PART 1: Fermentation result of GABA
Below are some pictures showing that the process of fermenting GABA.

After fermentation, we detect the data by using Chromatograph software.

Below are some detail information about the machine and methods we used.
MACHINE: Chromatograph software name:LCsolution 15C
                  SERIAL No:L20525031877CS
                  VOL TS:220V-240~50-60Hz 150VA


Before measuring GABA output, we first carried out the derivative steps:

1. Take standard sample solution and diluted sample and put 200ul in 1.5ml test tube

2. Add 100ul 1M triethylamine-acetonitrile solution and 100ul 0.1m phenyl isothiocyanate-acetonitrile solution successively, mix them evenly, and stand for 1h at room temperature.

3. Add 200ul n-hexane and mix it evenly. After vortex mixing, stand for 30min at room temperature, and stand for stratification

4. Use a syringe to absorb the derivativated lower liquid, and put it into liquid and vial after passing 0.22um organic phase filtration membrane.

Below is the result of fermentation 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 palm oil as substrate:
GABA was produced by fermentation with soybean oil as substrate”

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.

Based on these statistics, it is obvious that we have successfully produce GABA. Also, considering that normal synthesis pathways, which consisted of gadB(wild), of producing GABA usually produce GABA for 1mg/ml, so it is obvious that our gadB(mut) gene plays its special function and helps overproducing GABA!

PART 2: Fermentation result of
pCS-gadB(mut)-gdhA-fadD-fadL-T1 and pCS-gadB(mut)-gdhA-fadD-fadL-T1(∆fadR)
We constructed the plasmid pCS-gadB(mut)-gdhA-fadD-fadL-T1 and introduced it into E.coli. After fermentation, we detected the consumption of palm oil and the production of GABA.

PART 2.1: Detect the consumption of palm oil
We configure the Gas Chromatograph test group: 200µl fermentation broth (obtain 20g/l palm oil), 2000µl petroleum ether-diethyl ether, 100µl methanol-KOH. From the chart, we can see the amount of palm oil is decreasing, and it proves this composite part lac-fadD-fadL-T1 ( BBa_K3875001) (Wild Strain) can enhance the decomposition of fatty acids by beta oxidation in E.coli.

Then we cut fadR which represses the expression of above 15 genes, constructing ∆fadR+lac-fadD-fadL-T1(∆fadR Strain). We find the amount of palm oil decreasing more quickly, and the rate of decline literally improved.
We can notice that the ∆fadR strain shows a weaker culture performance than the wild strain in culture dishes. The value of OD600 of ∆fadR strain less than wild strain as well. However, in the beginning 24h, the ∆fadR strain consumes more palm oil than wild strain. That is a good explanation that our new construction is successful. Linking fadL and fadD and cutting fadR can literally enhance the decomposition of fatty acids by beta oxidation in E.coli.

PART 2.2: Detect the consumption of palm oil.
Production of GABA with pCS-gadB(mut)-gdhA-fadD-fadL-T1:
Production of GABA with pCS-gadB(mut)-gdhA-fadD-fadL-T1 (∆fadR):
As we can see from these two charts, we got a good production of GABA. However, the bacteria could not continue to produce GABA due to insufficient substrates and GABA's self-decomposition, so the production of GABA decreased after 48 hours.

Based on Part 2, it is obvious that we have successfully produces GABA while consuming fat.

PART 3: Production of 5-HTP

We coated E. coli with plasmids pCS-lac-trpB-p4h-pcd-T1 and pSA-lac-trpEG-AntrpC, and the results were as follows:
It is possible that the synthesis path of 5-HTP is too long, so the yield of 5-HTP is not high.

The fermentation production of 5-HTP in E. coli Nissle 1917 appeared rising trend, and the yield at 48h was about 2mg/L. Technically, blocking chorismate's entry into the branching pathway for synthesis of other substances could further increase the yield of 5-HTP. It’s also a question we needs to solve in the future.

Figure. 1 5-HTP fermentation curve with glycerol as substrate

PART 4: Toxin-antitoxin system
Nissle 1917, which was successfully imported into the plasmids, was cultured in the anaerobic workstation.

Figure. The engineered bacteria are cultured under oxygen-free conditions

Cultured 16h under aerobic conditions, no colonies were found to grow, which proved that there was no escape of engineering bacteria under aerobic conditions, which is in good compliance with the safety principle.

Figure. Aerobic conditions cultured 16h, engineering bacteria died

After constantly groping for reaction conditions, it was found that E coli. Nissle 1917 grew very slowly in an oxygen-free environment, requiring 48-72h growth in order to see the obvious colony of the crucible in the aerobic air environment, it is not possible to observe obvious traces of engineering bacteria growth, which can indicate the success of the suicide system.

Figure. The culture contrast between aerobic environment and anaerobic environmentd

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