Team:BJU China/Proof Of Concept
Proof of Concept
Overall Objective:
In the design of our project, we need to knockout TnaA gene in E.coli BL21 genome and complete the following metabolic pathways (Figure 1), achieving denovo synthesis tyrian purple ( 6,6'-dibromoindigo, 6BrIG) from tryptophan. Furthermore, we also need to realize the application of 6BrIG in dyes and semiconductors.
Figure 1. Metabolic pathway of denovo synthesis 6BrIG from tryptophan
In order to complete proof of our concept, we made two parts to present our project: (1) Dyeing of 6BrIG, (2) Sensitized solar cell hardware.
Part I Dying 6BrIG
As mentioned above, 6BrIG is originated from tryptophan and produced by engineered E.coli with series of enzymes. In order to achieve the goal for producing 6BrIG, we knocked out TanA gene and obtain our chassis ΔTnaA E.coli BL21 using CRISPR-Cas9 system in advance (details are shown on our results page ). Then our progress can be divided into 3 sections including the verification of enzymes validity, the production of Tyrian purple, and the dyeing production.
First, we verified enzymes' validity. We constructed two plasmids (Figure 2) to express enzymes TnaA, MaFMO and Fre-L3-SttH in the ΔTnaA E.coli BL21: Fre-L3-SttH on pACYC backbone and TanA-MaFMO on pET28b backbone. All of these enzymes were controlled by T7-lacO inducible promoter. We used PCR cloning and gene sequencing to verify the sequences (Figure 3).
Figure 2. Gene maps of two plasmids with enzymes
Figure 3. PCR verification of three enzyme genes
Next, we want to test the expression of these enzymes with IPTG induction. For better expression of enzymes, we optimize the induction condition of gene expression: three different temperatures (18℃, 30℃, 37℃) and inducer concentrations (0.1mM, 1mM, 5mM for Fre-L3-SttH and 0.1mM, 1mM for TanA-MaFMO ) were set for test. Additionally, since the 6-halogenase Fre-L3-SttH is a fusion enzyme (including core enzyme SttH，co-enzyme Fre and rigid linker L3) with higher solubility, we disrupted collected cells and tested both pellet and supernatant after induction.
Figure 4. SDS-PAGE Gel of Fre-L3-SttH (Left is precipitation, Right is supernatant)
From the results in Figure 4, we can see that Fre-L3-SttH was successfully expressed (arrow points). Among the three temperatures, the enzyme expressed better at 37℃ , and when comparing different inducer concentrations, the induction effect of 1mM was the best. Meanwhile, though we made a fusion enzyme to improve SttH expression solubility, part of the enzymes could not be dissolved after expression and remained in the precipitation.
We also successfully detected TnaA-MaFMO enzyme after IPTG induction using SDS-PAGE (Figure 5). In the gel picture, we can clearly see TanA-MaFMO bands (about 52.8 KDa). And we can find that TanA-MaFMO expresses significantly better at 30℃ and 37℃ than 18℃. When the inducer concentration is lower (0.1mM), the protein bands are more obvious, and even when there is no inducer, the gene also have a basic expression, that is also called leakage. The leakage may caused by high gene expression using high copy plasmid pET28b.
Figure 5. SDS-PAGE Gel of TanA-MaFMO
Then, we introduced the entire reaction system to the two sets ΔTnaA E.coli BL21 strains, and let them undergo fermentation, to ultimately meet the goal of producing 6BrIG from Tryptophan. From our final experimental results, we successfully got certain amount of 6BrIG from our designed pathway. The purple shown in the bottom of the test tube proved the production of Tyrian purple.
We also did a fermentation experiment on 6,6-dichloroindigo. According to the result, our manufacture of indigo derivatives was a great success.
Figure 6. Fermentation Results
Finally, with the manufacture of 6BrIG, we dyed string to show its application in the dyeing industry. You can see that this purple color looks pretty in the sun.
Figure 7. Tyrian purple dyed string
Part II Sensitized solar cells
With the help of professional laboratories, we tested some of the photosensitive properties of 6BrIG. The data obtained is shown in Figure 8. This test result is consistent with what we obtained in literature research, so we determined to do optical test.
Figure 8. Fluorescence intensity of 6BrIG
According to the measurement of 6BrIG fluorescence strength, its maximum absorption peak is around 530nm, which is in the visible light region. So we then applied hardware to measure its function and proved the application of sensitized solar cells. We used TiO2 hardware, adhering with the 6BrIG layer to test its effect.
Figure 9. Hardware for sensitized solar cells
Following protocol2 we constructed the final hardware and exposed it on the lab environment to measure its working function by combining with electric ring and thermometer. Here are the final results:
（Electric Ring）
(Thermometer)
As shown in the two presentation videos, even though the electric element still works unstable now, we still hold optimistic opinion that its potential application is very large in the future.
