Team:Manchester/Proof

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Proof of Concept

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 RFP 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 isolate and purify the 98kDa AlcR protein by SDS-PAGE. 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 or the size of the protein is too big to be seen on the gel. There is a possibility that the expressed AlcR protein can be seen on 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. 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.

Quantification

Figure 1: 22-hour growth curves of DH5α E. coli containing: (i) co-transformed (CT) (ii) control (Ctrl) plasmids grown in LB broth (negative control). CT= Co-transformed Plasmid 1 (CP3+alcR) + plasmid 2 (CP3+amilCP. Both CT and Ctrl sample groups were induced with different ethanol concentrations (0-5%) at mid-log phase, OD₅₈₀ = ± 0.4. The samples were measured every 30 minutes at OD₅₈₈ (λ_max) and OD₄₅₀ (λ_min) using the BMG Labtech FLUOstar Omega spectrophotometer, under constant temperature of 37°C. All points are the mean of 2 biological replicates for each plasmid, normalized to the blank with error bars = SD.

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 was 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 tried 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).

Figure 2: Absorbance spectrum readings (λ=350-650nm) of undiluted, purified CT and Ctrl samples, using the top optic of the BMG Labtech FLUOstar Omega spectrophotometer under constant temperature of 37°C.

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 by several other iGEM teams.


We then tried to spin down the aliquots and perform 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.

Figure 3: Absorbance spectrum readings (λ=350-650nm) of 1:10 diluted, purified CT and Ctrl samples, using the top optic of the BMG Labtech FLUOstar Omega spectrophotometer under constant temperature of 37°C.

Result shown in Figure 3 was 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 had as we did not expect to come as far to this stage. Additionally, due to the time constraint we did not proceed with this troubleshooting. However, based on the several failed attempts we propose that:

The baseline settings of our spectrophotometer for absorbance measurement were not fully configured.

Our purification strategy and technique might not be accurate as this was the very first time we performed this procedure.

The spectrophotometer’s filter used might be dirty or not suitable for the tests we ran.

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