iGEM 2019 — Team Jiangnan-China

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# Team:Jiangnan-China/Notebook

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Transfer *sfp* gene into *Bacillus
subtilis* 168

2019/05/13-2019/05/20

Restoring surfactin biosynthetic activity by integrating the heterologous sfp gene

|  |  |
| --- | --- |
| Participant | Huiting Yang, Xia Sun, Diqing Sheng |
| Protocol | [PCR](https://static.igem.org/mediawiki/2019/d/d7/T--Jiangnan-China--1PCR.pdf), [Agarose gel electrophoresis](https://static.igem.org/mediawiki/2019/3/3d/T--Jiangnan-China--1Agarose_gel_electrophoresis.pdf), [Competence](https://static.igem.org/mediawiki/2019/e/e6/T--Jiangnan-China--1competence.pdf) |
| 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 |

![](https://static.igem.org/mediawiki/2019/6/6b/T--Jiangnan-China----KMLT--_-PYZZXFWX%29%298-I.png)

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

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](https://static.igem.org/mediawiki/2019/a/ab/T--Jiangnan-China--2PCR.pdf), [Agarose gel electrophoresis](https://static.igem.org/mediawiki/2019/3/3d/T--Jiangnan-China--1Agarose_gel_electrophoresis.pdf), [Competence](https://static.igem.org/mediawiki/2019/1/1b/T--Jiangnan-China--2competence.pdf), [Plasmid extraction](https://static.igem.org/mediawiki/2019/b/bd/T--Jiangnan-China--2Plasmid_extraction.pdf) |
| 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. |

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

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](https://static.igem.org/mediawiki/2019/3/3e/T--Jiangnan-China--3PCR.pdf), [Agarose gel electrophoresis](https://static.igem.org/mediawiki/2019/3/3d/T--Jiangnan-China--1Agarose_gel_electrophoresis.pdf), [Competence](https://static.igem.org/mediawiki/2019/c/c5/T--Jiangnan-China--3competence.pdf), [Plasmid extraction](https://static.igem.org/mediawiki/2019/d/d5/T--Jiangnan-China--3Plasmid_extraction.pdf) |
| 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. |

![](https://static.igem.org/mediawiki/2019/a/a9/T--Jiangnan-China--3%E5%9B%BE%E7%89%871.png)
![](https://static.igem.org/mediawiki/2019/3/36/T--Jiangnan-China--3%E5%9B%BE%E7%89%872.png)

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

Replace promoter with *sacB* promoter

2019/09.01-2019/09.15

|  |  |
| --- | --- |
| Participant | Jingyi Yang, Shuangxue Fu. |
| Protocol | [PCR](https://static.igem.org/mediawiki/2019/9/97/T--Jiangnan-China--4PCR.pdf), [Agarose gel electrophoresis](https://static.igem.org/mediawiki/2019/3/3d/T--Jiangnan-China--1Agarose_gel_electrophoresis.pdf), [Competence](https://static.igem.org/mediawiki/2019/1/11/T--Jiangnan-China--4competence.pdf), [Plasmid extraction](https://static.igem.org/mediawiki/2019/a/a8/T--Jiangnan-China--4Plasmid_extraction.pdf) |
| 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 |

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

Fermentation of *Bacillus subtilis* D1

2019.05.26-2019.06.03

|  |  |
| --- | --- |
| Participant | Diqing Sheng, Jingyi Yang |
| Protocol | [Fermentation](https://static.igem.org/mediawiki/2019/a/a5/T--Jiangnan-China--5Fermentation.pdf), [HPLC](https://static.igem.org/mediawiki/2019/c/c1/T--Jiangnan-China--5HPLC.pdf), [Purification](https://static.igem.org/mediawiki/2019/b/bd/T--Jiangnan-China--5Purification.pdf) |
| 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. |

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

Fermentation of *Bacillus subtilis* D8

2019.07.18-2019.07.29

|  |  |
| --- | --- |
| Participant | Shuangxue Fu, Jingyi Zhang, Diqing Sheng, Jingyi Yang |
| Protocol | [Fermentation](https://static.igem.org/mediawiki/2019/c/c7/T--Jiangnan-China--6Fermentation.pdf), [HPLC](https://static.igem.org/mediawiki/2019/f/f4/T--Jiangnan-China--6HPLC.pdf), [Purification](https://static.igem.org/mediawiki/2019/f/f3/T--Jiangnan-China--6Purification.pdf) |
| 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. |

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

Fermentation of *Bacillus subtilis* D35

2019.08.23-2019.08.27

|  |  |
| --- | --- |
| Participant | Shuangxue Fu, Jingyi Zhang, Huiting Yang |
| Protocol | [Fermentation](https://static.igem.org/mediawiki/2019/0/0f/T--Jiangnan-China--7Fermentation.pdf), [HPLC](https://static.igem.org/mediawiki/2019/6/6d/T--Jiangnan-China--7HPLC.pdf), [Purification](https://static.igem.org/mediawiki/2019/f/ff/T--Jiangnan-China--7Purification.pdf) |
| 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. |

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

Fermentation of *Bacillus subtilis* D45

2019.09.17-2019.09.23

|  |  |
| --- | --- |
| Participant | Shuangxue Fu, Jingyi Zhang, Huiting Yang |
| Protocol | [Fermentation](https://static.igem.org/mediawiki/2019/0/06/T--Jiangnan-China--8Fermentation.pdf), [HPLC](https://static.igem.org/mediawiki/2019/e/ed/T--Jiangnan-China--8HPLC.pdf), [Purification](https://static.igem.org/mediawiki/2019/1/14/T--Jiangnan-China--8Purification.pdf) |
| Notes | 2019.09.17 Overnight activated *Bacillus subtilis* D45.   2019.09.18-2019.09.20 Inoculated Transfer the bacteria to the medium in conical flasks, and then ferment for 72h. Sample every 2 h.  2019.09.21-2019.09.22Measure the yield at different times with the HPLC phase.  2019.09.21 Centrifuge the fermentation broth to remove the bacteria, collect the supernatant and add 1:1 volume of ethanol.  2019.09.21-2019.09.23 After removing ethanol by rotary evaporation, adding hydrochloric acid solution, adjusting PH=2, and stationary precipitation for 4 hours. Collect centrifugal precipitation.  2019.09.23 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* D45 had a surfactin titer of 3,81g/l.  The purity is 97 percent. 18.3g purified surfactin was got. |

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

Emulsification experiment

2019/8/20-2019/8/30

|  |  |
| --- | --- |
| Participant | Huiting Yang, Yaxin Li |
| Protocol | [Emulsification](https://static.igem.org/mediawiki/2019/f/f8/T--Jiangnan-China--9Emulsification.pdf) |
| 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 |

After emulsification for 3h

![](https://static.igem.org/mediawiki/2019/d/d0/T--Jiangnan-China--nt3h.png)
![](https://static.igem.org/mediawiki/2019/c/c9/T--Jiangnan-China--40t3h.png)

![](https://static.igem.org/mediawiki/2019/3/33/T--Jiangnan-China--modelClose.png)

Oil displacement experiment

2019/9/1-2019/9/10

|  |  |
| --- | --- |
| Participant | Huiting Yang, Yaxin Li |
| Protocol | [Oil Displacement](https://static.igem.org/mediawiki/2019/4/46/T--Jiangnan-China--10Displacement.pdf) |
| Notes | 2019.9.1 Preparation of oil sample  2019.9.1 Preparation of surfactin solution  2019.9.1 Preparation of simulated formation water  2019.9.1-2019.9.2 Preparation of the first core  2019.9.3 Displace the oil of the first core  2019.9.4 Analyze the result and design the next experiment  2019.9.5-2019.9.9 Displacement experiment of the larger core |
| Results | 0.1g/L surfactin solution increase the oil displacement rate 9.23%.  0.3g/L surfactin solution increase the oil displacement rate 14.94% |


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# Team:Jiangnan-China/Experiments

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# Team:Jiangnan-China/Results

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# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)