pLacI-rbs-MarA-rbs-GFP-double terminator-pLsrA-rbs-LL-37
General Design Our design has two systems: The Antimicrobial Peptide System, which produces the peptide LL-37; and The Resistance and Expulsion System which produces the MarA protein, which activates the arcAB & TolC operons, activating the arcAB & TolC efflux pump. Antimicrobial Peptide System H. pylori and other pathogenic bacteria produce AI-2, a quorum sensing molecule. This signal is detected by the pLsrA promoter (BBa_K117002), which allows the transcription of LL37 (BBa_K1230001), which is traduced and then translated into the antimicrobial peptide LL37. Once LL37 is expelled by the ArcAB-TolC efflux pump, it can act against H. pylori. Resistance & Expulsion System This system is induced by promoter LacI (BBa_R0010), which allows the transcription of marA gene (BBa_K1230000), producing the protein MarA. MarA acts on the arcAB and tolC operons, which activates de ArcAB-TolC efflux pump, allowing the expulsion of LL37 peptide.
LL-37 has the codon usage for humans. Using E. coli codon usage may inhibit protein function by wrong folding.
2013 DNA distribution and synthesis by genscript.
The MarA protein acts as a transcriptional activator in E. coli. It allows the expression of the acrAB and tolC operons, which activate the AcrAB-TolC efflux pump, a mechanism that has been related with resistance to organic solvents, dyes, detergents, antibiotics such as chloramphenicol, tetracycline, novobiocin, erythromycin, fusidic acid and cloxacillin, as well as to cationic antimicrobial peptides, such as LL-37, HNP-2 and HBD-1. This composite part allows MarA expression in presence of IPTG, by using the LacI promoter and the RBS based on Elowitz repressilator (Part BBa_J04500)
We ligated part BBa_K875009 as a prefix and part BBa_E0840 as a suffix.
LL37 Generator under pLsrA promoter, with part BBa_E0840 as prefix.
This is an intermediate part. It can be used to produce an antimicrobial peptide in the presence of AI-2, a quorum-sensing molecule.
AcrAB-TolC multidrug efflux transport system activator from E. coli
The MarA protein acts as a transcriptional activator in E. coli, it allows the expression of the acrAB and tolC operons, which activate the AcrAB-TolC efflux pump, a mechanism that has been related with resistance to organic solvents, dyes, detergents, antibiotics such as chloramphenicol, tetracycline, novobiocin, erythromycin, fusidic acid and cloxacillin, as well as to cationic antimicrobial peptides, such as LL-37, HNP-2 and HBD-1.
Silent mutation (G->C) at base 132 to remove AarI and BspMI sites.
The human peptide LL-37 is a cationic antimicrobial peptide with activity against Gram-negative and Gram-positive bacteria. It has been shown to protect against Helicobacter pylori infection and its related gastritis, besides of inhibiting the growth of other pathogenic bacteria in the gastrointestinal tract such as Salmonella typhimurium and Escherichia coli.
LL-37 antimicrobial peptide generator with codon usage for E. coli, under the arabinose promoter with a GFP generator.
LL-37 has the codon usage for E. coli, this could change the folding activities of the peptide.
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.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
LsrA promoter (indirectly activated by AI-2)
The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Once uptaken into the cell, AI-2 will be phosphorylated under the effect of LsrK. Without the presence of AI-2, LsrA promoter is inhibited by the repressor, LsrR. Phosphorylated AI-2 will prevent LsrR from inhibiting this promoter, hence activating it so that the downstream sequence can be expressed. To prevent the cell from self-inducing this promoter, we must suppress its AI-2 producing capability. It can be done by knocking out LuxS, one of the vital gene required for producing AI-2. By doing that, LsrA promoter can only be activated under the presence of AI-2 from other sources than the host cell which is LuxS mutant (say, by contact with normal cells). More information on AI-2 quorum-sensing (QS) system please refer to: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1952038
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.
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</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 ("orig" in figure 4-14 of Ron Weiss 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 Brian Chow
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Conversion of the iGEM parts registry to SBOL2.1