Overview

In the process of engineering practice, we are constantly thinking about how to improve the synthesis of 2-PE and caffeine to make the project more consistent with real-world prototypes. We also think very carefully about biosafety. In each iteration and update, we are very grateful to the people who provide us with help and advice.

1 Synthesis of 2-phenylethyl alcohol (2-PE)

Stage 1	Stage 2	Stage 3
In the early stage of 2-PE synthesis and construction, we did not go well. Our 2-PE synthesis involves three genes: aroG, adh6 and aro10. In the process of constructing three genes on the same plasmid, we found that pET could not lay down all three genes at the same time. It can be speculated that the length of three genes placed on a plasmid is close to the length that the plasmid can bear, which may be because the molecular weight of the plasmid is too large, resulting in low transformation efficiency.	The construction of aroG, adh6 and aro10 genes on plasmid pET failed, so we temporarily used plasmid pCS to express aroG and plasmid pET to express adh6 and aro10. After the successful construction of pET-aro10-adh6 and pCS-aroG, 2-PE was synthesized by E. coli Nissle 1917 through induced expression and fermentation. After 72h fermentation, the content of 2-PE was detected.	The highest yield we obtained was 67.16mg/L. Compared with the maximum yield of JLU-China, the yield of BUCT was lower. Referring to the ideal yield of 130mg/L calculated by JLU-China, we believe that measures should be taken to increase the yield of 2-PE. First, add phenylalanine (Phe) to the substrate; Second, the genes applied by JLU-China in 2-PE synthesis were compared with our gene combination to select the optimal element combination.

Stage 1

Stage 2

Stage 3

In the early stage of 2-PE synthesis and construction, we did not go well.

Our 2-PE synthesis involves three genes: aroG, adh6 and aro10. In the process of constructing three genes on the same plasmid, we found that pET could not lay down all three genes at the same time.

It can be speculated that the length of three genes placed on a plasmid is close to the length that the plasmid can bear, which may be because the molecular weight of the plasmid is too large, resulting in low transformation efficiency.

The construction of aroG, adh6 and aro10 genes on plasmid pET failed, so we temporarily used plasmid pCS to express aroG and plasmid pET to express adh6 and aro10. After the successful construction of pET-aro10-adh6 and pCS-aroG, 2-PE was synthesized by E. coli Nissle 1917 through induced expression and fermentation.

After 72h fermentation, the content of 2-PE was detected.

The highest yield we obtained was 67.16mg/L. Compared with the maximum yield of JLU-China, the yield of BUCT was lower. Referring to the ideal yield of 130mg/L calculated by JLU-China, we believe that measures should be taken to increase the yield of 2-PE.

First, add phenylalanine (Phe) to the substrate; Second, the genes applied by JLU-China in 2-PE synthesis were compared with our gene combination to select the optimal element combination.

Unfortunately, due to the epidemic, we could not use our laboratory for a long time. In the future, we will continue to complete the new design.

2 Synthesis of Caffeine

Stage 1	Stage 2
We constructed pCS-TcAncCS-TcCS2-TCS1 based on methylated proteases selected from the literature, starting with Xanthine for methylation, and then using 3-methylxanthine to synthesize theobromine, a precursor of caffeine, and finally caffeine. After induction and fermentation, caffeine content was measured by HPLC. The maximum yield of theobromine and caffeine was 7.16mg/L and 1.83mg/L, respectively.	In reality, there was no condition to verify the yield of E. coli Nissle in practical application environment. We need to test the reasonableness of the yield through modeling and calculation. According to the results of the model, the caffeine production in the actual application environment can exceed the effective concentration threshold of 0.4mg/L. So in reality, our engineered bacteria can produce caffeine to promote the growth of hair follicles.

Stage 1

Stage 2

We constructed pCS-TcAncCS-TcCS2-TCS1 based on methylated proteases selected from the literature, starting with Xanthine for methylation, and then using 3-methylxanthine to synthesize theobromine, a precursor of caffeine, and finally caffeine.

After induction and fermentation, caffeine content was measured by HPLC. The maximum yield of theobromine and caffeine was 7.16mg/L and 1.83mg/L, respectively.

In reality, there was no condition to verify the yield of E. coli Nissle in practical application environment. We need to test the reasonableness of the yield through modeling and calculation.

According to the results of the model, the caffeine production in the actual application environment can exceed the effective concentration threshold of 0.4mg/L. So in reality, our engineered bacteria can produce caffeine to promote the growth of hair follicles.

3 Suicide system

Stage 1	Stage 2	Stage 3
The original suicide system requires iron concentration to control promoter expression. And the promoter controls the expression of the toxic protein MazF. We constructed this initial model in the hope of finding an economical, safe and effective donor of iron ions. We also consulted two doctors and took other teams' advice. Finally, we chosed to try the quorum sensing promoter because the safety of this prototype was not high enough.	We combined several suggestions and then built a quorum-sensing promoter-controlled suicide system. This promoter also controls the expression of MazF. In order to screen a reasonable bacterial density, we calculated on the basis of laboratory fermentation data. We then determined whether caffeine could be synthesized at the desired yield under real-world conditions to ensure that the bacterial density selection did not fall below that standard.	We considered the competitive relationship between E. coli Nissle 1917 and Malassezia grown under the same conditions. In our design, Nissle is required to inhibit Malassezia by competing for free fatty acids. According to calculation, when triggering OD=0.21, MazF would be expressed and cause Nissle to die. Meanwhile, under 0.21, the growth curve of Nissle is always higher than that of Malassezia, indicating that Nissle could inhibit Malassezia. The modeling and calculation based on the experimental data proved that our Nissle could not only ensure the beneficial effect of caffeine on hair follicles at 250CFU/mm², but also compete with Malassezia for survival resources at this density and affect the growth of Malassezia.

Stage 1

Stage 2

Stage 3

The original suicide system requires iron concentration to control promoter expression. And the promoter controls the expression of the toxic protein MazF. We constructed this initial model in the hope of finding an economical, safe and effective donor of iron ions. We also consulted two doctors and took other teams' advice.

Finally, we chosed to try the quorum sensing promoter because the safety of this prototype was not high enough.

We combined several suggestions and then built a quorum-sensing promoter-controlled suicide system. This promoter also controls the expression of MazF.

In order to screen a reasonable bacterial density, we calculated on the basis of laboratory fermentation data.

We then determined whether caffeine could be synthesized at the desired yield under real-world conditions to ensure that the bacterial density selection did not fall below that standard.

We considered the competitive relationship between E. coli Nissle 1917 and Malassezia grown under the same conditions. In our design, Nissle is required to inhibit Malassezia by competing for free fatty acids.

According to calculation, when triggering OD=0.21, MazF would be expressed and cause Nissle to die.

Meanwhile, under 0.21, the growth curve of Nissle is always higher than that of Malassezia, indicating that Nissle could inhibit Malassezia. The modeling and calculation based on the experimental data proved that our Nissle could not only ensure the beneficial effect of caffeine on hair follicles at 250CFU/mm², but also compete with Malassezia for survival resources at this density and affect the growth of Malassezia.