1.New document to the part,MazF and MazE
We decided to characterise two sequences that encode the mazE ( BBa_K302032) and mazF ( BBa_K823044) proteins. mazE is antitoxin for the mazF toxin and mazF is antitoxin for the mazE. mazEF is a toxin-antitoxin relation in E.coli and other bacteria that activates programmed cell death in response to malnutrition. Cell growth and viability are not affected when mazF and mazE are coexpressed.

MazF is toxin protein in MazF-MazE, toxin-antitoxin module. It was first registered in 2013 and used as an mRNA endonuclease. It kills bacteria without cracking them.

We received an exogenous mazF from the HAZU-China team and obtained an endogenous mazF from BW25113 via PCR, and by comparing the two effects, we finally decided to choose endogenous mazF.

Figure. 1 Exogenous mazF from HZAU-China

Figure. 2 mazE from BW25113

Because direct expression of MazF will kill bacteria, we construct the circuit ”lac-mazF-T1-lac-phyb-mazE-T1” and import the constructed plasmid carrier into Nissle 1917.The engineered bacteria were cultured under oxygen-free conditions at 37 degrees for 48h.

Figure. 3 MazE and MazF are expressed at the same time, and the engineered bacteria survive(Left). Only mazF is expressed, and the engineered bacteria die(Right)

2.New document to the part,FadL and FadD
β-oxidation of fatty acids in E.coli involves two key genes, fadL and fadD, encoding outer membrane protein and acyl-CoA synthetase, respectively. We use pCS27 as the expression vector of fadL and fadD, and the purpose of overexpressing these two genes is to solve the problem of consumption and transport of exogenous fatty acids in E. coli.

PCR amplification technique was used to obtain the endogenous fadL and fadD of E. coli, and then the two genes were transferred into the plasmid by enzyme ligation method as the composite part lac-fadD-fadL-T1 ( BBa_K3875001).

Finally the plasmid was transformed into E. coli Nissle.

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 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. We find the amount of palm oil decreasing more quickly, and the rate of decline literally improved.

3.New document to the part,vgb
Dissolved oxygen (DO) plays an important role in cell growth, especially in industry-scale microbial production. We find Vitreoscilla hemoglobin promoter (P vgb ) ,which was found to be the strongest among all 11 promoters(Wu, H., Wang, H., Chen, J. et al. Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant E.coli grown micro-aerobically. Appl Microbiol Biotechnol 98, 10013–10021 (2014).).
After the culture and sequencing verification of pZE-pvgb, the constructed plasmids will be imported into nissle 1917, tighten the lid to ensure low oxygen status, 37 degrees Celsius shaker culture 12-16h, found that the colonies grow and the size is obvious, after the extraction of plasmids sent sequencing found lac promoter loss, indicating that the construction of suicide system is not successful.

Figure. The growth of bacteria imported into the suicide system

4.New document to the part,phyb
phyb ,promoter of the hybO,is an oxygen-sensing promoter that initiates regulation under anaerobic conditions and inactivates under aerobic conditions.

This is one of three E.coli hydrogenases synthesized in response to different physiological conditions. HYD2 is involved in hydrogen uptake.

Catalytic activity:A + H2 = AH2
The expression is 100% under oxygen-free conditions, however, when it leaks with oxygen in the environment, the promoter fails and is a very strict promoter.
Thus, the gene inactivation of its connection, this year we used phyb ( BBa_K3875031) to initiate the regulation of mazE, so that the engineering bacteria in an oxygen-free environment to produce mazE and mazF resistance to help the normal growth and metabolism of engineering bacteria, and once leaking in the oxygen environment, the promoter inactivation, lack of mazE expression after the engineering bacteria can not succeed.

Vector construction success, and import it into Nissle 1917, observe the culture effect: in the anaerobic workstation, the medium grows colonies, aerobic conditions no longer grow. Individual aerobic culture 16h, aseptic growth traces.

Figure. There are no engineered bacteria growing slugs under aerobic conditions .No promoter leakage expression under aerobic conditions

The design perfectly fits our intestinal engineering bacteria designed around the human gut this year to survive GABA and 5-HTP.

Figure. Anaerobic and aerobic culture 48h difference

The whole BUCT team would like to thank our sponsors. Especially:
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