To begin our experiment of producing 6BrIG from tryptophan in the proposed pathway, we have to first remove the TnaA already present in the genome of E.coli using CRISPR-Cas9 system. This action is carried out to prevent the endogenous TnaA expression and thus the reduction of 6BrIG production. However, due to a faulty design in the gRNA, the off-target effect disabled the ability of the Cas9 to remove TnaA. Through this process, the sgRNA is bounded to the targeted gene and lastly knock it out. After the redesignation of the gRNA, the TnaA was successfully removed and inserted onto the MaFMO plasmid. We then did a PCR of the plasmid and the results of the agarose gel electrophoresis (AGE) showed that the gene is completely removed (The length of the plasmid before removal is 2514bp, while the length of tnaA is 1334bp. Therefore after removal the length of the plasmid should be 1180bp, which is consistent with the results shown in the picture). The professional sequencing process yielded the same result.
Figure 1. Mutation result of TnaA
While doing the mutation experiment, we also underwent cloning and protein expression of our target genes.
By using Gibson cloning, we infused Fre-L3-SttH, Fre-L3-PyrH, Fre-L3-RebH into pACYC plasmid. With IPTG induction, we analyze the protein expression of halogenase by SDS-PAGE method.
Figure 2. (a) SDS-PAGE of precipitated Stth protein. (b)SDS-PAGE of supernatant Stth protein. (c) SDS-PAGE electrophoresis of supernatant PyrH.(d) SDS-PAGE of precipitated PyrH. (e)SDS-PAGE of supernatant and precipitate of RebH protein. (f) SDS-PAGE of TnaA and MaFMO protein.
By changing the induct IPTG concentration and the induction temperature, we get the best induce condition for each halogenase. The best induce condition for SttH is 1mM 37℃(Figure 1a and Figure 1b), PyrH is 0.1mM 18℃(Figure 1c and Figure 1d), RebH is 0.1mM 37℃(Figure 1e) as shown by the arrow .
Additionally, we combined TnaA and MaFMO genes together and fused in pET28b plasmid. From Figure 1f, the induction of enzyme TnaA and MaFMO has no significant difference between IPTG concentration and temperature, so we decided to use 0.1mM IPTG and 18℃ to induce the protein for further fermentation analysis.
According to protocol 6 guidance on producing 6BrIG, we induced ΔTnaA E.coli with MaFMO gene transfered. We then centrifuged the induced bacteria and suspended with 0.6%(ω/V) glucose solution and added 6Br-indole to the final concentration of 1mM, 30℃ fermentation for 6h.
Figure 3. Solution after the reaction has a distinct purple color compared to the unreacted E. coli solution.
After 6h fermentation, we got the solution. Compared with the original solution(Figure 2. right EP tube), the final product has component with purple. In order to get a specific analysis of the final product, we extracted the component and sent to university to do MALDI-TOF spectra analysis.
Figure 4. The Extraction of fermented solution
We also produced 6BrIG from tryptophan as substrates. From the figure5 result, team 1 and 2 are the production result of 6BrIG comparing with control group. With the addition of Fre-L3-SttH reaction products, the final solution turns to purple.
Fig5. The fermentation of 6BrIG.
3. Sensitized Solar Cell Analysis
Figure 6. Fluorescence intensity of 6BrIG with the results of excitation photometry experiments
According to the measurement of 6BrIG fluorescence strength, the result is conformed with the statement of research report. We then applied hardware to measure its function. Here is the construction of the electrical circuit of sensitized solar cells with the combination of thermometers:
And here is the construction of the electrical circuit of sensitized solar cells with the combination of ring: