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BBa_K1074007
Promoter ctc(BBa_K143010) is a sigma factor B-dependent promoter in B. subtilis. Activated by endogenous sigma factor B under mild stress( nutrient stress response or physical stress response). amilCP(BBa_K592009), blue chromoprotein,naturally exhibits strong color when expressed. when bacterial grow to the post-log phase, nutrient and space stress arise,Promoter ctc should be activited. We assemble this gene circuit just to expect a visual signal when B.subtilis grow to the post-log phase.
BBa_K1074010
Spbc in SDP(sdpA,sdpB,spbC)(BBa_K1074009)operon is a killing factor of Bacillus subtilis. We put the SDP operon under the strong Regulated promoter Pgrac(BBa_K1074012) to see whether overexpression of spbc can induce the suicide of engineered Bacillus subtilis.
BBa_K1074011
We construct this gene circuit as our kill switch to kill the engineered Bacillus subtilis for the safety purpose.Killing is mediated by the exported toxic protein SpbC. Extracellular SpbC induces the synthesis of an immunity protein, SdpI, which protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor, SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SpbC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein. The kill switch is based on a high-copy vector fused with promoter for operon sdpIR and coding sequence for protein SpbC. When SpbC toxins are sensed, they will be captured by Immunity Protein SdpI at the membrane, enabling SdpI to sequester SdpR. As a result, repression on promoter SdpIR is released and more SpbC will be produced. Trapped in this endless loop, the SpbC producing cells fails to cope with enormous toxin SpbC and doomed after eliminating their siblings. Eventually, the group of engineered Bacillus subtilis is destroyed instead of sporulating.

Plasmid Parts

BBa_K1074001 PHT43 is a E.coli-B.subtilis shuttle vector allowing high-level expression of secreted proteins in B.subtilis. It is based on the strong σA-dependent promoter preceding the groE operon of B. subtilis which has been converted into an efficiently controllable (IPTGinducible)promoter by addition of the lac operator. An efficient Shine-Dalgarno (SD) sequence as well as a multiple cloning site (BamH I, Xba I, Aat II, Sma I) were also inserted. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding an α-amylase was fused to the SD sequence.

RBS Parts

BBa_K1074015 is the hybrid promoter for the reporter node, which can be activated as well as inhibited.
BBa_K1031011 is the transcriptional factor that can activate its cognate promoter, which is used as the activate node in the band-pass filter.
BBa_K1031013 BBa_K1031014 BBa_K1031015 BBa_K1031016 are RBS library to tune the expression of C1, under the control of pSal promoter, which is a crucial part for band-pass filter.

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