Antinorovirus antibody, Antihorizontal transfer mechanism, Purification and secretion tag, tripartat
Parts were received from the iGEM registry and Team Trieste
This combinatory part is a self contained secretion system that combines a secretion and purification tag, a tripartite pump that is specific to the sec tag, a antinorovirus particle like antibody, Tse2 toxin mediated horizontal gene transfer mechanism. Tripartite SecI pump These are the three essential parts prtD, prtE, prtF, that make up the Type I secretion system from Erwinia chrysanthemi.This part is made of 3 genes: prtD, prtE, and prtF, that constitute a type I Erwinia chrysanthemi secretion system. The Pump is expressed from a strong constitutive promoter, BBa_k206000, and has the translational terminator BBa_B0014. In pSB1C3. Used to secrete proteins containing prtB C-terminal tag. The prtB C-terminal tag is built into the protein generator [BBa_K1175012]. Any protein of interest can be inserted into the protein generator and then secreted when used in conjunction with this secretion system. Secretion and purification tag This part BBa_K1175012 A composite part formed between BBa_K206000 strong pBAD promoter, BBa_B0034 strong RBS, and BBa_K215001 a purification and secretion tag; a secretion system designed by Washington iGEM team 2009 BBa_K215001. This tag can be used to secrete proteins of interest by inserting them into a specific NheI cut site flanked by C terminus His tag and an N terminus Secretion tag. Both tags can be removed by a TEV protease. The secretion tag is also specific to the tripartite pump. Antinorovirus like particle antibody This construct BBa_K875004 is designed for the expression of an already described engineered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. The antibody is expressed in a single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA-scFv 54.6 antinorovirus, Histidine tag (6HIS), Terminator (B0015). T5cumate repressed Tse2 mediated, antihorizontal transfer mechanism This construct BBa_K1175003 is a modification of the part designed by iGEM2012 Team Trieste it has a T5cumate inducible promoter suppressed by the CymR gene integrated genome into the chassis E.coli Nissle 1917. This part will not be suppressed by CymR in the bacterial cells that have taken up the plasmid.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Double terminator consisting of BBa_B0010 and BBa_B0012
prtD_prtE_prtF from erwinia chrysanthemi
Gene Bank ID: Erwinia chrysanthemi M60395.1
This is the 3 genes: prtD, prtE, prtF, that are from a type I Erwinia chrysanthemi secretion system.
Synthesized to be bio-bricked, keeps original protein sequence and ribosome binding site.
Secretion Signal and Streptavidin Binding Tags
Nanotag (bases 1-48): From Lamla and Erdmann (http://www.ncbi.nlm.nih.gov/pubmed/14680960). Secretion tag (last 181 AA): Gene Bank ID Erwinia chrysanthemi M60395.1
This part is the vector into which afp is inserted to tag it for purification using the IPP system. It contains an NheI restriction site that allows for the insertion of the desired protein coding sequence (NheI is compatible with XbaI and SpeI). This Target Vector part contains two tags: a secretion tag that causes the protein to be exported to the extracellular space, and a nanotag that causes the protein to bind to stretavidin (or some analog). For this part to function properly, it must be combined with a promoter and RBS, and used in E.coli strain UW5alpha, which has two other plasmids containing the other genes required for proper secretion and display of the protein to be purified on the outer membrane.
NheI was chosen as the protein insertion site because it is compatible with digested XbaI and SpeI restriction sites. The TEV sites and HIS tags flanking the NheI site are present to separate the inserted protein from the added tags. GSA linkers were used to separate the various features of the part.
T5LacO-LPP-OmpA-scFv (scFv 54.6 antinorovirus)-6HIS-TT
DNA synthesis. 1.???Transport and anchoring of 8-lactamase to the external surface of Escherichia coli??? J. A. Francisco, C. F. Earhart and G. Georgiou. Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2713-2717, April 1992 Biochemistry. 2. ???Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands??? K. Ettayebi and M. E. Hardy. Published: 31 January 2008 in Virology Journal 2008, 5:21?
This construct is designed for the expression of an already descibed engeneered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. The antibody is expressed in a single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA-scFv 54.6 antinorovirus, Histidine tag (6HIS), Terminator (B0015).
This coding sequence was optimized for the expression in prokaryotic cells. It has a Histidine tag.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type (<partinfo>I13453</partinfo>) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
Medium RBS based on Ron Weiss thesis. Strength considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-1" in figure 4-14 of thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Cho
1) The cumate gene-switch: a system for regulated expression in mammalian cells Alaka Mullick, Yan Xu, Ren?? Warren, Maria Koutroumanis, Claire Guilbault, Sophie Broussau, F??lix Malenfant, Lucie Bourget, Linda Lamoureux, Rita Lo, Antoine W Caron, Amelie Pilotte and Bernard Massie ( BMC Biotechnology 2006, 6:43 doi:10.1186/1472-6750-6-43 ) 2. Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains Young J. Choi, Lyne Morel, Teffanie Le Fran??ois Denis Bourque, Lucie Bourget, Denis Groleau, Bernard Massie and Carlos B. M??guez (Applied and Environmental Microbiology, Aug. 2010, p. 5058???5066 Vol.76 doi:10.1128/AEM.00413-10)
￼￼￼This promoter is composed by phage T5 promoter and Cumate operator. It is repressed in the presence of CymR protein which binds the Cumate Operator.
We added the standard iGEM prefix and suffix in order to clone it into the standard iGEM vector pSB1C3
A toxic protein originating from pseudomonas aeruginosa that has been shown to arrest growth in both prokaryotic and eukaryotic cells when expressed intracelluarly. It is a substrate p. aruginosas type 6 secretion system
mention something about promoter needs to have no leaky expression for succesful cloning
Matthew Coyne, Ingrid Swanson, Jesa Landis, Matthew Harger
Transcriptional terminator consisting of a 64 bp stem-loop.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
This is one of the 3 genes: prtD, prtE, prtF, that are from a type I Erwinia chrysanthemi secretion system.
Synthesized to be bio-bricked, keeps original protein sequence.
Synthesized to be bio-bricked, keeps original protein sequence and rhibosome binding site.
Conversion of the iGEM parts registry to SBOL2.1