iGEM 2017 — Team NKU_China

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# Team:NKU China/Notebook

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###### Additional Links

* NKU\_China

![](https://static.igem.org/mediawiki/2017/1/16/T--NKU_China--backgroundgc1.png)

[*BcsA*-knockout *Enterobacteria* sp. FY-07](#BE)

[Plasmids](#Plasmids)

[Functional Identification](#FI)

[Basic Parts](#BPC)

Laboratory Notes

1. Construction of *BcsA*-knockout *Enterobacteria* sp. FY-07

In order to express cellulose in *Enterobacteria* sp. FY-07 in a controllable way, we should first knock out the *BcsA* gene in *Enterobacteria* sp. FY-07. Therefore, *Enterobacteria* sp. FY-07 will not produce cellulose under natural environmental condition.

Week 2 (2017.7.10-7.16)

To begin with, we amplified the upstream and downstream of *BcsA* gene in *Enterobacteria* sp. FY-07 through PCR.
Next, we combined the upstream and downstream of *BcsA* gene by overlapping PCR and the overlapped PCR products were ligated into plasmid pTsk1.
Then pTsk1-Δ*BcsA* was transformed into S17 and positive clones were selected.

Week 3 (2017.7.17-7.23)

We screened the selected clones using PCR and introduced pTsk1-Δ*BcsA* to *Enterobacteria* sp. FY-07 through conjugative transfer.
The positive pTsk1-Δ*BcsA* transformants were cultured at 42℃ with chloramphenicol to select single-crossover clones.

Week 4 (2017.7.24-7.30)

We screened the single-crossover strains using PCR and then cultured right strains in LB medium and passaged every 12 hours for 4 generations.

Week 5 (2017.7.31-8.6)

We screened the selected clones using PCR and introduced pTsk1-Δ*BcsA* to *Enterobacteria* sp. FY-07 through conjugative transfer.
The positive pTsk1-Δ*BcsA* transformants were cultured at 42℃ with chloramphenicol to select single-crossover clones.

Week 6-8 (2017.8.7-8.20)

We reapeated the conjugative transfer and the following procedures, and successfully got double-crossover clones, which was verified using PCR.
Finally, we obtained our aimed strain – *BcsA*-knockout *Enterobacteria* sp. FY-07.
The *BcsA*-knockout *Enterobacteria* sp. FY-07 strain was cultured and made competent for future use.

2. Construction of Plasmids

Week 1-3 (2017.7.3-7.23)

Initially we planned to construct pBBR1-*rhlABC*-*fimS*-*lacI*-*lacO*-*BcsA* and pBad30-cm-*fimE*. Then first we cloned some single fragments by PCR for preparation.

*rhlABC*

We cloned *rhlABC* gene from pET28a-*rhlABC* and gained the fragment through gel extraction purification. But the concentration was very low, just 22.314 ng/μL.

![](https://static.igem.org/mediawiki/2017/6/60/T--NKU_China--notebook1.png)
![](https://static.igem.org/mediawiki/2017/2/2e/T--NKU_China--notebook2.png)
![](https://static.igem.org/mediawiki/2017/8/86/T--NKU_China--notebook3.png)

*lacI*-*lacO*

We cloned *lacI* gene from *E.coli* K12 genome. After this we used the product as template and changed a new downstream primers to gain *lacI*-*lacO*.

![](https://static.igem.org/mediawiki/2017/7/7c/T--NKU_China--notebook4.png)
![](https://static.igem.org/mediawiki/2017/6/63/T--NKU_China--notebook5.png)

![](https://static.igem.org/mediawiki/2017/3/3b/T--NKU_China--notebook6.png)
![](https://static.igem.org/mediawiki/2017/b/b1/T--NKU_China--notebook7.png)

*BcsA*

*BcsA* gene was cloned from FY-07 genome.

![](https://static.igem.org/mediawiki/2017/b/b5/T--NKU_China--notebook8.png)
![](https://static.igem.org/mediawiki/2017/b/b5/T--NKU_China--notebook9.png)
![](https://static.igem.org/mediawiki/2017/d/db/T--NKU_China--notebook10.png)

*fimS*

*fimS* was cloned from *BL*21 and was amplified by PCR.

![](https://static.igem.org/mediawiki/2017/3/39/T--NKU_China--notebook11.png)
![](https://static.igem.org/mediawiki/2017/d/d9/T--NKU_China--notebook12.png)
![](https://static.igem.org/mediawiki/2017/9/97/T--NKU_China--notebook13.png)

*fimE*

*fimE* gene was cloned from *BL*21 and was amplified by PCR.

![](https://static.igem.org/mediawiki/2017/5/56/T--NKU_China--notebook14.png)
![](https://static.igem.org/mediawiki/2017/a/af/T--NKU_China--notebook15.png)

Week 4-6 (2017.7.24-8.13)

Because every time we cloned the *rhlABC* gene through PCR, the product is mixed with other non target products. In addition, the concentration of the fragment through gel extraction purification was always low. So this week we tried to find a way to improve this condition.

Using existing fragments as templates

We tried to use existing *rhlABC* as templates, but sadly the products were only non-targeted products with 1000-2000bp.

![](https://static.igem.org/mediawiki/2017/0/0a/T--NKU_China--notebook16.png)
![](https://static.igem.org/mediawiki/2017/3/34/T--NKU_China--notebook17.png)

Using temperature gradient to find the best Tm

The PCR system and PCR reaction condition was the same as above. The best Tm we found is 50.7℃. However, we cloned it again using this Tm, the concentration of the product was still frustrating, just 18.699 ng/μL.

Nested PCR

There was no aim product and the result of nucleic acid electrophoresis showed that the template could have some mistakes.

![](https://static.igem.org/mediawiki/2017/1/1e/T--NKU_China--notebook18.png)

Verify the pET28a-*rhlABC*

Later we picked up several bacteria on the plate and cultured them, then we tried verify PCR with T7 primer. The result was that there were no aim strips. So we redesigned the primers and planned to get *rhlABC* through overlapping *rhlAB* and *rhlC*.

![](https://static.igem.org/mediawiki/2017/1/12/T--NKU_China--notebook19.png)

Week 7-9 (2017.8.14-9.3)

This week we tried to get some combined fragments using existing genes through overlap extention PCR(OE PCR).

*fimS*-*lacI*(*lacO*)

We successfully got the aim products.

![](https://static.igem.org/mediawiki/2017/6/65/T--NKU_China--notebook20.png)

① Tm=65℃ 12cycles
②adding primers, Tm=61℃ 18cycles

*rhlABC*-*fimS*

We successfully got the aim products.

![](https://static.igem.org/mediawiki/2017/c/cb/T--NKU_China--notebook21.png)

① Tm=60℃ 12cycles
②adding primers, Tm=65℃ 18cycles

*lacI*(*lacO*)-*BcsA*

We successfully got the aim products.

![](https://static.igem.org/mediawiki/2017/f/fe/T--NKU_China--notebook22.png)

① Tm=60℃ 12cycles
②adding primers, Tm=61℃ 35cycles

![](https://static.igem.org/mediawiki/2017/e/ea/T--NKU_China--notebook23.png)

4.*rhlABC*-*fimS*-*lacI*-*lacO*-*BcsA*

In the same way, we got *fimS*-*lacI*-*lacO*-*BcsA*. Then, We have tried different combinations (*rhlABC*-*fimS* and *lacI*(*lacO*)-*BcsA*; *rhlABC* and *fimS*-*lacI*-*lacO*-*BcsA*) and different mix(STAR, LA and phanta mix), but didn’t get the aim product.

Week 10 (2017.9.4-9.10)

We continued OE PCR, simultaneously, we went on to construct pBad30-cm-*fimE*.

Both pBad30 and cm segments were treated with restriction enzyme. Later cm gene was ligated to linear pBad30 and then we got pBad30-cm. In the same way, we got pBad30-cm-*fimE* and transformed it into DH5α. But when we use verify PCR to test the *fimE* gene, we didn’t get the aim strip.

Week 11 (2017.9.11-9.17)

To verify the feasibility of *fimS* first, we went on to construct pBBR1-*rfp*-*fimS*-*lacI*-*lacO*-*gfp*.We successfully got *rfp*, *gfp* and *rfp*-*fimS*-*lacI*-*lacO*, but failed to overlap the whole segment-- *rfp*-*fimS*-*lacI*-*lacO*-*gfp*.

Sum up this result and previous experience, we desided to make some adjustments in our design.

1. The distance between *lacI* and *lacO* is too close, which may affect the function. So we desided to construct pBBR1-*rhlABC*-*fimS*-*lacO*-*BcsA* and pBad30-*fimE*-cm-*lacI*.

2. There was a solution that the *BcsA* and *gfp* genes were digested and ligated to pK18, and the *lacO*-*BcsA*/*GFP* could be obtained if PCR started from the *lacO* position before the pK18 cloning site.

3. We redesigned the primers.

Week 12 (2017.9.18-9.24)

We redesigned the primers for Gibson Assembly and successfully constructed pBBR1-*rhlABC*-*fimS*-*lacO*-*BcsA*, pBBR1-*rfp*-*fimS*-*lacO*-*gfp* and pBad30-*fimE*-cm-*lacI*.

3. Functional Identification of Our Design

Week 1-2 (2017.7.3-7.16)

We constructed pBBR-*rhlABC* and introduced the plasmid into wild-type *Enterobacter* sp. FY-07. Through Diesel Emulsification Experiment，we verified that *Enterobacter* sp. FY-07 had the emulsification effect after overexpressing pBBR-*rhlABC*

![](https://static.igem.org/mediawiki/2017/4/4a/T--NKU_China--notebook24.png)

Week 9 (2017.8.28-9.3)

We verified that *BcsA*-knockout *Enterobacter* sp. FY-07 didn’t produce cellulose under general condition, while overexpression of *BcsA* can rescue the expression of cellulose in *BcsA*-knockout *Enterobacter* sp. FY-07.

![](https://static.igem.org/mediawiki/2017/8/83/T--NKU_China--notebook25.png)

4. Basic Parts Construction

Week 13-16 (2017.9.25-10.22)

1. The *lyase*, *fimS* and *fimE* genes are cloned in the same way as week 1.

2. Then both these segments and pSB1C3 vector were treated with restriction enzymes. After that, the three gene were separately ligated to linear pSB1C3, and the ligation products were separately transformed into DH5α.

![](https://static.igem.org/mediawiki/2017/d/d1/T--NKU_China--notebook26.png)
![](https://static.igem.org/mediawiki/2017/9/95/T--NKU_China--notebook27.png)

3. Verification PCR was performed to select the positive clones. The positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

![](https://static.igem.org/mediawiki/2017/c/c1/T--NKU_China--sponsor.png)

## Contacts

## Address

Nankai University
No.94 Weijin Road, Nankai District
Tianjin, P.R.China 300071

Copyright © Team NKU-China 2017

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# Team:NKU China/Experiments

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###### Additional Links

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Experiment

2017.7.10-8.20 Construction of *BcsA*-knockout *Enterobacteria* sp.FY-07

In order to make the production of cellulose become controllable, we knocked out the BcsA gene (the coding gene of the A subunit of the cellulose synthase) in FY-07. It was observed that FY-07 knocked out the BcsA gene no longer produced cellulose. When we replenish the *BcsA* gene into FY-07, FY-07 began to produce cellulose again. This phenomenon can be clearly observed on Congo red media. On the Congo red medium, when the bacteria produce cellulose, the colonies will show red.

2017.7.3-9.24 Construction of engineered Enterobacter *sp. FY-07*

Our project is aiming to engineer *Enterobacter* sp. FY-07 (which is separated from oilfield produced water and able to produce cellulose naturally), so as to controllably produce rhamnolipid and cellulose.

2017.7.3-9.3 Functional Test of Our Design

We have test the functional of gene *rhlABC*, *BcsA* and *fimS* switch. The results are roughly in accordance with our expectation.

2017.9.25-10.22 Basic Parts Construction

The lyase, *fimS* and *fimE* genes are cloned by PCR. Then both these segments and pSB1C3 vector were treated with restriction enzymes. After that, the three gene were separately ligated to linear pSB1C3, and the ligation products were separately transformed into DH5α. Verification PCR was performed to select the positive clones. The positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

![](https://static.igem.org/mediawiki/2017/c/c1/T--NKU_China--sponsor.png)

## Contacts

## Address

Nankai University
No.94 Weijin Road, Nankai District
Tianjin, P.R.China 300071

Copyright © Team NKU-China 2017

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

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# Team:NKU China/Results

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[JUDGING FORM](https://igem.org/2017_Judging_Form?team=NKU_China)

# NKU\_China

# 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.

##### You should also 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 be on part pages in the Registry.
* 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)

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