In stage 1, vector used is the pRSF-Duet1 from own storage clone and inserts included are the RpfC, RpfG, Clp-PspF, sigma 54, CBS, eforRed, synthesised by the INTEGRATED DNA TECHNOLOGIES (IDT). Protocols used are on the protocols page. With the aid of PCR, the fragments was amplified for further cloning processes. The optimal conditions, which is enclosed in the protocol page, were found after numerous tests.

Figure 1: 1% agarose gel showing the results of PCR. Right) the bands of RpfC and RpfG. Middle) the bands of Clp-PspF and sigma 54. Left) the bands of CBS and eforRed.

Then we performed restriction digest for afterward insert ligation. To prevent self-ligation, double digestion and alkaline phosphatase were used. The ligated plasmids were then successfully transformed into E. coli.

Figure 2: 1% agarose gel showing the results of restriction digestion. Lanes 3 to 4 are the uncut control and single cut control, showing the viability of enzyme used. Lanes 6 to 8 are the double cut vector, pRSFduet. Lanes 10 to 15 are the double-cut inserts.

After transformation, colonies were picked and colony PCR were performed to verify the existence of inserts in clones.

Figure 3: 1% agarose gel showing the results of colony PCR. 3 colonies were picked for each insert. At least one successful were obtained for each insert. Correct band sizes and locations were labelled. The faint bands at the bottom were suspected to be the primer dimers. Regarding the lanes of CBS, the CBS bands were overlapped with the primer dimers.

Stage 3

In stage 3, PCR was carried out to change the restriction sites of the constructs. Construct A, the RpfC-RpfG insert, would contain the EcoRI and PstI sites. Construct B, the Clp_pspF_TAD-sigma 54 insert, would contain NdeI and XhoI sites. Construct C, CBS-eforRed insert, would contain XbaI and SpeI sites. After PCR, restriction digestion, ligation and transformation were followed. The existence of positive clones was confirmed by colony PCR (figure 4).

Figure 4a: 1% agarose gel; Clones containing construct A were obtained. Clone 1, 2, 3, 5, 6, 8, 10, 11, 12, 13, 15, 16 showed positive results.

Figure 4b: 1% agarose gel; Clones containing construct B were obtained. All clones picked showed positive results.

Figure 4c: 1% agarose gel; Clones containing construct C were obtained. Clone 1, 2, 3, 4 showed positive results.

Stage 5

In stage 5, construct A, B and C were ligated in series in the same plasmid, forming the final cloning product. (figure 5)

Figure 5: 1% agarose gel showing the result of colony PCR. Clones containing construct ABC were obtained. Clones from 1 to 16, except 8, showed positive results.

Protein expression

After stages of cloning, western blotting was performed using the iBind system from Life Technologies to see if the inserts can be successfully expressed after being induced. Clones were induced by 1mM IPTG at 32oC and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr.

Clones were induced by 1mM IPTG at 32℃ and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, Clp-PspF, RpfG, RpfC proteins were confirmed with successful expression at all time points. Results of blots probing Clp (figure 6a) and RpfG (figure 6b) proteins showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells. For the blot probing RpfC (figure 6c) protein, clones collected at 16hr and 20hr showed highest target protein quantity instead.

Figure 6a: Western blot analysis of Clp protein expression using Myc-Tag (9B11) Mouse mAb (1:2000).

Figure 6b: Western Blot analysis of RpfG protein expression using Sigma F7425 ANTI-FLAG® antibody (1:5000).

Figure 6c: Western Blot analysis of RpfC protein expression using Sigma Aldrich Monoclonal Anti-HA antibody(H9658) (1:20000).

Codon optimization confirmation

Figure 7: Nitrocellulose membrane stained by Pierce™ Reversible Protein Stain Kit after protein transfer from SDS-PAGE gel.

Following confirmation of protein expression, codon optimization of RpfG was performed. RpfG overexpression can be clearly seen in clones after codon optimization. The amount of RpfG proteins significantly increased compared to that in the old clones. The amount also rose with a longer incubation time of IPTG. Comparing Figure 1b and Figure 2, we can deduce that RpfG expression reached saturation at 12hr and started degradation afterward.

eforRed signal generation - Dosage and time dependent

Figure 8a:

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Figure 8a -8h shows cells that were induced by different concentrations of IPTG, ranging from 0 mM to 10 mM. In general, red-colored pellets could be shown after 2 hours incubation with IPTG.

eforRed signal generation - Time dependent

Figure 9: eforRed expression level in response to 1mM IPTG induction from the induction time of 0 to 18 hour. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). Two star signs (**) indicate significance at p < 0.01. Three star signs (***) indicate significance at p < 0.001. Four star signs (****) indicate significance at p < 0.0001.

Expression eforRed was significantly increased, starting from 6 hours after induction, while it reached maximum at 10 hours after induction and started decline.

c-di-GMP suppressive drug assay

Figure 10: The graph showed the fold change in eforRed expression after various concentration of sulfathiazole treatment. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). Two star signs (**) indicate significance at p < 0.01. Three star signs (***) indicate significance at p < 0.001. Four star signs (****) indicate significance at p < 0.0001.

The cells were first induced for 4 hours by 1mM IPTG. Then, it was followed by the 16-hour treatment by 0-800g/mL of sulfathiazole. The cells reached the maximum increase in eforRed expression at 25ug/mL drug treatment, followed by decrease in expression level at higher drug concentrations. Results showed a significant increase in all treatment groups. The decrease in expression might be due to the cytotoxic activity of sulfathiazole.

Figure 11: The graph showed the fold change in eforRed expression after various concentration of azathioprine treatment. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). Two star signs (**) indicate significance at p < 0.01. Four star signs (****) indicate significance at p < 0.0001.

The cells were first induced for 4 hours by 1mM IPTG. Then, it was followed by the 16-hour treatment by 0-400g/mL of azathioprine. The cells reached the maximum increase in eforRed expression at 100ug/mL drug treatment, followed by decrease in expression level at higher drug concentrations. Results showed significant increase in all treatment groups. The decrease in expression might be due to the cytotoxic activity of azathioprine.

Azathioprine and Sulfathiazole are indirect suppressor to cyclic-di-GMP, treatment of those suppressive drug to bacteria culture can reduce the cyclic-di-GMP level and lead to activation of the pspF-Clp and by-pass the RpfC/RpfG two-component system in theory .The result from rt-qPCR data below showed the above synthetic biological system was functional, indicated by the significant up-regulation of reporter mRNA.

DSF treatment assay

Figure 12: The graph showed the fold change in eforRed expression after various concentration of DSF treatment. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). One star sign (*) indicates significance at p < 0.05.

As DSF is the extracellular signaling molecule secreted by the Xcm bacteria, the aim of our product is to detect the existence of DSF and release color signal. The DSF treatment assay helped us ascertain whether the engineered E. Coli starts transcription under environment with DSF. Results indicated that at 1uM DSF concentration, eforRed transcript significantly increased in the bacteria, proving that the bacteria responsed to DSF molecules.

The cells were first induced for 4 hours by 1mM IPTG. Then, it was followed by the 16-hour treatment by 0-1000uM of DSF. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation.

Team:Hong Kong-CUHK/Results

Wet Lab-Cloning

Stage 1