Team:BUCT/Implementation
Overview
In this year, our project aims to help modern people to solve the problem of excessive pressure in the fast-paced life of work and study, and achieve a healthier life. We hope to accelerate the consumption of fatty acids by engineering bacteria, and produce GABA and 5HTP, which can help improve people's mental quality, and help people get healthier. Based on the brain-gut axis theory, our project has full feasibility and is likely to be translated into reality in the future, potentially providing better health care products for people.
Overview of the overall design
Production of GABA
According to our design, a system based on the engineered bacteria E.coli Nissle 1917 will be developed to accelerate the consumption of fatty acids and efficiently produce GABA and 5HTP with precursors produced by beta oxidation reactions, which can survive and be effective in the human gut environment.
After constructing plasmid pCS-gadB(mut)-gdhA-T1, we transfer the plasimid into DH5a and culture for 12 hours, then we can pick individual colonies and shake flask to cultivate under the circumstance that the temperature is 37℃. Then we performed enzyme validation.
Meanwhile, electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit. Then, we eletrotransformation the plasmid into E.coli Nissele 1917, and culture it for 12 hours. Then we select a single colony to continue to cultivate and prepare for fermentation.
The temperature of fermentation is 30℃ and we fermented for 60h.
After constructing plasmid pCS-gadB(mut)-gdhA-T1, we transfer the plasimid into DH5a and culture for 12 hours, then we can pick individual colonies and shake flask to cultivate under the circumstance that the temperature is 37℃. Then we performed enzyme validation.
Meanwhile, electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit. Then, we eletrotransformation the plasmid into E.coli Nissele 1917, and culture it for 12 hours. Then we select a single colony to continue to cultivate and prepare for fermentation.
The temperature of fermentation is 30℃ and we fermented for 60h.
Below are the statistics after Chromatograph test and it shows that we have successfully produced GABA from simple carbon sources:
GABA was produced by fermentation with glycerol as substrate:
GABA was produced by fermentation with glycerol as substrate:
GABA was produced by fermentation with palm oil as substrate:
GABA was produced by fermentation with soybean oil as substrate:
Analysis:
As we can see from the three charts, we overproduce GABA up to 5.276g/L when the substrate is glycerol. And overproduce GABA up to 4.807g/L when the substrate is palm oil and up to 8.315g/L when the substrate is soybean oil.
Although we have successfully overproduce GABA, the question is that the amount of GABA is too much and human body can not undertake it. So maybe it is a good choice to overproduce GABA outside the human body and just in the factory.
As we can see from the three charts, we overproduce GABA up to 5.276g/L when the substrate is glycerol. And overproduce GABA up to 4.807g/L when the substrate is palm oil and up to 8.315g/L when the substrate is soybean oil.
Although we have successfully overproduce GABA, the question is that the amount of GABA is too much and human body can not undertake it. So maybe it is a good choice to overproduce GABA outside the human body and just in the factory.
Production of 5-HTP
After amplifying individual genes and plasmid pCS and pSA through PCR technology, we constructed pCS-lac-trpB-pcd-p4h-T1 and pSA-lac-trpE-trpG-AntrpC-T1 by enzymatic slicing, respectively, and identified two plasmids by enzymatic slicing. The two plasmids were then converted into E.coli Nissle 1917, cultured at a constant temperature of 12h at 37 degrees C, and then inoculated into a shaker bottle with three colonies, fermented at 30 degrees C with glycerin as a carbon source, sampled every 12h, fermented 60h. After the sample was made, the 5-HTP of the fermented sample was measured in liquid phase, and the data results were as follows:
Our output is not very high in the future we consider knocking out the bypass metabolism of branch acids to increase the metabolic flux of tryptophan in order to try to increase the output of 5-HTP.
Toxin-antitoxin system
In terms of biosafety, we designed a TA system to prevent leakage of engineered bacteria by taking advantage of the sensitivity of phyb promoter to changes in oxygen concentration. The used toxin gene and antitoxin gene were both common mazEF. In particular, mazEF and phyb both came from E.coli itself. Thus, the risk of interference from foreign genes to the engineered bacteria itself will be avoided. We used an oxygen-free glove box to create an oxygen-free environment that mimics the intestinal environment, in which the phyb promoter works properly, produces mazE, and neutralizes with mazF toxin to allow the engineered bacteria to grow and metabolize normally.
Please link here to gain more information
Please link here to gain more information
Challenges
However, there are some challenges to our project.
We also need to test the time period and delivery area of the strains inoculated in the intestinal environment to know what maximum efficiency they can achieve. In addition, under natural conditions, our engineered bacteria are at risk of plasmid loss and gene transfer.
On the one hand, engineered bacteria that lack plasmids cannot produce GABA and 5HTP efficiently and normally, so they cannot perform their intended function. Horizontal gene transfer, on the other hand, may interfere with the normal flora in the gut. We think this problem can be avoided in the future by integrating constructed circuits into genomes with genome editing tools like CRISPR-CAS.
On the one hand, engineered bacteria that lack plasmids cannot produce GABA and 5HTP efficiently and normally, so they cannot perform their intended function. Horizontal gene transfer, on the other hand, may interfere with the normal flora in the gut. We think this problem can be avoided in the future by integrating constructed circuits into genomes with genome editing tools like CRISPR-CAS.
The whole BUCT team would like to thank our sponsors. Especially:
Tel: 86+18718806286
Address: No.15 North Third Ring East Road,Chaoyang District,Beijing,P.R.China. Beijing University of Chemical Technology
BUCT--iGEM
BUCT--iGEM
