Engineer cycle
First iteration
Design
Research
The main chemical constituent of Tyrian Purple: 6, 6'-dibromoindigo(6BrIG), is a halide derivative of indigo. Therefore, we tried finding a way to produce indigo at first. Fortunately, we found that some previous iGEM teams( 2013 iGEM team Berkeley, 2019 iGEM team GreatBay_SZ, 2020 iGEM team SZU-China et al.) have already built the pathway from tryptophan to indigo. So we decided to reconstruct the indigo pathway based on their work.
Figure 1 Scheme of indigo production pathway
Build
Plasmid construction
We synthesize the sequence of TnaA and MaFMO with pET28a vectors through Genscript.
Figure 2 Plasmid Maps of pET28a-TnaA and pET28a-MaFMO
Strain establishment
Once received the DNA dry powder of pET28a-TnaA and pET28a-MaFMO from Genscript, we diluted them with ddH2O and use DH5alpha to clone the plasmid. Then we transformed BL21 strains with the cloned plasmids to establish expression strains.
Figure 3 Transformant colony on the LB agar
Test
Expression verification
To gain the enzyme we need to produce indigo, the BL21 strain which harbored pET28a-TnaA and pET28a-MaFMO was inoculated to LB medium and incubated. Continue the culture for about 4 hours after induction with IPTG when the OD reach 0.6-0.8. SDS-PAGE was loaded with the denatured culture sample and results are as follows. We also determine the optimal induction time of these enzymes.
Figure 4 MaFMO(right) expression results under induction time gradient:1: 0h, 2: 9h, 3: 10.5h, 4: 12h, 5: 13.5h, 6: 15h, 7: 16.5h, 8: 18h, 9: 19.5h; TnaA(left) expression results under induction time gradient:
0: 0h, 1: 4h, 2: 5h, 3: 6h, 4: 7h, 5: 8h, 6: 9h, 7: 10h, 8: 11h
Growth curve measurement
We measured the growth curve of the two strain to get the optimal growth condition. In this experiment, we took samples every half an hour and tracked the growth of the strain continuously for 36 hours.
Figure 5 Growth curve of TnaA and MaFMO
Indigo production in vivo
For MaFMO need reducing power like NADH to exert its catalytic function1, we take a strategy of in vivo reaction. We centrifuged the culture of logarithmic period to cells, and use their resuspension to conduct the in vivo reaction. You could find more experiment details here.The results is listed below.
Figure 6 LB medium with produced indigo in culturetube
Learn
In our first iteration, we have successfully construct a pathway to produce indigo using E.colias chassis. Although we hadn't measured the actual yield of the strain, the result of the in vivo experiment show that our strategy is feasible. The second iteration would realize the preliminary production of 6,6'-dibromoindigo.
Second iteration
Design
After we produced Tyrian purple, we want to simplify the reaction process and improve the yield of producing. We bought 6-Br-indole and did a series of solubility test, found 6-Br-indole is very difficult to dissolute even in absolute ethyl alcohol(only dissolved in DMSO). Based on this we suspected that due to insolubility of 6-Br-indole, the transmembrane transport was limited，so we used high-efficiency assembly method——In-Fusion to connect TnaA and MaFMO in one plasmid.
Here is the brief reaction steps:
Bulid
We connected the sequence of TnaA and MaFMO with pET28a vectors through In-Fusion.
Figure 7 Plasmid Maps of pET28a-TnaA- MaFMO
Test
First, we verified the expression of TnaA-MaFMO at 30℃
Figure 8 SDS-PAGE analysis of TnaA-MaFMO
Second, we added 6-Br-Trp to whole cell reaction and obtained excellent production.
Figure 9 Some image to verify the infusion reaction
Third ,We compared two reaction systems by Microplate Reader, and found this improvement was more productive.
Figure 10 Microplate Reader analysised of produced Tyrian purple.
TnaA knockout strain
Potential side reaction in the first step may reduce the final productivity, which we have explained in the our description page. Hence we knocked out TnaA gene of the E.coli strain we used in the first steps by CRISPR/Cas9. Here are the PCR result to validate the knock-out.
Figure 11 PCR products using the upstream and downstream sequence contained primers
Learn
We had achieved our goal to simply the second step of the whole-cell reaction and increase 6BrIG production.
Safety and risk assessment
On April 15, 2021, a new biosecurity law was passed in China, making biological information security to the national level. We strictly obey the legal approval process, carry out a series of safety monitoring such as testing chemical drugs in dye process before putting our production into market. We should try our best to ensure that our products don’t do harm to humans and environment.
References
[1] Fabara, A.N., Fraaije, M.W. An overview of microbial indigo-forming enzymes. Appl Microbiol Biotechnol 104, 925–933 (2020). doi.org/10.1007/s00253-019-10292-5
[2] Lee J, Kim J, Song JE, et al. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol. 2021; 17(1):104-112. doi:10.1038/s41589-020-00684-4