Team:H14Z1 Hangzhou/Collaborations
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Collaborations
Collaborations with SZU-China
This year, we made a collaboration with SZU-China in Shenzhen University. We helped them make some new content of their WeChat-based Mini Program. We gave them detailed feedback after using it, and shared our familiar experience with them about how to promote it to more and more high schools. In return, they did much work to screen several compatible strains, which were used by us to screen the suitable strain to produce our “smart yogurts ” combined with our constructed L. lactis. They went to the supermarket, bought various bands and different conditions of yogurt for them, including Classy·Kiss and CHENGUANG DAIRY, finally isolated several local strains, which were mailed to us for our strain screening plan.
Collaborations with XMU-china team
In the present project, since the oral table administration of GSH and SAM have some disadvantages, such as low stability and short life span, here we tried to develop a novel in-vivo strategy of produce and deliver them simultaneously by using NICE system. In the experiment, two-functional GSH synthetase gene (gshF) and SAM synthetase gene (metK) were in tandem inserted into the expression vector (pNZ8148), and the resulted plasmid (pNZ8148-SG) was employed to construct the target vector pNZ8148-SGC by introducing adhesion factor gene (cwaA). This target vector was transformed to get recombinant Lactococcus lacti, which was employed to produce our “smart yogurt”.
The project is outlined with two stages：
Demonstrate the function of SC-L7Ae-mRFP1 by H14Z1_Hangzhou
As team XMU-China’s HSFCM data shown, OmpA-ST inside OMVs could be detected. To validate the ST/SC conjugation inside OMVs, they linked BBa_K2623022/BBa_K2623024 with BBa_K2623025 (NG5) to form BBa_K2623028 and BBa_K2623030 respectively. Hence, they need to demonstrate the SC-L7Ae-mRFP1 inside E. coli BL21 to validate its presence inside E. coli BL21 and therefore OMVs. We measured the RFP intensity versus OD600 value (RFP intensity/OD600) of E. coli transfected with NG5 to test the function of NG5.
Experiment procedure is:1) Transform plasmid pSB1C3-NG5 into E.coli BL21(DE3);2) Inoculate 5 ml LB with monocolony from the plate, and grow the cells overnight at 37 ℃，200 rpm;3) Inoculate 50 ml of LB with pre-culture in a dilution of 1:100 and grow at 37℃，200rpm, and keep another 50ml LB as negative control;4) Grow the culture until OD600 is 0.6，add 50ul 0.5M IPTG solution, and keep another 50ml LB as negative control;5) Measure the fluorescence and OD600 every 2 hours. Keep another 50ml LB as negative control.
The data and curve of RFP intensity/OD600 were collected in the following:
Demonstrate the function of SC-L7Ae-mRFP1 by H14Z1_Hangzhou
XMU-China gave a good demonstrative example for our smart yogurt conception by preparing our special yogurt and determined the cell number and contents of two liver-protective agents. The process and results were described in the following:
1) Yogurt production process
We provided them the yogurt formula ( raw milk 93%, white granulated sugar 7%) and two strains (L. lactis NZ9000 and L. lactis NZ9000/pGMcA). They did the following steps for the process: 1) Heating raw milk to 55-60 oC, add a quantity of sugar, then stir then for 10 minutes at 3000 rpm, finally heat up to 65 oC; 2) keep the culture medium at 65 oC for two-stage homogenization (5/20MPa), then do the sterilization at 90 oC for 5 minutes. Then cool down to 30 oC for the next inoculation; 3) Inoculate the milk-containing medium with suitable amount of L. lactis NZ9000 or L. lactis NZ9000/pGMcA, meanwhile the mixed amino acids were supplemented suitabley. Each fermentation process was maintained for 12 hour at 30 oC, and both of the end pH was around 4.6. Several samples were taken out carefully at some time points.
2) Product analysis
The comparison was made for product quality by using different strains. The results showed that the recombinant strain (NZ9000/pGMcA) had a little lower growth rate with regard the original host, but the function of the combinatory module was fully confirmed by measuring the content of the yogurts. The original host did produce GSH during the whole process, but the recombinant cells raised GSH concentration up to 11.8mg/l, which is quite comparable to the data in our laboratory. They also proved that the original host can produce a small amount of SAM during its growth process, but the introduction of our composite module did improved the SAM biosynthesis greatly. Moreover, high density (1.0 x 109/ml) of the strain NZ9000/pGMcA gave the good potential for their continuous biosynthesis of GSH and SAM in human gastrointestinal tract.
