Addition of a New Basic Part: P_rhl/lac
In this project, we added a new promoter part to the registry: P_rhl/lac (Part: BBa_K4708001).
P_rhl/lac is a hybrid promoter that, in our project, drives the expression of RhlI and fluorescence proteins (Chen, et. al., 2015). The promoter is constitutively on unless LacI is present and the cell is induced with IPTG or a quorum signaling molecule that promotes LacI expression.
In our project, P_rhl/lac is inserted in a CFP expressing cell to study cell communication: pC165 (Figure 1). The cell with this promoter can act as a receiver cell in a quorum signaling pathway if LacI is targeted and induced by the signaling molecule (repressing the promoter's activity) (Figure 2a). The cell can also act as a sender in a quorum signaling pathway, since it promotes the expression of RhlI, producing C4HSL (Figure 2b).
Figure 1. The plasmid, pC165, in which P_rhl/lac is inserted. In pC165, P_rhl/lac drives the expression of CFP and RhlI. Kanamycin acted as the selectable marker. To turn off constitutive expression, LacI must be in the native genome or inserted via a second plasmid and induced with IPTG.
Figure 2. Two 2-pathway options within the CFP/YFP synthetic pathway. (A) Pathway 1 involves the CFP cell acting as the receiver. When activated with IPTG, the receiver will be on, constitutively fluorescing CFP. Only when the YFP sender cell produces 3-OHC14HSL (once LacI is inhibited by IPTG, driving expression of CinI) will the sender cell turn off by driving LacI expression and repressing CFP. (B) Pathway 2 involves the YFP cell acting as the receiver. The CFP sender cell will produce C4HSL via RhlI, triggering the receiver cell to produce YFP fluorescence oscillations.
Characterizing P_rhl/lac
We first characterized the plasmid containing P_rhl/lac, pC165, by creating a growth curve (Figure 3). The pC165 cell entered its log phase (the phase in which we want to begin experimentation) at hour 3 and growth leveled off after hour 6. Additional fluorescence data was added to the registry and can be found under Results page.
Figure 3. pC165 growth curve. We took cells from a glycerol stock and grew in LB media and kanamycin overnight. Cells were transferred to 3mL LB and kanamycin (1:1000 Kanamycin:LB) and data was recorded for 7 hours.
References
Chen, Y., Kim, J. K., Hirning, A. J., Josić, K., & Bennett, M. R. (2015). Emergent genetic oscillations in a synthetic microbial consortium. Science, 349(6251), 986-989.