Since we are working with insulin and diabetes, we wanted our bacteria to produce insulin in response to the glucose concentration in the media. To do that, we designed a control system using the carbon catabolite repression of Lactococcus lactis and a sRNA regulation.

Carbon catabolite repression (CCR) is a regulatory mechanism by which bacteria regulate the expression of functions for the use of secondary carbon sources in the presence of a preferred carbon source. Whereas the transcription factor cyclic AMP receptor protein (CRP) is responsible for CCR in gram-negative bacteria, in gram-positive bacteria the CCR is mediated by the carbon catabolite control protein A (CcpA). In L. lactis, a transcriptomic analysis revealed that Ccpa regulates genes of carbon metabolism and also its own expression [1].

To cause CCR, CcpA must bind to a specific palindromic sequence in the promoters regions of catabolic operons, called catabolite responsive elements (cre sites) [2]. Figure 1 shows the general CCR in gram-positive bacteria.

Figure 1. Phosphorylation of HPr occurs when intracellular concentrations of fructose-1,6-bisphosphate (FBP) and ATP are high, which reflects the presence of preferred carbon sources. The phosphorylated HPr(His-P) binds to the CcpA protein and this complex binds to cre site on the DNA and then represses the transcription of catabolic genes [2].

The CCR mechanism in L. lactis works as an inverter (NOT gate): when there is glucose, the gene expression is repressed, and when there is no glucose, the gene expression is activated. In order to regulate the expression of insulin in our genetically engineered machine by glucose we needed to invert this system, so we designed a circuit using the CCR mechanism regulating the expression of a small RNA (sRNA) that blocks the translation of insulin. That way, when there is glucose, the sRNA will not be expressed and the insulin will be expressed. When there is no glucose, the sRNA will be expressed and it will repress the expression of insulin.

In order to be able to test and detect the signak from our sytem, we used the sfGFP(Bs) (BBa_K2270010) as gene reporter, previously inserted into the pSEUDO plasmid [4], which was used in our constructions. So, we designed the sRNA to target the sequence of the sfGFP(Bs) mRNA from the RBS sequence to the start codon to ensure its specificity to the target and an efficient hybridization.

To design the sRNA, we used the software RiboMaker, which gave us the sequence, hybridization energy values and the possible secondary structure of the sRNA. Analyzing the data, we selected the best two results and then performed a new analysis using the software NUPACK. By this new analysis we were able to choose the best sRNA and build our final construction. To learn more, see our modeling section.

Our final construction (BBa_K2270008) was then composed by:

• the gal operon promoter from L. lactis, which contains the cre site and has already been well studied [3];
• the designed sRNA;
• the terminator from Bacillus subtilis rrnb;

Figure 2 shows our construction and its mechanism of action.

Figure 2. Our glucose-responsive control system. (A) When glucose is present the carbon catabolite repression system will repress the sRNA expression, leaving the GFP mRNA free to be translated into protein. (B) When glucose is absent, there will be no carbon catabolite repression and the sRNA will be expressed. The sRNA then will hybridizes to the GFP mRNA and block its translation.

References
[1] Zomer et al. Time-Resolved Determination of the CcpA Regulon of Lactococcus lactis subsp. cremoris MG1363. Journal of Bacteriology. vol. 189, p. 1366-1381. American Society for Microbiology, 2007.
[2] Görke, B.; Stülke, J. Carbon catabolite repression in bacteria: many ways to make the mos out of nutrients. Nature Reviews: Microbiology. vol. 6, p. 613-624. Macmillan Publishers Ltd, 2008.
[3] Luesink et al. Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Molecular Microbiology. vol. 30, n. 4, p. 789-798. Blackwell Science Ltd, 1998. [4] Pinto el al. pSEUDO, a genetic integration standard of Lacotococcus lactis. Applied and Environmental Microbiology. vol. 77, n. 18, p. 6687-6690, 2011.