After successfully characterizing our choice of constitutive promoters and chromoproteins, we proceeded to obtain a working model of our mechanism. We first built plasmid 1 and plasmid 2 (Figure 1: How it works?) using the iGEM 3A assembly method.

Plasmid 1

Figure 1: Schematic representation of the assembly of Plasmid 1.

This plasmid was constructed to obtain a constant expression of the AlcR protein. As seen in the figure, the constitutive promoter and the alcR were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Ampicilin/Carbenicilin resistance. Upon obtaining positive confirmation of the transformants, we proceeded to transform the ligated product into BL21, a protein expression strain.

Figure 2: SDS-PAGE gel showing protein expression results. As shown by boxes in the gel, the 27kDa mRFP1 protein can be clearly seen. However, no proteins can be seen at the 98kDa region where the AlcR protein is expected to be present.

After successfully obtaining transformants with the BL21 strain, we proceeded to overexpress the protein and attempted to show the expressed 98kDa AlcR protein on an SDS-PAGE (Figure 2). Unfortunately, after several attempts, we failed to see any visible protein band on the SDS-PAGE. This could be due to many factors, one of them being that the amount of expressed protein is not enough to be visualised on a SDS-PAGE. There is a possibility that the expressed AlcR protein could be seen by doing a Western-Blot analysis. However, the lack of a His-tag on the alcR gene did not allow us to explore this option.

Plasmid 2

Figure 3: Schematic representation of the assembly of Plasmid 2.

This plasmid is crucial in proving that our mechanism works (Figure 3). It consists of the alcA promoters (BBa_K2092002 and BBa_K2092003), which has the AlcR binding site, and chromoproteins to produce a visible colour change. Similar to Plasmid 1, the alcA promoters and the chromoproteins were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Chloramphenicol resistance.

Co-Transformation

Just two weeks before Wiki Freeze and the deadline for vector submission, we attempted to prove that our model works. To do so, we co-transformed Plasmid 1 (CP3+alcR) and Plasmid 2 (PalcA(var)+amilCP) and fortunately succeeded! Despite the short time available, we managed to test our model on our FLUOstar Image plate reader, using the parameters provided in the iGEM registry ( BBa_K592009 ) (λ_max=588nm) and research paper (λ_min=450nm). We compared the dynamics of co-transformed (CT) colonies with one of our new BioBricks, CP3+amilCP, as a positive control (Ctrl) under the influence of different ethanol concentrations (0-5% (v/v) with 1% increment). The parameters used for ethanol induction were based on a similar study which tested the same alc regulon system in E. coli K-12.

Quantification

Figure 1 indicates that all of the E. coli CT and Ctrl plasmids had normal growth curve patterns over the 22 hours period with no detectable growth in the negative control. Our data analysis based on the 588/450 OD ratio showed that there was no difference between the dynamics of chromoprotein expression by CT and Ctrl plasmids - even when the blue colour on Ctrl wells were clearly visible by naked eye! Sadly, we also did not observe a blue colour change in our CT samples despite being induced by different ethanol concentrations.

We attempted to troubleshoot our parameters by extracting the AlcR protein from CT plasmid and amilCP blue chromoprotein from Ctrl plasmid using a BugBuster protein extraction kit. The purified samples were then used to perform an absorbance spectrum test (λ=350-650nm).

Again, despite being able to see the blue colour of the extracted amilCP chromoprotein, there was no detectable peak at 588nm (λ_max) in Figure 2 as previously reported.


We then spun down the aliquots and performed a 1:10 dilution using the supernatant and LB broth as diluent to see if we could identify the suitable parameters to validate our model.

Results shown in Figure 3 were similar to Figure 2 as no peak could be detected at 588nm (λ_max).


We explored all of the alternatives using the limited resources that we have as we did not expect to come this far with the lab work. Additionally, due to the time constraint we did not proceed with troubleshooting the problem. However, based on the several failed attempts we propose that:

• The parameters used to quantify the chromoproteins are not correct.
• Our purification strategies and technique may not be accurate as this is the first time we are performing this procedure.

Pilot Experiment

It was decided to test our system using glucose oxidase while doing the experimental work involving synthesis and purification of alcohol oxidase. Similar to alcohol oxidase, glucose oxidase can be readily applied to cell free colorimetric assays.

As the name suggests glucose oxidase is involved in oxidation of glucose into D-gluconolactone and H₂O₂ as a by-product. In subsequent reaction horseradish peroxidase is used to oxidise its substrate ABTS in the presence of H₂O₂ that’s acts as an oxidising agent. This two-step reaction results in the formation of the green product that absorbs light at 420 nm.

• Plasmid 1
• Plasmid 2
• Co-Transformation
• Quantification
• Pilot Experiment