Team:NDC-HighRiverAB/MethodologyandResults
Methodology And Results
In order to reach our goal of breaking down fats, which are a major component of buildups in wastewater systems, we created a novel biobrick which would allow our bacteria to produce an esterase under control of an inducible-promoter.
Design
- First, we researched an esterase-producing gene that could be expressed in E.coli. We decided to use the EstA gene, which is found in pseudomonas aeruginosa.
- For our promoter, we decided to use pLAC, which is lactose-inducible and is naturally found in E.coli.and was available in the registry (Part:BBa_B0011).
- We chose the B0034 RBS (Part:BBa_B0034).
- Finally, we chose the double terminator B0015, which is the most common terminator and is known to be reliable.(Part:BBa_B0015).
- After we had chosen all of our biobrick parts, we had them synthesized together, creating our new biobrick: (Part:BBa_K2694000).
Figure 1: Our part BBa_K2694000
Qualitative Testing
Once we received our DNA, we transformed it into DH5α E.coli. We then did a plasmid switch to clone our part into PSB1C3 for submission to the Registry. To test our new part, we grew up cultures of our bacteria overnight. We then subcultured them for three hours before beginning the testing.
We first did a qualitative test. From the literature, we found that we could use 4-nitrophenyl palmitate and 4-nitrophenyl octanoate to test our esterase activity. Both substrates, when cleaved by an esterase, would go from clear to green, allowing us to monitor the cleavage activity visually. We set-up a time-course assay where we mixed 1mM of each nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer. We also tested DH5⍺ cells without any plasmid as well as the ester mixture with no cells as controls. Figure 2 Shows the results from this experiment.
Figure 2: The top row shows centrifuge tubes containing the 4-nitrophenol ester reaction mixture for the palmitate (a) and octanoate (b) esters with no cells added. The middle row shows the response for a negative control where DH5⍺ cells without any plasmid were added to reaction mixtures containing 4-nitrophenyl-palmitate (c) or 4-nitrophenyl-octanoate (d). The bottom row shows the response for the BBa_K2694000 circuit transformed into DH5⍺ cells when added to reaction mixtures containing 4-nitrophenyl-palmitate (e) or 4-nitrophenyl-octanoate (f). All reaction mixtures were 1mM of the nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer.
From these results, we concluded that our bacteria was indeed cleaving the substrates, resulting in the green color. The clear control remained in the controls, showing that it was our part specifically that was inducing this change.
Quantitative Testing
Once we had shown that our bacteria could cleave the substrates to some extent, we wanted to try to show this more quantitatively. In collaboration with the University of Calgary’s iGEM team, we designed an experiment to test our reaction in a time course, using their spectrophotometer, in order to more accurately determine the color change.
Graph 1 shows all our data for the absorbance values (measured at 400nm) for BBa_K2694000 breaking down 4-nitrophenyl octanoate with and without IPTG. Our control started at 0 and ended at 0.002. All other samples increased for about 660 seconds and then started to level out. This graph shows that our part, BBa_k2694000 in E.coli was producing the EstA protein because the solution got progressively more green with the introduction of our bacteria, but without the bacteria the control did not change colour.
Graph 1: Absorbance Values for the Breakdown of 4-Nitrophenyl Octanoate by BBa_K2694000 With and Without IPTG.
Graph 2 shows the averages of the results in Graph 1. Based on these averages, the IPTG did not appear to have an effect on our reaction under these conditions. Our bacteria passively produces the repressor for pLac, but since we added more pLac to the system, there may not have been enough of the repressor to inhibit the production of our EstA, which may explain why IPTG did not have any effect on the reaction.
Graph 2: Average Absorbance Rates for the Breakdown of 4-Nitrophenyl Octanoate using BBa_K2694000 With and Without IPTG.
Graph 3 shows all our data for the absorbance values (measured at 400nm) for BBa_K2694000 breaking down 4-nitrophenyl palmitate with and without IPTG. Our control started at 0 and ended at 0.002 (same control as for Graph 1. All other samples did not increase significantly. This shows that BBa_K2694000 is not good at breaking down the 4-nitrophenyl palmitate under these conditions. As shown in our qualitative data in the section above, however, it does breakdown the palmitate. In this protocol we only used 100uL of cells as compared to 500-1000uL; maybe with more cells we would see the reaction happening faster.
Graph 3: Absorbance Values for the Breakdown of 4-Nitrophenyl Palmitate by BBa_K2694000 With and Without IPTG.
Graph 4 shows the averages of the results in Graph 3. Based on these averages, the IPTG did not appear to have an effect on our reaction under these conditions.
Graph 4: Average Absorbance Values for the Breakdown of 4-Nitrophenyl Palmitate by BBa_K2694000 With and Without IPTG.
Graph 5 shows the comparison of our absorbance values for the reaction of BBa_K2694000 with 4-nitrophenyl octanoate and 4-nitrophenyl palmitate with IPTG. This shows that our part is better at breaking down the 4-nitrophenyl octanoate.
Graph 5: Absorbance Values for the Breakdown of 4-Nitrophenyl Octanoate and 4-Nitrophenyl Palmitate by BBa_K2694000 With IPTG.
Graph 6 shows the average values from Graph 5, again showing the difference between how our part breaks down these different lengths of fatty acids attached to the 4-nitrophenol group. Under these conditions, our part is better at breaking down the shorter chain esters, like octanoate.
