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.
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 IPTG inducible T5 lac operator (BBa_K875002) regulates the expression of the Tse2 toxin (BBa_K314200).
beta-galacturonidase (YesZ) from Bacillus Subtilis 168
yesZ The enzyme acts on the terminal end of side chains of Rhamnogalacturonan I Pectin, releasing the free galactose. This gene has been isolated from the bacterium Bacillus subtilis subtilis 168. The enzyme yesZ has beta-galactosidase (beta-galacturonidase) activity, cleaving (1→4)-β-D-galactans to produce single galactose molecules from RG I Pectin. Usage and Biology Pectin is a component of the primary cell wall of plants. As galactose can be metabolized by the human body and by animals, it is an alternative to the usual target of nutrition/energy boosts in biotechnology, glucose. (1) http://www.ncbi.nlm.nih.gov/pubmed/17449691 (2) http://subtiwiki.uni-goettingen.de/wiki/index.php/YesZ (3) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694227/ (4) http://aem.asm.org/content/73/12/3803 (5) http://www.tandfonline.com/doi/full/10.1080/15583724.2011.615962#tabModule
No design considerations were needed, although we did add a second stop codon.
Strong pBAD promoter BBa_K206000 attached to SecI pump BBa_K215104
iGEM registry, acquired from University of Washington 2009 iGEM team from the genomic DNA of Erwinia chrysanthemi
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_K215002]. Any protein of interest can be inserted into the protein generator and then secreted when used in conjunction with this secretion system. For full characterization data on this part, please see the University of Washington 2009 iGEM project page and Wisconsin Lutheran College 2013 iGEM project page.
We had to remove the RBS and in the future will be removing all Bative RBSs from the sequence to be replaced with BBa_B0034
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.
Double terminator consisting of BBa_B0010 and BBa_B0012
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
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.
This promoter is composed by phage T5 promoter and Lac operator. It is repressed in the presence of the lac repressor protein. Trieste Team 2012 used this inducible promotor in the first phase of their project to test every construct that could have been toxic for the host strain, if expressed in a constitutive way.
This basic part has been designed in order to have a prefix and a suffix according to the standard Biobrick structure.
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
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
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