Team:Macquarie Australia/Improve
IMPROVE
Original Biopart: [BBa_K2471001]
Improved Biopart: [BBa_K3151008]
The secondary messenger used within our system is cyclic-di-GMP, which interacts with it in two main ways:
- Degradation by the EAL phosphodiesterase domain of the hydrogenase, the function of which is modulated by the presence of molecular hydrogen.
- Binding to the riboswitch to prevent the transcription of GFP
Figure 1: Hypothetical mode of action of the sense/response system of our biosensor. The hydrogenase will have its phosphodiesterase domain activated in the presence of molecular hydrogen. This will lead to a decrease in intracellular cyclic-di-GMP, preventing the riboswitch from forming the terminator loop structure and allowing the transcription of eGFP.
The native activity of cyclic-di-GMP as a secondary messenger in bacteria is primarily to regulate the transition between motile and sessile biofilm forming behaviour. Synthesis of cyclic-di-GMP itself is regulated by the stationary-phase sigma factor RpoS (σS), which when induced results in an increase in cyclic-di-GMP concentration, and subsequent downstream increase in the production of cellulose and curly fimbriae, the primary components of Escherichia coli biofilm.
In order to develop assays for the phosphodiesterase activity of our system, we took an existing Biopart [BBa_K2471001] which encodes for the yhjH phosphodiesterase, and improved it by replacing its T7 promoter with an inducible Lac promoter, as well as adding a double terminator. This improved part allows us to not only control the expression of the phosphodiesterase with the addition of IPTG, but to use the part in strains which do not use a T7 RNA polymerase.
Our plasmid, along with a plasmid containing the original T7+yhjH Biopart, were transformed into the E. coli strains DH5α, Nissle 1917, and BL21(DE3). By inducing the yhjH phosphodiesterase activity with IPTG (1 mM) we can cause a decrease in cellular concentrations of cyclic-di-GMP, and can measure the intensity of this activity by observing the decreased biofilm formation capability of the cells.
We assayed the phosphodiesterase activity of the transformed cells through spotting the transformants onto LB plates supplemented with Congo Red and Coomassie Blue (link). The Congo Red stains the cellulose produced by the cells, while the Coomassie Blue binds to the curly fimbriae (both products of biofilm formation). This effect is particularly noticeable in the Nissle 1917 cells, which produces less curly fimbriae but significantly higher levels of cellulose than wild-type E. coli.
The spot assay shows a significant reduction in cellulose synthesis in Nissle 1917 cells transformed with the Lac+yhjH compared with negative control, and little to no reduction in cells transformed with the T7+yhjH (Figure 2). No significant change was seen in any of the DH5a cells (Figure 4). No significant difference in colony morphology was observed between negative control BL21(DE3) and BL21(DE3) cells transformed with T7+yhjH, though some reduction in Congo-Red staining was observed in BL21(DE3) cells transformed with Lac-yhjH (Figure 3), which indicates a reduction in cellulose biosynthesis and therefore an increase in phosphodiesterase activity.
Figure 2: Spot Assay of the negative control Nissle 1917 (A), Nissle 1917 with T7+yhjH (B), and Nissle 1917 with Lac+yhjH (induced with 1 mM IPTG) (C). Plate observed under blue light to enhance visualisation of Congo Red-stained cellulose.
Figure 3: Spot Assay of the negative control BL21(DE3) (A), BL21(DE3) with T7+yhjH (B), and BL21(DE3) with Lac+yhjH (induced with 1 mM IPTG) (C). Plate observed under white light to enhance visualisation of colony morphology.
Figure 4: Spot Assay of the negative control DH5a (A), DH5a with T7+yhjH (B), and DH5a with Lac+yhjH (induced with 1 mM IPTG) (C). Plate observed under white light to enhance visualisation of colony morphology.
To better characterise the effect of the Lac promoter in the context of our biosensor, we combined the Lac+yhjH construct with our two riboswitch+eGFP constructs, one with and one without the terminator stem, into new plasmid backbones. These are referred to as yhjH+ROFF [BBa_K3151029] and yhjH+RON [BBa_K3151030] respectively. The expression of eGFP by the yhjH+RON part should not be affected by the concentration of cyclic-di-GMP, so inducing the phosphodiesterase ought to have no effect on the fluorescence of the transformants. The expression of eGFP by the yhjH+ROFF part should be repressed by the concentration of cyclic-di-GMP, so inducing the phosphodiesterase should increase the intensity fluorescence of the transformants.
This effect was assayed by performing a spot assay on LB plates supplemented with Congo-Red and Coomassie Blue. After 24 hours of incubation, the plates were observed under blue light to visualise the eGFP expression (Figure 5). Our model of the mode of action of the induced phosphodiesterase was correct in both cases; induction had no effect on the yhjH+RON cells, while inducing the yhjH+ROFF cells resulted in a significant increase in expression of eGFP.
Figure 5: LB agar plate supplemented with Congo Red and Coomassie blue, spotted with yhjH+RON induced (A), yhjH+ROFF induced (B), yhjH+RON uninduced (C), and yhjH+ROFF uninduced (D).
We initally attempted to perform the same microtitre plate biofilm adherence capacity assay that was performed to characterise the original T7+yhjH Biopart. We expected to see a reduction in biofilm formation in cells with active phosphodiesterase activity. However, after many attempts we found that the assay was prone to error and the results were not reproducible.
Figure 5: Microtitre plate biofilm adherence capacity assay results performed on all constructs containing the yhjH. These include the yhjH containing the Lac promoter and double terminator in DH5a and Nissle 1917, yhjH without the Lac promoter and double terminator in DH5a and Nissle 1917, yhjH+RON/ROFF in DH5a and Nissle 1917, as well as negative control DH5a and Nissle 1917. Induction performed with 1 mM IPTG.
In conclusion, our newly designed composite part is a significant improvement over the original Biopart. Not only does it have inducible phosphodiesterase activity, but it can also be expressed in a variety of E. coli strains which do not possess T7 RNA polymerase, including the incredibly common DH5α lab strain.