iGEM 2016 — Team NKU_China

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# Team:NKU China/Notebook

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Team☞☟

[Team](https://2016.igem.org/Team%3ANKU_China/Team)
[Collaborations](https://2016.igem.org/Team%3ANKU_China/Collaborations)

Project☞☟

[Description](https://2016.igem.org/Team%3ANKU_China/Description)
[Experiments](https://2016.igem.org/Team%3ANKU_China/Experiments)
[Model](https://2016.igem.org/Team%3ANKU_China/Model)
[Proof of Concept](https://2016.igem.org/Team%3ANKU_China/Proof)
[Demonstrate](https://2016.igem.org/Team%3ANKU_China/Demonstrate)

HP☞☟

[HP](https://2016.igem.org/Team%3ANKU_China/Human_Practices)
[Silver](https://2016.igem.org/Team%3ANKU_China/HP/Silver)
[Gold](https://2016.igem.org/Team%3ANKU_China/HP/Gold)

[Safety](https://2016.igem.org/Team%3ANKU_China/Safety)
[Attributions](https://2016.igem.org/Team%3ANKU_China/Attributions)
[Parts](https://2016.igem.org/Team%3ANKU_China/Parts)
[InterLab](https://2016.igem.org/Team%3ANKU_China/InterLab)

Notebook☞☟

[Lab Notes](https://2016.igem.org/Team%3ANKU_China/Notebook)
[Protocols](https://2016.igem.org/Team%3ANKU_China/Protocols)

you can not see me!

50μL PCR system ×2

| 2× Taq Master Mix | 25μL |
| C2-F | 2μL |
| C2-R | 2μL |
| p-C2 | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 15 sec | 30 cycles |
| 72oC | 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| *C2-luxS* | 4μL |
| ddH2O | 12μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| T-*lsrACDB* | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

100μL methylation system

| 10× BamH Ⅰ methyltransferase Buffer | 10μL |
| BamH Ⅰ methyltransferase | 1μL |
| S-adenosylmethionine | 0.5μL |
| pWH-*C2-luxS* | 80μL |
| ddH2O | 8.5μL |
| Total | 100μL |
| Reaction condition: 37oC for 1 hour | |

Groups divided in this experiment

| GR286 | wild strain as control group |
| GR286Δ*luxS* | GR286 without *luxS* gene |
| pWH-*luxS* | *luxS* overexpression plasmid in GR286; without induced by xylose |
| pWH-*luxS* &plus; xyl | *luxS* overexpression plasmid in GR286; induced by xylose |
| pWH1520 | empty plasmid in GR286 as control group |
| pHT-*lsrACDB* | *lsrACDB* overexpression plasmid in GR286Δ*luxS* |
| pHT-01 | empty plasmid in GR286Δ*luxS* as control group |

![](image/Notebook/notebook-6-13-1.png)

Selection of positive clones by colony PCR

(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-*C2-luxS* into GR286)

Laboratory Notes

☞☟ Week1 (May 16–May 22)

* [begin](#fragment1-1)
* [➀](#fragment1-2)
* [➁](#fragment1-3)
* [➂](#fragment1-4)

In order to make sure the efficiency of our "consumer", we should first knock out the *luxS* gene in our engineering bacteria GR286(a simplified strain of *Bacillus amyloliquefaciens* LL3). We used a markerless gene replacement method to knock out the *luxS* gene.

Construction of targeting vector : the upstream and downstream of *luxS* gene were combined by over-lapping PCR and ligated into plasmid pKSU.

pKSU-Δ*luxS* was transformed into GR286, and positive clones were selected.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| pKSU-F | 1μL |
| pKSU-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 1 min 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/e/e9/T--NKU_China--notebook-1-5-1.png)

Selection of positive clones by PCR

The transformants were cultured at 42oC with chloramphenicol to select single-crossover clones.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| *luxS*-up-F | 1μL |
| *luxS*-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/d/de/T--NKU_China--notebook-1-5-2.jpeg)

Selection of single-crossover clones by PCR

(No.1-4 are single-crossover strains,
No.5 is positive control.)

☞☟ Week2 (May 23–May 29)

* [➀](#fragment2-1)
* [➁](#fragment2-2)

The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.

The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| *luxS*-up-F | 1μL |
| *luxS*-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/3/3d/T--NKU_China--notebook-1-5-3.png)

Selection of double-crossover clones by PCR

(No.1-5 are experimental groups, No.6 is wild GR286. The result showed that we failed to get the double-crossover clones.)

☞☟ Week3 (May 30–Jun 05)

* [➀](#fragment3-1)
* [➁](#fragment3-2)
* [➂](#fragment3-3)

Transformants were cultured at 42oC with chloramphenicol again and the single-crossover clones were selected successfully.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| *luxS*-up-F | 1μL |
| *luxS*-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/9/93/T--NKU_China--notebook-1-5-4.png)

Selection of single-crossover clones by PCR

(No.1&2&4 are single-crossover strains,No.5 is positive control. )

The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.

The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. We finally obtained our aimed strain—GR286Δ*luxS*.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| *luxS*-up-F | 1μL |
| *luxS*-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 56oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-1-5-5.png)

Selection of Δ*luxS* clones by PCR

(The No.4 is the aimed strain&horbar;GR286Δ*luxS*)

☞☟ Week4 (Jun 06–Jun 12)

* [➀](#fragment4-1)
* [➁](#fragment4-2)
* [➂](#fragment4-3)
* [➃](#fragment4-4)
* [➄](#fragment4-5)
* [➅](#fragment4-6)

The GR286Δ*luxS* strain was cultured and made competent for future use.

The *lsrACDB* gene from *Bacillus thuringiensis* was cloned and ligated to T-vector.

50μL PCR system ×2

| 2× Taq Master Mix | 25μL |
| *lsrACDB*-F | 2μL |
| *lsrACDB*-R | 2μL |
| Bacterium solution | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 57oC | 30 sec | 30 cycles |
| 72oC | 4 min 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| *lsrACDB* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

The T-*lsrACDB* was transformed into DH5α and plate was coated, and then positive clones were selected.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| M13F | 1μL |
| M13R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 59oC | 30 sec | 30 cycles |
| 72oC | 4 min 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/7/72/T--NKU_China--notebook-1-5-6.png)

Selection of positive clones by PCR

(No. 3&4 are positive results)

After restriction enzyme digestion verification, the positive clones were then sequenced. Unfortunately, the sequencing result showed some mutations inside the target gene.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| T-*lsrACDB* | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/9/91/T--NKU_China--notebook-1-5-7.png)

Restriction enzyme digestion verification

(No.1 are *lsrACDB* fragement, No.2 are linearized T-vector.)

The gene cloning process was repeated but there were still some mutations.

We finally decided to request the gene company to synthesize the lsrACDB gene.

☞☟ Week5 (Jun 13–Jun 19)

* [begin](#fragment5-1)
* [➀](#fragment5-2)
* [➁](#fragment5-3)
* [➂](#fragment5-4)
* [➃](#fragment5-5)

This week, we started to construct another controller―supplier.

A strong promoter *C2* was cloned from former kit and *luxS* gene was cloned from GR286.

50μL PCR system ×2

| 2× Taq Master Mix | 25μL |
| C2-F | 2μL |
| C2-R | 2μL |
| p-C2 | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 15 sec | 30 cycles |
| 72oC | 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

50μL PCR system ×2

| 2× Taq Master Mix | 25μL |
| *luxS*-F | 2μL |
| *luxS*-R | 2μL |
| Bacterium solution | 1μL |
| GR286 | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 59oC | 30 sec | 30 cycles |
| 72oC | 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/d/dc/T--NKU_China--notebook-1-5-8.png)

PCR cloning product of promoter *C2* and gene *luxS*

(NO.1&2 are *C2*, No.3&4 are *luxS*)

Two segments were fused together by fusion PCR and ligated into T-vector. After that, the vector was transformed into DH5α.

50μL PCR system ×2

| 2× Taq Master Mix | 25μL |
| C2-F | 2μL |
| *luxS*-R | 2μL |
| C2 | 2μL |
| *luxS* | 2μL |
| ddH2O | 17μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 59oC | 30 sec | 30 cycles |
| 72oC | 40 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| *C2-luxS* | 4μL |
| ddH2O | 12μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Positive clones were selected by colony PCR.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| M13-F | 1μL |
| M13-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 59oC | 30 sec | 30 cycles |
| 72oC | 40 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/c/c9/T--NKU_China--notebook-1-5-9.png)

PCR cloning product of promoter *C2* and gene *luxS*

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| T-*lsrACDB* | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/f/fb/T--NKU_China--notebook-1-5-10.png)

Restriction enzyme digestion verification

(No.1 are linearized T-vector, No.2 are *C2-luxS* fragment.)

☞☟ Week6 (Jun 20–Jun 26)

* [➀](#fragment6-1)
* [➁](#fragment6-2)
* [➂](#fragment6-3)
* [➃](#fragment6-4)
* [➄](#fragment6-5)

The sequencing result showed that there was a correct strain and thus it could be used for the following experiments. We obtained the correct plasmid T-*C2-luxS* from DH5α. Then the fragment *C2-luxS* was obtained by digestion and gel extraction.

40μL digestion system

| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| T-*C2-luxS* | 25μL |
| ddH2O | 9μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

The *C2-luxS* fragment was ligated to linearized plasmid pWH1520, and then the ligation product was transformed into DH5α.

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| *C2-luxS* | 5μL |
| ddH2O | 11μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

The plasmid pWH-*C2-luxS* was extracted from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into *E.coli* JM110.

The plasmid pWH-*C2-luxS* was extracted from JM110,and then it was treated with BamH Ⅰ methylase.

100μL methylation system

| 10× BamH Ⅰ methyltransferase Buffer | 10μL |
| BamH Ⅰ methyltransferase | 1μL |
| S-adenosylmethionine | 0.5μL |
| pWH-*C2-luxS* | 80μL |
| ddH2O | 8.5μL |
| Total | 100μL |
| Reaction condition: 37oC for 1 hour | |

The plasmid was transformed into GR286 by electroporation.[Failed]

![](https://static.igem.org/mediawiki/2016/e/e0/T--NKU_China--notebook-6-13-1.png)

Selection of positive clones by colony PCR

(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-*C2-luxS* into GR286)

☞☟ Week7 (Jun 27–Jul 03)

* [➀](#fragment7-1)
* [➁](#fragment7-2)
* [➂](#fragment7-3)

This week, we tried to use different voltages to transform the plasmid. Sadly, all of these attempts rendered negative results.

We considered whether the *luxS* gene is toxic for GR286, and the bacteria tends to reject the gene when a strong promoter is inserted upstream of it. So, we planned to use inducible promoter to reconstruct our expression vector.

The plasmid pWH1520 contains a strong *xylA* promoter originating from *Bacillus megaterium*, and transcription initiated by this promoter is xylose-inducible. Also, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers.

![](https://static.igem.org/mediawiki/2016/e/eb/T--NKU_China--notebook-6-13-2.png)

☞☟ Week8 (Jul 04–Jul 10)

* [➀](#fragment8-1)
* [➁](#fragment8-2)
* [➂](#fragment8-3)
* [➃](#fragment8-4)

*luxS* gene was cloned from GR286 using our new primers.

50μL PCR system ×2

| 2× Taq Master Mix | 25μL |
| YD-luxS-F | 2μL |
| YD-luxS-R | 2μL |
| Bacterium solution | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 40 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/3/3a/T--NKU_China--notebook-6-13-3.png)

PCR cloning product of gene *luxS*

The *luxS* fragment was purified by gel extraction, and ligated into linearized pWH1520. Then the vector was transformed into DH5α.

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| pWH1520 | 25μL |
| ddH2O | 9μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pWH1520 | 1μL |
| *luxS* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Positive clones were selected by colony PCR.

20μL PCR system

| 2× Taq Master Mix | 10μL |
| pWH-F | 1μL |
| pWH-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 40 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/4/49/T--NKU_China--notebook-6-13-4.png)

Selection of positive clones by colony PCR

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| pWH-*luxs* | 10μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/3/35/T--NKU_China--notebook-6-13-5.png)

Restriction enzyme digestion verification

(No.1 are linearized pWH1520, No.2 are *luxS* fragments.)

☞☟ Week9 (Jul 11–Jul 17)

* [➀](#fragment9-1)
* [➁](#fragment9-2)
* [➂](#fragment9-3)
* [end](#fragment9-4)

The sequencing result showed there's three positive strains. So one positive strain was chosen to be used for the following experiments. The pWH-*luxS* plasmid was extracted from the chosen strain. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.

the plasmid pWH-*luxS* was extracted from JM110,and it was treated withBamH Ⅰ methylase.

100μL methylation system

| 10× BamH Ⅰ methyltransferase Buffer | 10μL |
| BamH Ⅰ methyltransferase | 1μL |
| S-adenosylmethionine | 0.5μL |
| pWH-*C2-luxS* | 80μL |
| ddH2O | 8.5μL |
| Total | 100μL |
| Reaction condition: 37oC for 1 hour | |

The plasmid was transformed into GR286 by electroporation, and positive clones were selected.

![](https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-6-13-6.png)

Selection of positive clones by colony PCR

The construction of supplier was accomplished!

☞☟ Week10 (Jul 18–Jul 24)

* [➀](#fragment10-1)
* [➁](#fragment10-2)

We have received the product of synthetic *lsrACDB* gene. We first used restriction-ligation method to ligate *lsrACDB* to plasmid pWH1520, but we failed to select positive after several tries.

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pWH1520 | 1μL |
| *lsrACDB* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Considering that the *lsrACDB* gene is a large fragment (4500bp), we used ClonExpress technique to clone the gene again to improve the efficiency of ligation. The *lsrACDB* sequence was divided into two parts and they were cloned separately. Then the two segments were ligated to the plasmid pWH1520 and the recombinant vector was transformed into DH5α. After that, verification PCR was used to select the positive clones. However, we didn't get a good result.

![](https://static.igem.org/mediawiki/2016/1/17/T--NKU_China--notebook-6-13-7.png)

Selection of positive clones by colony PCR

(No.6 is positive control, No.1-5 are experimental groups)

☞☟ Week11 (Jul 25–Jul 31)

* [➀](#fragment11-1)

We learnt a new method called circular polymerase extension cloning (CPEC) for high-throughput cloning of complex and combinatorial DNA libraries, and we decided to use this method to try to ligate our *lsrACDB* gene. It's encouraging that we succeeded to ligate the *lsrACDB* gene to the plasmid pHT-01.

![](https://static.igem.org/mediawiki/2016/c/c2/T--NKU_China--notebook-6-13-8.png)

Selection of positive clones by colony PCR

(No.3-6 are positive clones)

![](https://static.igem.org/mediawiki/2016/7/7a/T--NKU_China--notebook-6-13-9.png)

Restriction enzyme digestion verification

(No.2&3 are positive results.)

☞☟ Week12 (Aug 1–Aug 7)

* [➀](#fragment12-1)
* [➁](#fragment12-2)

Since we have already successfully constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller".

Groups divided in this experiment

| GR286 | wild strain as control group |
| GR286Δ*luxS* | GR286 without *luxS* gene |
| pWH-*luxS* | *luxS* overexpression plasmid in GR286; without induced by xylose |
| pWH-*luxS* &plus; xyl | *luxS* overexpression plasmid in GR286; induced by xylose |
| pWH1520 | empty plasmid in GR286 as control group |
| pHT-*lsrACDB* | *lsrACDB* overexpression plasmid in GR286Δ*luxS* |
| pHT-01 | empty plasmid in GR286Δ*luxS* as control group |

![](https://static.igem.org/mediawiki/2016/a/ad/T--NKU_China--notebook-6-13-10.png)

OD600 of different groups at specific times

![](https://static.igem.org/mediawiki/2016/0/00/T--NKU_China--notebook-6-13-11.png)

Growth curve of GR286 and GR286Δ*luxS*

![](https://static.igem.org/mediawiki/2016/a/a9/T--NKU_China--notebook-6-13-12.png)

Growth curve of pWH-*luxS*, pWH-*luxS* &plus; xyl and pWH1520

![](https://static.igem.org/mediawiki/2016/6/6b/T--NKU_China--notebook-6-13-13.png)

Growth curve of pHT-*lsrACDB* and pHT-01

Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence of *Vibrio harveyi* reporter strain BB170.

![](https://static.igem.org/mediawiki/2016/a/a4/T--NKU_China--notebook-6-13-14.png)

Fluorescence intensity of different groups at specific times

![](https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-15.png)

Relative fluorescence intensity of different groups

☞☟ Week13 (Aug 8–Aug 14)

* [➀](#fragment13-1)
* [➁](#fragment13-2)

For our consumer, we should also overexpress the *lsrK* and *lsrFG* gene for phosphorylating and degrading phosphorylated AI-2. We used ClonExpress technique to clone the two genes and ligate them to plasmid pHT-01 successfully.

![](https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-16.png)

Restriction enzyme digestion verification

(No.1&3&4 are positive results. )

We co-cultured the supplier with BB170 and tested the fluorescence intensity to explore the function of supplier. (negative result)

![](https://static.igem.org/mediawiki/2016/f/f1/T--NKU_China--notebook-6-13-17.png)

Fluorescence intensity of BB170 and co-culture medium

☞☟ Week14 (Aug 15–Aug 21)

* [begin](#fragment14)
* [outline](#fragment14-0)
* [1.1](#fragment14-1-1)
* [1.2](#fragment14-1-2)
* [1.3](#fragment14-1-3)

Since the controller we constructed with GR286 did not demonstrate positive results, we decided to substitute GR286 with *Escherichia coli* whose AI-2 metabolic pathway had been elucidated as the chassis of our controller.

* The strains to be constructed are shown in the tables below.

  AI-2 supplier

  |  | LuxS | Mtn |
  | pTrcHisB | Native | Native |
  | pLuxS | Induced | Native |
  | pLuxS-Mtn | Induced | Induced |

  AI-2 consumer

  |  | LsrACDB | LsrK | LsrFG |
  | pTrcHisB | Native | Native | Native |
  | pLsrACDB | Induced | Native | Native |
  | pLsrFG | Native | Native | Induced |
  | pLsrK | Native | Induced | Native |
  | pLsrACDBFG | Induced | Native | Induced |
  | pLsrACDBK | Induced | Constitutive | Native |
  | plsrACDBFGK | Induced | Constitutive | Induced |
* The sequences of *lsrACDB*, *lsrFG*, *lsrK*, *luxS*, *mtn* gene in *E.coli K12* were searched in NCBI, and primers were designed according to the gene sequences. The primers used in the construction of AI-2 controller are shown in the table below.

  Primers used in the construction of AI-2 controller

  | Primer name | Sequence |
  | *lsrACDBFG*1-F | CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC |
  | *lsrACDBFG*1-R | TGTTTGGCGTTTCCGGCAGCGGTGCGGAGAGC |
  | *lsrACDBFG*2-F | TGTTTGGCGTTTCCGCGATTG |
  | *lsrACDBFG*2-R | GCACTCTCACACCACGTTGCATGGCGCGTTTC |
  | *lsrACDBFG*3-F | GTGGTGTGAGAGTGCTGAC |
  | *lsrACDBFG*3-R | ACCAGCTGCAGATCTCGAGCTCGTCACGGCATCAACCCATTGAAC |
  | *lsrACDB*1-F | CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC |
  | *lsrACDB*1-R | ACCGGAACCGCCGAGCAAACTAATGCCGCCCAGCAC |
  | *lsrACDB*2-F | CTCGGCGGTTCCGGTGCGAT |
  | *lsrACDB*2-R | ACCAGCTGCAGATCTCGAGCTCGTCAGAAATCGTATTTGCCG |
  | *luxS*-F | ATGACGATAAGGATCCGAGCTCGATGCCGTTGTTAGATAGCTTC |
  | *luxS*-R | GGACTCCCCCGGGGGACTAAATGTGCAGTTCCTGCAACTTC |
  | *mtn*-F | TCCCCCGGGGGAGTCCTCTCCCGCGTGAGAAATAC |
  | *mtn*-R | TATGGTACCAGCTGCAGATCTCTTAGCCATGTGCAAGTTTCTG |
  | *luxS*-Fd | GGAATTCCCTAAATGTGCAGTTCCTGCAACTTC |
  | *luxS*-Rv | GGGGTACCCCATGCCGTTGTTAGATAGCTTCACAG |
  | *lsrK*-Fd | GGGGTACCCCATGGCTCGACTCTTTACC |
  | *lsrK*-Rv | GGAATTCCCTATAACCCAGGCGCTTTCC |
  | *lsrFG*-Fd | CGGGATCCCGATGGCAGATTTAGACGATATTAAAGATGG |
  | *lsrFG*-Rv | GAAGATCTTCTCACGGCATCAACCCATTGAAC |
  | *lsrK*-F | AACTGCAGAACCAATGCATTGGTTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCAAAGAGGAGAAAATGGCTCGACTCTTTACC |
  | *lsrK*-R | TTCGAACTATAACCCAGGCGCTTTCC |
  | BK-JC-F | TTTCAGTGTATGTCGCGGATGC |
  | GK-JC-F | TTGCGCTTCGATGTCTTACAGG |
  | pTrc-JC-F | TGGGCACTCGACCGGAATTATC |
  | pTrc-JC-R | GCTACTGCCGCCAGGCAAATTC |

1. Construction of pLuxS-Mtn

*luxS* gene was cloned from *E.coli K12*.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *luxS*-F | 2μL |
| *luxS*-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 63oC | 30 sec | 30 cycles |
| 72oC | 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/f/f7/T--NKU_China--notebook-14-17-01.png)

PCR cloning product of gene *luxS*

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *mtn*-F | 2μL |
| *mtn*-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 63oC | 30sec | 30 cycles |
| 72oC | 45 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/6/66/T--NKU_China--notebook-14-17-02.png)

PCR cloning product of gene *mtn*

ClonExpress technique was used to ligate *luxS* and *mtn* genes to plasmid pTrcHisB. The recombinant vector pTrc-*luxS*-*mtn* was transformed into *E.coli K12*. After that, verification PCR was performed to select the positive clones.

20μL PCR system

| PrimeSTAR Max Premix(2×) | 10μL |
| pTrc-JC-F | 1μL |
| pTrc-JC-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30sec | 30 cycles |
| 72oC | 1 min 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/e/e4/T--NKU_China--notebook-14-17-03.png)

Selection of positive clones by PCR

(NO.3&5&8 are positive results)

3 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| Sac Ⅰ | 1μL |
| Bgl Ⅱ | 1μL |
| pTrc-*luxS*-*mtn* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/8/83/T--NKU_China--notebook-14-17-04.png)

Restriction enzyme digestion verification

(No.1 are linearized pTrcHisB, No.2 are *luxS*-*mtn* fragment. )

☞☟ Week15 (Aug 22–Aug 28)

* [outline](#fragment15-0)
* [1.1](#fragment15-1-1)
* [1.2](#fragment15-1-2)
* [1.3](#fragment15-1-3)
* [1.4](#fragment15-1-4)
* [2.1](#fragment15-2-1)
* [2.2](#fragment15-2-2)
* [2.3](#fragment15-2-3)
* [3.1](#fragment15-3-1)
* [3.2](#fragment15-3-2)
* [3.3](#fragment15-3-3)

1. Construction of pLuxS
2. Construction of pLsrACDB
3. Construction of pLsrACDBFG

*luxS* gene was cloned from *E.coli K12*.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *luxS*-Fd | 2μL |
| *luxS*-Rv | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 60oC | 30 sec | 30 cycles |
| 72oC | 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/b/bc/T--NKU_China--notebook-14-17-05.png)

PCR cloning product of gene *luxS*

The *luxS* gene was purified by gel extraction. Then, both the *luxS* segment and pTrcHisB vector were treated with restriction enzyme. After that, *luxS* gene was ligated to linearized pTrcHisB, and the ligation product was transformed into *E.coli K12*.

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| Kpn Ⅰ | 2μL |
| EcoR Ⅰ | 2μL |
| *luxS* | 25μL |
| ddH2O | 7μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| Kpn Ⅰ | 2μL |
| EcoR Ⅰ | 2μL |
| pTrcHisB | 20μL |
| ddH2O | 12μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pTrcHisB | 1μL |
| *luxS* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Verification PCR was performed to select the positive clones.

20μL PCR system ×2

| 2× Taq Master Mix | 10μL |
| pTrc-JC-F | 1μL |
| pTrc-JC-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30sec | 30 cycles |
| 72oC | 50 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/6/65/T--NKU_China--notebook-14-17-06.png)

Selection of positive clones by PCR

One positive strain was chosen to be cultured overnight and plasmid was extracted. After restriction enzyme digestion verification, the positive clone was sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| Kpn Ⅰ | 1μL |
| EcoR Ⅰ | 1μL |
| pTrc-*luxS* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/c/ca/T--NKU_China--notebook-14-17-07.png)

Restriction enzyme digestion verification

(No.1 is linearized pTrcHisB &plus; *luxS*, No.2 is plasmid pTrc-*luxS*. )

The *lsrACDBFG* sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrACDB*1-F | 2μL |
| *lsrACDB*1-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrACDB*2-F | 2μL |
| *lsrACDB*2-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 60oC | 30 sec | 30 cycles |
| 72oC | 2 min 20 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/b/b6/T--NKU_China--notebook-14-17-08.png)

PCR cloning product of segment *lsrACDB1*,*lsrACDB1*2

Verification PCR was performed to select the positive clones.

![](https://static.igem.org/mediawiki/2016/e/e1/T--NKU_China--notebook-14-17-09.png)

Selection of positive clones by PCR

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| Sac Ⅰ | 1μL |
| pTrc-*lsrACDB* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/5/5d/T--NKU_China--notebook-14-17-10.png)

Restriction enzyme digestion verification

The *lsrACDBFG* sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrACDBFG*1-F | 2μL |
| *lsrACDBFG*1-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrACDBFG*2-F | 2μL |
| *lsrACDBFG*2-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrACDBFG*3-F | 2μL |
| *lsrACDBFG*3-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/6/69/T--NKU_China--notebook-14-17-11.png)

PCR cloning product of segment *lsrACDBFG*1,*lsrACDBFG*2, *lsrACDBFG*3

Verification PCR was performed to select the positive clones.

![](https://static.igem.org/mediawiki/2016/9/97/T--NKU_China--notebook-14-17-12.png)

Selection of positive clones by PCR

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| Sac Ⅰ | 1μL |
| pTrc-*lsrACDB* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/3/33/T--NKU_China--notebook-14-17-13.png)

Restriction enzyme digestion verification

(No.1-4 are linearized pTrcHisB &plus; *lsrACDBFG*, No.5 is plasmid pTrc-*lsrACDBFG*.)

☞☟ Week16 (Aug 29–Sep 04)

* [outline](#fragment16-0)
* [1.1](#fragment16-1-1)
* [1.2](#fragment16-1-2)
* [1.3](#fragment16-1-3)
* [1.4](#fragment16-1-4)
* [2.1](#fragment16-2-1)
* [2.2](#fragment16-2-2)
* [2.3](#fragment16-2-3)
* [2.4](#fragment16-2-4)
* [3.1](#fragment16-3-1)
* [3.2](#fragment16-3-2)
* [3.3](#fragment16-3-3)
* [3.4](#fragment16-3-4)

1. Construction of pLsrFG
2. Construction of pLsrACDBK
3. Construction of pLsrACDBFGK

*lsrFG* gene was cloned from *E.coli K12*.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrFG*-Fd | 2μL |
| *lsrFG*-Rv | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 1 min 15 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/5/5c/T--NKU_China--notebook-14-17-14.png)

PCR cloning product of gene *lsrFG*

The *lsrFG* gene was purified by gel extraction. Then, both the *lsrFG* segment and pTrcHisB vector were treated with restriction enzyme. After that, *lsrFG* gene was ligated to linearized pTrcHisB, and the ligation product was transformed into *E.coli K12*.

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| Bgl Ⅱ | 2μL |
| *lsrFG* | 25μL |
| ddH2O | 7μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| Bgl Ⅱ | 2μL |
| pTrcHisB | 20μL |
| ddH2O | 12μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| *luxSFG* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Verification PCR was performed to select the positive clones.

20μL PCR system ×2

| 2× Taq Master Mix | 10μL |
| pTrc-JC-F | 1μL |
| pTrc-JC-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 1 min 30 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/5/5b/T--NKU_China--notebook-14-17-15.png)

Selection of positive clones by PCR

(NO. 1&3 are positive results)

Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| Bgl Ⅱ | 1μL |
| pTrc-*lsrFG* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/b/b0/T--NKU_China--notebook-14-17-16.png)

Restriction enzyme digestion verification

*lsrK* gene was cloned from *E.coli K12*.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrK*-F | 2μL |
| *lsrK*-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 55oC | 30 sec | 30 cycles |
| 72oC | 1 min 40 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/8/85/T--NKU_China--notebook-14-17-17.png)

PCR cloning product of gene *lsrK*

The *lsrK* gene was purified by gel extraction. Then, both the *lsrK* segment and pTrc-*lsrACDB* vector were treated with restriction enzyme. After that, *lsrK* gene was ligated to linearized pTrc-*lsrACDB*, and the ligation product was transformed into *E.coli K12*.

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| Pst Ⅰ | 2μL |
| BstB Ⅰ | 2μL |
| *lsrK* | 25μL |
| ddH2O | 7μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| Pst Ⅰ | 2μL |
| BstB Ⅰ | 2μL |
| pTrc-*lsrACDB* | 25μL |
| ddH2O | 7μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pTrc-*lsrACDB* | 1μL |
| *lsrK* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Verification PCR was performed to select the positive clones.

20μL PCR system ×2

| 2× Taq Master Mix | 10μL |
| BK-JC-F | 1μL |
| pTrc-JC-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/c/cb/T--NKU_China--notebook-14-17-18.png)

Selection of positive clones by PCR

(NO. 1&3 are positive results)

Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| Pst Ⅰ | 1μL |
| BstB Ⅰ | 1μL |
| pTrc-*lsrACDBK* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/5/56/T--NKU_China--notebook-14-17-19.png)

Restriction enzyme digestion verification

*lsrK* gene was cloned from *E.coli K12*.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrK*-F | 2μL |
| *lsrK*-R | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 55oC | 30 sec | 30 cycles |
| 72oC | 1 min 40 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/8/85/T--NKU_China--notebook-14-17-17.png)

PCR cloning product of gene *lsrK*

The *lsrK* gene was purified by gel extraction. Then, both the *lsrK* segment and pTrc-*lsrACDBFG* vector were treated with restriction enzyme. After that, *lsrK* gene was ligated to linearized pTrc-*lsrACDBFG*, and the ligation product was transformed into *E.coli K12*.

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| Pst Ⅰ | 2μL |
| BstB Ⅰ | 2μL |
| *lsrK* | 25μL |
| ddH2O | 7μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

40μL digestion system ×2

| 10× FastDigest Buffer | 4μL |
| Pst Ⅰ | 2μL |
| BstB Ⅰ | 2μL |
| pTrc-*lsrACDBFG* | 20μL |
| ddH2O | 12μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pTrc-*lsrACDFGB* | 1μL |
| *lsrK* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Verification PCR was performed to select the positive clones.

20μL PCR system ×2

| 2× Taq Master Mix | 10μL |
| GK-JC-F | 1μL |
| pTrc-JC-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/7/78/T--NKU_China--notebook-14-17-20.png)

Selection of positive clones by PCR

(NO.3 is positive result)

Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| Pst Ⅰ | 1μL |
| BstB Ⅰ | 1μL |
| pTrc-*lsrACDBKFGK* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/e/e2/T--NKU_China--notebook-14-17-21.png)

Restriction enzyme digestion verification

☞☟ Week17 (Sep 05–Sep 11)

* [outline](#fragment17-0)
* [1.1](#fragment17-1-1)
* [1.2](#fragment17-1-2)
* [1.3](#fragment17-1-3)
* [1.4](#fragment17-1-4)

1. Construction of pLsrK

*lsrK* gene was cloned from *E.coli K12*.

50μL PCR system ×2

| PrimeSTAR Max Premix(2×) | 25μL |
| *lsrK*-Fd | 2μL |
| *lsrK*-Rv | 2μL |
| *E.coli K12* | 2μL |
| ddH2O | 19μL |
| Total | 50μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 55oC | 30 sec | 30 cycles |
| 72oC | 1 min 40 sec |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/2/24/T--NKU_China--notebook-14-17-22.png)

PCR cloning product of gene *lsrK*

The *lsrK* gene was purified by gel extraction. Then, both the *lsrK* segment and pTrc-*lsrACDBFG* vector were treated with restriction enzyme. After that, *lsrK* gene was ligated to linearized pTrc-*lsrACDBFG*, and the ligation product was transformed into *E.coli K12*.

40μL digestion system ×2 ×2

| 10× FastDigest Buffer | 4μL |
| Kpn Ⅰ | 2μL |
| EcoR Ⅰ | 2μL |
| *lsrK* | 25μL |
| ddH2O | 7μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

40μL digestion system ×2 ×2

| 10× FastDigest Buffer | 4μL |
| Pst Ⅰ | 2μL |
| BstB Ⅰ | 2μL |
| pTrc-HisB | 20μL |
| ddH2O | 12μL |
| Total | 40μL |
| Reaction condition: 37oC for 40 min | |

20μL ligation system

| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pTrcHisB | 1μL |
| *lsrK* | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16oC overnight | |

Verification PCR was performed to select the positive clones.

20μL PCR system ×2

| 2× Taq Master Mix | 10μL |
| pTrc-JC-F | 1μL |
| pTrc-JC-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |

}

PCR reaction condition

| 94oC | 10 min |  |
| 94oC | 30 sec |  |
| 58oC | 30 sec | 30 cycles |
| 72oC | 2 min |  |
| 72oC | 10 min |  |
| 16oC | ∞ |  |

![](https://static.igem.org/mediawiki/2016/3/30/T--NKU_China--notebook-14-17-23.png)

Selection of positive clones by PCR

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system

| 10× FastDigest Buffer | 2μL |
| Kpn Ⅰ | 1μL |
| EcoR Ⅰ | 1μL |
| pTrc-*lsrK* | 6μL |
| ddH2O | 10μL |
| Total | 20μL |
| Reaction condition: 37oC for 40 min | |

![](https://static.igem.org/mediawiki/2016/2/22/T--NKU_China--notebook-14-17-24.png)

Restriction enzyme digestion verification

☞☟ Week18 (Seq 12–Seq 18)

* [➀](#fragment18-1)
* [➁](#fragment18-2)

We detected the expression of several overexpressed genes at the mRNA level by RT-PCR, and obtained positive results.

![](https://static.igem.org/mediawiki/2016/f/f8/T--NKU_China--notebook-18-20-01.png)

Relative expression of several overexpressed genes at the mRNA level

SDS-PAGE was performed to detect the expression of these genes at the protein level.

![](https://static.igem.org/mediawiki/2016/d/d2/T--NKU_China--notebook-18-20-02.png)

pTrcHisB, pLuxS, pLuxS-Mtn, pLsrACDB, pLsrACDBFG SDS-PAGE

☞☟ Week19 (Seq 19–Seq 25)

* [➀](#fragment19-1)
* [➁](#fragment19-2)

Cultured media of controllers were tested for the presence of AI-2 by inducing luminescence of *Vibrio harveyi* reporter strain BB170.

![](https://static.igem.org/mediawiki/2016/4/4c/T--NKU_China--notebook-18-20-03.jpg)

Relative fluorescence intensity of suppliers at specific times

![](https://static.igem.org/mediawiki/2016/f/f7/T--NKU_China--notebook-18-20-04.jpg)

Relative fluorescence intensity of consumers at specific times

HPLC was performed to detect the production of AI-2 of the strain pLuxS.

![](https://static.igem.org/mediawiki/2016/d/d9/T--NKU_China--notebook-18-20-05.png)

HPLC result of AI-2 production of luxS overexpression strain and negative control

☞☟ Week20 (Seq 26–Oct 02)

* [➀](#fragment20-1)
* [➁](#fragment20-2)

Biofilm studies and evaluation:
pTrcHisB, pLuxS, pLuxSMtn, pLsrACDB, pLsrACDBFG, pLsrACDBK, pLsrACDBFGK were diluted to OD~0.05 and reinoculated at a total volume of 200 μL at a 1:1 (v/v) ratio. IPTG (1 mM) was added at OD ~ 0.4, and biofilms were cultured for ~24 h (&plus;/−30 min) at 30 oC in static conditions. After incubation, optical density was read on a plate reader at 600 nm. The supernatant was gently decanted, and each well was washed 3 times with 300 μL of sterile PBS to detach loosely adhered cells. The plate was then incubated at 60 oC with the lid off for 60 min, and afterwards, 250 μL of 0.1% crystal violet was added to each well and incubated for 15 min at room temperature. Crystal violet stain was aspirated with a pipette and excess stain was washed off by gently submerging and mixing in a tray filled with distilled water until washings were free of the stain. After the microplate was airdried, the dye was resolubilized by adding 250 μL of 95% ethanol, and incubated at room temperature with shaking for 30 min. The optical density of each well stained with crystal violet was measured at 540 nm.

![](https://static.igem.org/mediawiki/2016/3/33/T--NKU_China--notebook-18-20-06.jpg)

Relative biofilm production of suppliers

![](https://static.igem.org/mediawiki/2016/8/8b/T--NKU_China--notebook-18-20-07.jpg)

Relative biofilm production of consumers

Cultured media of controllers were tested for the presence of AI-2 by HPLC. AI-2 output and uptake profiles of controllers were drawn according to the HPLC results.

![](https://static.igem.org/mediawiki/2016/e/ed/T--NKU_China--notebook-18-20-08.jpg)

AI-2 output profiles of suppliers

![](https://static.igem.org/mediawiki/2016/0/04/T--NKU_China--notebook-18-20-09.jpg)

AI-2 uptake profiles of consumers

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