern.
NOTE- the sequence as listed here is wrong. Because part K165007 is supplied on a Silver lab vector (Bba_J63009 or V0002) with a modified biobrick prefix, the scar between ompA and Gli-1 is non-standard. The G at position 840 is not there. This has been verified by sequencing. Deleting the G keeps Gli-1 in frame with ompA. Part length is 1364bp. The sequence was generated automatically and I couldn't figure out how to edit it.
Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1
TliA has four glycinerich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins.Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, EGF-TliA was excreted into the culture supernatant.Whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF which has 3 disulfide bonds in E. coli supplemented with ABC transporter. Activity domain (residues 1???268) and secretion/chaperon domain (residues 279???476). In our experiment, we observed that TliA fused proteins were excreted to supernatant culture succesfully by detecting lipase activity with tributyrin and spectrophotometric detection with the substrate p-nitrophenyl phosphate at 420 nm. At the experiments, Tlia fused proteins were excreted ten-fold more with ABC transporter PrtDEF of Erwinia chrysanthemi.
The sequence was optimized for E.coli with Biobrick restriction sites
The thermostable lipase (TliA) of P. fluorescens SIK W1 is comprised of 476 amino acids and has the characteristic C-terminal signal sequence recognized by the ABC transporter.
This part encodes E coli protein Ag43, with an additional 6xHis tag for easy purification and quantification. Ag43 expression is known to cause E coli cells to adhere together, and the 6xHis tag was added to allow for convenient study of variation of E coli aggregation with respect to Ag43 expression levels.
Ag43 is an outer membrane E coli protein, so it has a N-terminal 52 amino acid long signal peptide sequence, which is cleaved after Ag43 pro-protein is transported into the periplasmic space. Since strong dependence has been demonstrated on the signal peptide cleavage on the signal peptide-protein junction, the 6xHis tag should be added after Ala53, the residue immediately after the signal peptide sequence.
This comes from Gibson assembly of fragments from a previous biobrick, BBa_K1352000 by Team:Aberdeen_Scotland, 2014.
This is designed as the positive control for protein presentation on the outer membrane.
His tag is inserted between OmpA and mCherry to facilitate detection after expression.
OmpA is cloned from genomic DNA of BL21(DE3) E. Coli strain. mCherry is from the Clontech plasmid pmCherry-C1.
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.
This is a fusion protein with Ag43, a protein from E coli tagged with a 6xHis tag on the N-terminal and sfGFP tagged on C-terminal of the pro-protein. Ag43 causes cell-cell adhesion in E coli, and the 6xHis and sfGFP tags are present to allow convenient study dependence of Ag43 mediated aggregation on Ag43 expression levels, which can be determined from the sfGFP fluoresence and/or quantification of 6xHis tag. The 6xHis tag is included to allow protein concentration estimation in case the sfGFP fails to fold properly.
The location for the 6xHis tag is the same as that for BBa_K2168000(Ag43 tagged with 6xHis on N-terminal under pBAD/araC promoter) and uses the same design principles. For independent folding, a flexible GGGGSGGGGS linker was added between Ag43 and sfGFP, and the linker was choosen to be not very hydrophilic or hydrophobic, since Ag43 lies very close to lipid membranes.
This part was made from Gibson assemblies from fragments of two previous parts - BBa_K1352000 and BBa_I746908, which code for Ag43 and sfGFP respectively.
This is a fusion between ompA and the DNA binding protein Gli-1. It should be present on the outer surface of cells and bind the Gli-! recognition sequence there. This will also allows cells to be anchored to glass slide with the DNA attached.
Silver prefix on Gli-1 allowed us to fuse it to ompA in frame.