Team:Jiangnan-China/Notebook

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Knock out the competitive pathway large gene cluster of surfactin synthesis

2019/06/05-2019/07/18

Reducing competition by knocking out the biofilm formation-related genes

Participant	Xia Sun, Shuangxue Fu, Jingyi Zhang, Huiting Yang, Diqing Sheng, Jingyi Yang
Protocol	PCR, Agarose gel electrophoresis, Competence, Plasmid extraction
Notes	Gene traceless elimination of Bacillus subtilis D1 by Cre-lox system. 2019.06.05 Reactivate the target positive transformants carrying resistance genes. 2019.06.06-2019.06.15 Competence and transform plasmids carrying resistance genes. 2019.06.16 Screen on the corresponding resistance plate and verify the transformants by PCR. 2019.06.17 Culture overnight with the corresponding resistance in the LB test tube. 2019.06.18 Delimit the positive transformants obtained from the second transformation to the LB non-resistant plate and cultured at 55 C for more than 18 hours. 2019.06.19-20 Verify whether the resistance genes were eliminated or not in the obtained single colony by PCR and preserve the strain at - 80 ℃. Gene Traceless Elimination of Bacillus by Cre-lox System 2019.06.20 Reactivate the target positive transformants carrying resistance genes. 2019.06.21-2019.06.28 Competence and transform plasmids carrying resistance genes 2019.06.29 Screen on the corresponding resistance plate and verify the transformants by PCR 2019.06.30Culture overnight with the corresponding resistance in the LB test tube. 2019.07.01-2019.07.02 Delimit the positive transformants obtained from the second transformation to the LB non-resistant plate and cultured at 55℃for more than 18 hours 2019.07.03 Verify whether the resistance genes were eliminated or not in the obtained single colony by PCR and preserve the strain at - 80 ℃.
Results	In the Bacillus subtilis D1 strain background, we knocked out a part of pps gene cluster, and obtained mutants Bacillus subtilis D2. In the Bacillus subtilis D2 strain background, we knocked out the other parts of pps gene cluster, and obtained Bacillus subtilis D8.

Enhance YcxA gene expression and transfer efflux genes LiaH and YerP into Bacillus subtilis D8

2019/08/01-2019/08/20

Participant	Jingyi Yang, Shuangxue Fu, Jingyi Zhang
Protocol	PCR, Agarose gel electrophoresis, Competence, Plasmid extraction
Notes	2019.08.01 Reactivate the target positive transformants carrying resistance genes. 2019.08.02-2019.08.03 Competence and transform plasmids carrying resistance genes. 2019.08.04 Screen on the corresponding resistance plate and verify the transformants by PCR. 2019.08.05 Culture overnight with the corresponding resistance in the LB test tube. 2019.08.06 Delimit the positive transformants obtained from the second transformation to the LB non-resistant plate and cultured at 55 C for more than 18 hours. 2019.08.06 Verify whether the resistance genes were eliminated or not in the obtained single colony by PCR and preserve the strain at - 80 ℃. 2019.08.07Reactivate the target positive transformants carrying resistance genes. 2019.08.07-2019.08.08Competence and transform plasmids carrying resistance genes. 2019.08.09-2019.08.15Screen on the corresponding resistance plate and verify the transformants by PCR. 2019.08.16 Culture overnight with the corresponding resistance in the LB test tube. 2019.08.17Delimit the positive transformants obtained from the second transformation to the LB non-resistant plate and cultured at 55℃ for more than 18 hours. 2019.08.18-2019.08.20 Verify whether the resistance genes were eliminated or not in the obtained single colony by PCR and preserve the strain at - 80 ℃.
Results	YcxA gene was overexpressed in Bacillus subtilis D7 by replacing the native promoter with the strong promoter P43 successfully. Efflux genes LiaH and YerP were transferred into Bacillus subtilis D8. Bacillus subtilis D35 was got.

Participant	Huiting Yang, Xia Sun, Diqing Sheng
Protocol	PCR, Agarose gel electrophoresis, Competence
Notes	2019.05.13-15 PCR the sfp gene fragment and agarose gel electrophoresis 2019.05.15 Activated the strain Bacillus subtilis 168 2019.05.16-2019.05.20 competence：transfer the sfp gene into Bacillus subtilis 168 2019.05.21 PCR and agarose gel electrophoresis and strain preservation
Results	According to the glue map, the sfp gene was successfully transferred into Bacillus subtilis 168. The resulting recombinant strain was named Bacillus subtilis D1

Participant	Jingyi Yang, Shuangxue Fu.
Protocol	PCR, Agarose gel electrophoresis, Competence, Plasmid extraction
Notes	2019.09.01 Reactivate the target positive transformants carrying resistance genes. 2019.09.02-2019.09.06Competence and transform plasmids carrying resistance genes. 2019.09.09 Screen on the corresponding resistance plate and verify the transformants by PCR. 2019.09.10 Culture overnight with the corresponding resistance in the LB test tube. 2019.09.11Delimit the positive transformants obtained from the second transformation to the LB non-resistant plate and cultured at 55 C for more than 18 hours. 2019.09.12-2019.09.15Verify whether the resistance genes were eliminated or not in the obtained single colony by PCR and preserve the strain at - 80 ℃.
Results	SacB promoter was transferred into Bacillus subtilis D35. Bacillus subtilis D45 was got

Participant	Diqing Sheng, Jingyi Yang
Protocol	Fermentation, HPLC, Purification
Notes	2019.05.26 Overnight activated Bacillus subtilis D1. 2019.05.27-2019.05.29 Inoculated Transfer the bacteria to the medium in conical flasks, and then ferment for 72h. Sample every 2 h. 2019.05.30-2019.06.01Measure the yield at different times with the HPLC phase. 2019.05.30 Centrifuge the fermentation broth to remove the bacteria, collect the supernatant and add 1:1 volume of ethanol. 2019.06.01 After removing ethanol by rotary evaporation, adding hydrochloric acid solution, adjusting PH=2, and stationary precipitation for 4 hours. Collect centrifugal precipitation. 2019.06.02-2019.06.03 Use PH=2 hydrochloric acid solution to wash the precipitation by oscillation, and remove the supernatant by centrifugation. After two repetitions we can get the desired product. The product is frozen to freeze at - 80℃ refrigerator, and then put into freeze dryer for vacuum freeze-drying.
Results	Bacillus subtilis D1 had a surfactin titer of 0.38 g/l. The purity is 97 percent. 1.5g purified surfactin was got.

Participant	Shuangxue Fu, Jingyi Zhang, Diqing Sheng, Jingyi Yang
Protocol	Fermentation, HPLC, Purification
Notes	2019.07.18 Overnight activated Bacillus subtilis D8. 2019.07.19-2019.07.22 Inoculated Transfer the bacteria to the medium in conical flasks, and then ferment for 72h. Sample every 2 h. 2019.07.23-2019.07.25 Measure the yield at different times with the HPLC phase. 2019.07.26 Centrifuge the fermentation broth to remove the bacteria, collect the supernatant and add 1:1 volume of ethanol. 2019.07.27-2019.07.28 After removing ethanol by rotary evaporation, adding hydrochloric acid solution, adjusting PH=2, and stationary precipitation for 4 hours. Collect centrifugal precipitation. 2019.07.29 Use PH=2 hydrochloric acid solution to wash the precipitation by oscillation, and remove the supernatant by centrifugation. After two repetitions we can get the desired product. The product is frozen to freeze at - 80℃ refrigerator, and then put into freeze dryer for vacuum freeze-drying.
Results	Bacillus subtilis D8 had a surfactin titer of 0.96 g/l. The purity is 97 percent. 4.7g purified surfactin was got.

Participant	Shuangxue Fu, Jingyi Zhang, Huiting Yang
Protocol	Fermentation, HPLC, Purification
Notes	2019.08.23 Overnight activated Bacillus subtilis D35. 2019.08.23-2019.08.25 Inoculated Transfer the bacteria to the medium in conical flasks, and then ferment for 72h. Sample every 2 h. 2019.08.26-2019.08.27 Measure the yield at different times with the HPLC phase. 2019.08.24 Centrifuge the fermentation broth to remove the bacteria, collect the supernatant and add 1:1 volume of ethanol. 2019.08.24-2019.08.26 After removing ethanol by rotary evaporation, adding hydrochloric acid solution, adjusting PH=2, and stationary precipitation for 4 hours. Collect centrifugal precipitation. 2019.08.27 Use PH=2 hydrochloric acid solution to wash the precipitation by oscillation, and remove the supernatant by centrifugation. After two repetitions we can get the desired product. The product is frozen to freeze at - 80℃ refrigerator, and then put into freeze dryer for vacuum freeze-drying.
Results	Bacillus subtilis D35 had a surfactin titer of 1.92 g/l. The purity is 97 percent. 7.8g purified surfactin was got.

Participant	Huiting Yang, Yaxin Li
Protocol	Emulsification
Notes	2019.8.20 Preparation of oil sample 2019.8.20 Preparation of surfactin solution 2019.8.20 Mix 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L surfactin solution and 3g/L Petroleum sulfonate with the oil sample,and oscillating test tube respectively 2019.8.20-2019.8.22 Observing the phenomenon once in a while 2019.8.23 Analyze the result and design the next experiment 2019.8.24-2019.8.28 Emulsification experiment of 0.04g / L, 0.06g / L, 0.08g/L, 0.09g / L surfactin solution and 0.3g/L Petroleum sulfonate 2019.8.29-2019.8.30 Analyze the result
Results	Surfactin solution has good emulsifying effect and thermal stability