iGEM 2015 — Team Amoy

# Team:Amoy/Notebook

Aomy/Project

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μl ddH2O to solve the dry powder.
2. Suck 10μl of plasmid into 50μl of competent cell for transformation.

![](https://static.igem.org/mediawiki/2015/8/87/Amoy-Notebook_Node41_figure1.jpg)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm.
5. Plasmid extraction.

Product:

Plasmid of promoter\_J23100

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μl ddH2O to solve the dry powder
2. Suck 10μl of plasmid into 50μl of competent cell for transformation

![](https://static.igem.org/mediawiki/2015/d/d9/Amoy-Notebook_node42-1.jpeg)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of ampicillin for 12h, 37℃, 200rpm.
5. Plasmid extraction.

Product:

Plasmid of promoter\_LacI

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μl ddH2O to solve the dry powder
2. Suck 10μl of plasmid into 50μl of competent cell for transformation

![](https://static.igem.org/mediawiki/2015/6/64/Amoy-Notebook_node43-1.JPG)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of ampicillin for 12h, 37℃, 200rpm
5. Plasmid Extraction

Product:

Plasmid of RBS\_B0034

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μl ddH2O to solve the dry powder
2. Suck 10μl of plasmid into 50μl of competent cell for transformation

![](https://static.igem.org/mediawiki/2015/c/cd/Amoy-Notebook_node44-1.jpeg)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of ampicillin for 12h, 37℃, 200rpm
5. Plasmid Extraction

Product:

Plasmid of RBS\_B0032

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μl ddH2O to solve the dry powder
2. Suck 10μl of plasmid into 50μl of competent cell for transformation

![](https://static.igem.org/mediawiki/2015/e/e4/Amoy-Notebook_node45-1.jpeg)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm
5. Plasmid Extraction

Product:

Plasmid of RBS\_B0030

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μL ddH2O to solve the dry powder.
2. Suck 10μL of plasmid into 50μL of competent cell for transformation.

![](https://static.igem.org/mediawiki/2015/c/cf/Amoy-Notebook_node33-1.JPG)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm.
5. Plasmid Extraction

Product:

Plasmid of Plac\_RBS\_B0034

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μL ddH2O to solve the dry powder.
2. Suck 10μL of plasmid into 50μL of competent cell for transformation.

![](https://static.igem.org/mediawiki/2015/1/1a/Amoy-Notebook_node34-1.JPG)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm.
5. Plasmid Extraction

Product:

Plasmid of PJ23100\_RBS\_B0030

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μl ddH2O to solve the dry powde
2. Suck 10μl of plasmid into 50μl of competent cell for transformation

![](https://static.igem.org/mediawiki/2015/d/d5/Amoy-Notebook_node48-1.JPG)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm
5. Plasmid Extraction

Product:

Plasmid of terminator\_B0015

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μl ddH2O to solve the dry powde
2. Suck 10μl of plasmid into 50μl of competent cell for transformation

![](https://static.igem.org/mediawiki/2015/5/5c/Amoy-Notebook_node49-1.jpeg)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm
5. Plasmid Extraction

Product:

Plasmid of terminator\_B1006

Purpose:

PCR amplification for gene\_*leudh* and double enzyme digestion

Steps:

1. PCR amplification system for gene\_*leudh*

|  |  |
| --- | --- |
| Components | Volume |
| ddH2O | 16μL |
| *leudh*-F | 1.5μL |
| *leudh*-R | 1.5μL |
| pUB18-*leudh* | 1μL |
| Prime Star | 20μL |

2. Electrophoresis of PCR products of gene\_*leudh*
3. Double enzyme digestion of gene\_*leudh*

|  |  |
| --- | --- |
| Components | Volume |
| Gene | 20μL |
| ddH2O | 20μL |
| buffer | 10μL |
| EcoR I | 5μL |
| Spe I | 5μL |

4. Cycle purity of digested gene\_*leudh*

Product:

Digested gene\_*leudh*

Purpose:

PCR amplification for gene\_*fdh* and double enzyme digestion

Steps:

1. PCR amplification system for gene\_*fdh*

|  |  |
| --- | --- |
| Components | Volume |
| ddH2O | 16μL |
| *fdh*-F | 1.5μL |
| *fdh*-R | 1.5μL |
| pUB18-*fdh* | 1μL |
| Prime Star | 20μL |

2. Electrophoresis of PCR products of gene\_*fdh*
3. Double enzyme digestion of gene\_*fdh*

|  |  |
| --- | --- |
| Components | Volume |
| Gene | 50μL |
| ddH2O | 10μL |
| buffer | 10μL |
| EcoR I | 5μL |
| Spe I | 5μL |

4. Cycle purity of digested gene\_*fdh*

Product:

Digested gene\_*fdh*

Purpose:

Ligation of gene\_*leudh* and terminator

Steps:

1. Double enzyme digestion of gene\_*leudh* and terminator\_B0015
2. Electrophoresis analysis of double digested result of plasmid.
3. Extract double digested gene\_*leudh*.
4. Cycle purity of digested terminator.
5. Ligate under 16℃ for 8 hours.
6. Transformation

![](https://static.igem.org/mediawiki/2015/3/34/Amoy-Notebook_node35-1.jpeg)

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
8. Extract the plasmids.
9. Electrophoresis analysis of plasmids.

Product:

Plasmid of gene\_*leudh* and terminator\_B0015

Purpose:

Ligation of gene\_*fdh* and terminator

Steps:

1. Double enzyme digestion of gene\_*fdh* and terminator\_B0015
2. Electrophoresis analysis of double digested result of plasmid.
3. Extract double digested gene\_*fdh*.
4. Cycle purity of digested terminator.
5. Ligate under 16℃ for 8 hours.
6. Transformation

![](https://static.igem.org/mediawiki/2015/0/01/Amoy-Notebook_Node36_figure1.JPG)

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
8. Extract the plasmids.
9. Electrophoresis analysis of plasmids.

Product:

Plasmid of gene\_*fdh* and terminator\_B0015

Purpose:

Ligation of gene\_*leudh* and terminator

Steps:

1. Double enzyme digestion of gene\_*leudh* and terminator\_B1006
2. Electrophoresis analysis of double digested result of plasmid.
3. Extract double digested gene\_*leudh* and terminator.
4. Ligate under 16℃ for 8 hours.
5. Transformation

![](https://static.igem.org/mediawiki/2015/c/c1/Amoy-Notebook_node37-1.jpeg)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids.
8. Electrophoresis analysis of plasmids.

Product:

Plasmid of gene\_*leudh* and terminator\_B1006

Purpose:

Ligation of gene\_*fdh* and terminator

Steps:

1. Double enzyme digestion of gene\_*fdh* and terminator\_B1006
2. Electrophoresis analysis of double digested result of plasmid.
3. Extract double digested gene\_*fdh* and terminator.
4. Ligate under 16℃ for 8 hours.
5. Transformation

![](https://static.igem.org/mediawiki/2015/1/18/Amoy-Notebook_node38-1.jpeg)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids.
8. Electrophoresis analysis of plasmids.

Product:

Plasmid of gene\_*fdh* and terminator\_B1006

Purpose:

Ligation of isolated circuit with RBS\_B0032 and gene\_*leudh*

Steps:

1. Double enzyme digestion of Plac\_B0032 and LeuDH\_T.
2. Electrophoresis analysis of double digested result.
3. Extract double digested products.
4. Ligate under 16℃ for 8 hours.
5. Transformation
![](https://static.igem.org/mediawiki/2015/5/57/Amoy-Notebook_node21-2.JPG)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids.
8. Electrophoresis analysis of plasmids.

Product:

Isolated circuit with RBS\_B0032 and gene\_*leudh*

Purpose:

Ligation of isolated circuit with RBS\_B0034 and gene\_*leudh*

Steps:

1. Double enzyme digestion of Plac\_B0034 and LeuDH\_TT
2. Electrophoresis analysis of double digested result.

![](https://static.igem.org/mediawiki/2015/e/e2/Amoy-Notebook_Node2_figure1.png)

3. Extract double digested products.
4. Ligate under 16℃ for 8 hours.
5. Transformation

![](https://static.igem.org/mediawiki/2015/4/43/Amoy-Notebook_node24-2.JPG)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids
8. Electrophoresis analysis of plasmids

![](https://static.igem.org/mediawiki/2015/e/e3/Amoy-Notebook_Node22_figure3.png)

Product:

Isolated circuit with RBS\_B0034 and gene\_*leudh*

Purpose:

Ligation of isolated circuit with RBS\_B0034 and gene\_*fdh*

Steps:

1. Double enzyme digestion of Plac\_B0032 and LeuDH\_T
2. Electrophoresis analysis of double digested result

![](https://static.igem.org/mediawiki/2015/e/e2/Amoy-Notebook_Node2_figure1.png)

3. Extract double digested products.
4. Ligate under 16℃ for 8 hours.
5. Transformation

![](https://static.igem.org/mediawiki/2015/3/3e/Amoy-Notebook_node23-2.JPG)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids.
8. Electrophoresis analysis of plasmids.

![](https://static.igem.org/mediawiki/2015/d/d7/Amoy-Notebook_Node23_figure3.png)

Product:

Isolated circuit with RBS\_B0034 and gene\_*fdh*

Purpose:

Ligation of isolated circuit with RBS\_B0030 and gene\_*leudh*

Steps:

1. Double enzyme digestion of PJ23100\_B0030 and LeuDH\_T
2. Electrophoresis analysis of double digested result.
3. Extract double digested products.
4. Ligate under 16℃ for 8 hours.
5. Transformation
![](https://static.igem.org/mediawiki/2015/f/f8/Amoy-Notebook_Node24_figure2.JPG)

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
8. Extract the plasmids.
9. Electrophoresis analysis of plasmids.

![](https://static.igem.org/mediawiki/2015/c/c9/Amoy-Notebook_Node24_figure3.png)

Product:

Isolated circuit with RBS\_B0030 and gene\_*leudh*

Purpose:

Ligation of promoter and RBS\_B0032

Steps:

1. Double enzyme digestion of Plac and RBS\_B0032
2. Electrophoresis analysis of double digested result of plasmid

![](https://static.igem.org/mediawiki/2015/5/50/Amoy-Notebook_Node42_figure1.png)

3. Extract double digested Plac
4. Cycle purity of digested RBS\_B0032
5. Ligate under 16℃ for 8 hours.
6. Transformation

![](https://static.igem.org/mediawiki/2015/7/7a/Amoy-Notebook_node32-2.jpeg)

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
8. Extract the plasmids.
9. Electrophoresis analysis of plasmids.

![](https://static.igem.org/mediawiki/2015/f/fa/Amoy-Notebook_Node42_figure3.png)

Product:

Plasmid of Plac linked with RBS\_B0032

Purpose:

Ligation of the final circuit with RBS\_B0032

Steps:

1. Double enzyme digestion of circuits with RBS\_B0032\_gene\_*leudh* and RBS\_B0034\_gene\_*fdh*.
2. Electrophoresis analysis of double digested result.

![](https://static.igem.org/mediawiki/2015/2/2c/Amoy-Notebook_Node11_figure1.png)

3. Extract double digested products.
4. Ligate under 16℃ for 8 hours.
5. Transformation

![](https://static.igem.org/mediawiki/2015/b/b9/Amoy-Notebook_node11-2.JPG)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids.
8. Electrophoresis analysis of plasmids.
9. Verify the results by double enzyme digestion.

![](https://static.igem.org/mediawiki/2015/8/80/Amoy-Notebook_Node11_figure3.png)

Product:

Final circuit of RBS\_B0032

Purpose:

Ligation of the final circuit with RBS\_B0034

Steps:

1. Double enzyme digestion of circuit with RBS\_B0034\_gene\_*leudh* and RBS\_B0034\_gene\_*fdh*.
2. Electrophoresis analysis of double digested result.

![](https://static.igem.org/mediawiki/2015/5/51/Amoy-Notebook_Node12_figure1.png)

3. Extract double digested products.
4. Ligate under 16℃ for 8 hours.
5. Transformation

![](https://static.igem.org/mediawiki/2015/0/01/Amoy-Notebook_node12-2.jpeg)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids.
8. Electrophoresis analysis of plasmids.

![](https://static.igem.org/mediawiki/2015/4/4e/Amoy-Notebook_Node12_figure3.png)

9. Verify the results by double enzyme digestion.

![](https://static.igem.org/mediawiki/2015/9/9f/Amoy-Notebook_Node12_figure4.png)

Product:

Final circuit of RBS\_B0034

Purpose:

Ligation of the final circuit with RBS\_B0030

Steps:

1. Double enzyme digestion of circuit with RBS\_B0030\_gene\_*leudh* and RBS\_B0034\_gene\_*fdh*.
2. Electrophoresis analysis of double digested result

![](https://static.igem.org/mediawiki/2015/a/ae/Amoy-Notebook_Node13_figure1.png)

3. Extract double digested products.
4. Ligate under 16℃ for 8 hours.
5. Transformation

![](https://static.igem.org/mediawiki/2015/1/18/Amoy-Notebook_node13-2.JPG)

6. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
7. Extract the plasmids.
8. Electrophoresis analysis of plasmids.

![](https://static.igem.org/mediawiki/2015/2/29/Amoy-Notebook_Node13_figure3.png)

9. Verify the results by double enzyme digestion.

![](https://static.igem.org/mediawiki/2015/c/c7/Amoy-Notebook_Node13_figure4.png)

Product:

Final circuit of RBS\_B0030

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μL ddH2O to solve the dry powder.
2. Suck 10μL of plasmid into 50μL of competent cell for transformation.

![](https://static.igem.org/mediawiki/2015/c/cf/Amoy-Notebook_node33-1.JPG)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm.
5. Plasmid Extraction

Product:

Plasmid of Plac\_RBS\_B0034

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20μL ddH2O to solve the dry powder.
2. Suck 10μL of plasmid into 50μL of competent cell for transformation.

![](https://static.igem.org/mediawiki/2015/1/1a/Amoy-Notebook_node34-1.JPG)

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h, 37℃, 200rpm.
5. Plasmid Extraction

Product:

Plasmid of PJ23100\_RBS\_B0030

Purpose:

Ligation of promoter and RBS

Steps:

1. Double enzyme digestion of PJ23100 and RBS\_B0034.
2. Cycle purity of digested products.
3. Ligate under 16℃ for 8 hours.
4. Transformation

![](https://static.igem.org/mediawiki/2015/1/18/Amoy-Notebook_node31-1.jpeg)

5. Pick 10 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol. Culture at 37℃,200rpm for 12~14 hours.
6. Extract the plasmids.
7. Electrophoresis analysis of plasmids.

Product:

Plasmid of PJ23100 linked with RBS\_B0034

![](https://static.igem.org/mediawiki/2015/2/26/Amoy-MenuBar-bg.jpg)

[![](https://static.igem.org/mediawiki/2015/f/fb/Title1.png)](https://2015.igem.org/Team%3AAmoy)

* [TEAM](https://2015.igem.org/Team%3AAmoy/Team)
  + [Member](https://2015.igem.org/Team%3AAmoy/Team#member_student)
  + [Amoy](https://2015.igem.org/Team%3AAmoy/Member/Amoy)
  + [Attributions](https://2015.igem.org/Team%3AAmoy/Attributions)
* [PROJECT](https://2015.igem.org/Team%3AAmoy/Project)
  + [Background](https://2015.igem.org/Team%3AAmoy/Project/Background)
  + [Description](https://2015.igem.org/Team%3AAmoy/Description)
  + [Methods](https://2015.igem.org/Team%3AAmoy/Project/Methods)
  + [Results](https://2015.igem.org/Team%3AAmoy/Project/Results)
  + [Discussion](https://2015.igem.org/Team%3AAmoy/Project/Discussion)
  + [Future Work](https://2015.igem.org/Team%3AAmoy/Project/FutureWork)
* [NOTEBOOK](https://2015.igem.org/Team%3AAmoy/Notebook)
  + [Notebook](https://2015.igem.org/Team%3AAmoy/Notebook/Notebook)
  + [Protocols](https://2015.igem.org/Team%3AAmoy/Notebook/Protocol)
  + [Parts](https://2015.igem.org/Team%3AAmoy/Parts)
  + [Gallery](https://2015.igem.org/Team%3AAmoy/Notebook/Gallery)
* [INTERLAB](https://2015.igem.org/Team%3AAmoy/Interlab)
* [NEWSLETTER](https://2015.igem.org/Team%3AAmoy/Newsletter)
  + [Introduction](https://2015.igem.org/Team%3AAmoy/Newsletter#title)
  + [Contribution](https://2015.igem.org/Team%3AAmoy/Newsletter/Contribution)
  + [Discussion](https://2015.igem.org/Team%3AAmoy/Newsletter/Discussion)
  + [Links](https://2015.igem.org/Team%3AAmoy/Newsletter/Links)
* [PRACTICES](https://2015.igem.org/Team%3AAmoy/Practices)
  + [Promotion](https://2015.igem.org/Team%3AAmoy/Practices/Promotion)
  + [Talk](https://2015.igem.org/Team%3AAmoy/Practices/Talk)
  + [Communication](https://2015.igem.org/Team%3AAmoy/Practice/Communication)
  + [Collaborations](https://2015.igem.org/Team%3AAmoy/Collaborations)
* [JUDGING](https://2015.igem.org/Team%3AAmoy/Judging)
  + [Medal Criteria](https://2015.igem.org/Team%3AAmoy/Judging/Medal)
  + [Acknowledgement](https://2015.igem.org/Team%3AAmoy/Judging/Acknowledgement)
* [SAFETY](https://2015.igem.org/Team%3AAmoy/Safety)

[#### Notebook](https://2015.igem.org/Team%3AAmoy/Notebook)

* [Notebook](https://2015.igem.org/Team%3AAmoy/Notebook/Notebook)
* [Protocols](https://2015.igem.org/Team%3AAmoy/Notebook/Protocol)
* [Parts](https://2015.igem.org/Team%3AAmoy/Parts)
* [Gallery](https://2015.igem.org/Team%3AAmoy/Notebook/Gallery)

NOTEBOOK

Initially, they used isolated enzymes, which can be disadvantageous for the reason that enzymes are always destabilized in the isolation and purification process. What's more, the cofactor NADH is rather an expensive raw material, which will enhance the cost of L-*tert*-leucine production. So scientists introduced whole-cell biocatalysts to L-*tert*-leucine production. Whole-cell biocatalysts could stabilize enzymes and reduce the addition level of cofactor NADH.
In the path of building our biobricks, we divided the circuits into two modules. One is promoter linked with RBS and the other is gene linked with terminator. The dendrogram below is our experiments detail. Click each bottom for more information.

![](https://static.igem.org/mediawiki/2015/f/f2/Amoy-Footer_amoylogo.png)

CONTACT US

**Email:** igemxmu@gmail.com

**Website:** [2015.igem.org/Team:Amoy](https://2015.igem.org/Team%3AAmoy)

**Address:** Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P.R.China 361005

[![](https://static.igem.org/mediawiki/2015/1/1f/Amoy-Footer_xmulogo.png)](http://www.xmu.edu.cn/)
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