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From 2013.igem.org

Quorum Sensing

Quorum sensing is a system that controls population density using stimulus and response. These types of systems can be commandeered for use in synthetic biology and re-engineering to control gene expression in genetic circuits that are implanted in bacteria. The bacteria utilize quorum sensing to coordinate gene expression. To do so, the bacteria produce and secrete signaling molecules. These bacteria have receptors that detect the signaling molecule. When the signaling molecule binds to the receptor, it induces gene expression. The presence of one or more proteins will induce a promoter that controls the production of signal proteins, such as fluorescent proteins, or of other functional genes to link synthetic genetic circuits together.

A LuxIR-type quorum sensing system has been detected in Chromobacterium violaceum (Stauff et al., 2011). The goal is to introduce a new LuxR/I-like quorum sensing system to synthetic biology via MoClo. Chromobacterium violaceum, a gram-negative bacteria found in flora from water and soil in tropical and subtropical regions, uses the CviR/I system. The CviR/I system is homologous to the LuxR/I system. This can be done by cloning and characterizing CviR/I and pVioA, a promoter with CviR binding site) into E.coli.

New Level 0 Parts

We have successfully made three new parts while working on the QS project. CvI and CviR were submitted to the Registry as Level 0 MoClo Parts. The pVioA promoter was confirmed after the DNA submission deadline and was not included in our submission.

All three parts were sequence verified. Our next steps involve testing the CviI/CviR genes for function and checking them for cross-reactivity with LuxI/LuxR.

pVioA as a Constitutive Promoter

We have successfully characterized the pVioA promoter as a constitutive promoter controlling RFP expression (first transcriptional unit in diagram above). For more information on how we ran our characterization experiment, please check out our Data Collected page.

The graphs below show MEFL converted RFP measurements (done using the TASBE tools). The data is binned by fluorescence intensity and each bin also includes the cell count. The bottom graph shows the same data as the top one above but it is ordered by cell count, from highest (dark purple) to lowest (pale purple).

We can now see that within the population of cells, the smallest binned group shows the highest level of expression. This may be due to higher copy counts of plasmids within that subpopulation of cells.

We plan to continue with this work and hope to have an update on our progress at the Jamboree!

References

[1] Stauff, D.L., and Bassler, B.L. (2011) "Quorum Sensing in Chromobacterium violaceum: DNA Recognition and Gene Regulation by the CviR Receptor." Journal of Bacteriology 193(15):3871-3878. doi: 10.1128/JB.05125-11.