Difference between revisions of "Team:Amoy/Notebook"

@@ Line 647: / Line 647: @@
 <p class="detail_h1">Steps:</br></p>
 <p class="detail_p">
-. Double enzyme digestion of circuit with RBS_B0032 and gene_<i>ldh</i> and gene_<i>fdh</i> circuit.</br>
+. Double enzyme digestion of circuit with RBS_B0032_gene_<i>ldh</i> and RBS_B0034_gene_<i>fdh</i> circuit.</br>
 . Electrophoresis analysis of double digested result</br></p>
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/1/1f/Amoy-Notebook_Node32_figure1.jpg" />
 <p class="detail_p"></br>
 . Extract double digested product</br>
-. Ligate under 16℃ for 8 hours</br>
+. Ligation under 16℃ for 8 hours</br>
 . Transformation</br></p>
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/8/87/Amoy-Notebook_Node41_figure1.jpg" />
 <p class="detail_p"></br>
-. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol</br>
+. 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.</br>
 . Extract the plasmids</br>
 . Electrophoresis analysis of plasmids</br>
@@ Line 662: / Line 662: @@
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://2015.igem.org/File:Amoy-Notebook_Node11_figure3.jpg" />
 <p class="detail_h1">Product:</br></p>
-<p class="detail_p">final circuit of RBS_B0032</br></p>
+<p class="detail_p">final circuit of RBS_B0032</br></br></br></p>
 </div>
 </div>
@@ Line 671: / Line 671: @@
 <div style="width: 80%; margin-left: 10%;">
 <p class="detail_h1">Purpose:</br></p>
-<p class="detail_p">make connection of the final circuit with RBS_B0032</br></p>
+<p class="detail_p">make connection of the final circuit with RBS_B0034</br></p>
 <p class="detail_h1">Steps:</br></p>
 <p class="detail_p">
-. Double enzyme digestion of circuit with RBS_B0032 and gene_ldh and gene_fdh circuit.</br>
+. Double enzyme digestion of circuit with RBS_B0034_gene_<i>ldh</i> and RBS_B0034_gene_<i>fdh</i> circuit.</br>
 . Electrophoresis analysis of double digested result</br></p>
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/1/1f/Amoy-Notebook_Node32_figure1.jpg" />
@@ Line 683: / Line 683: @@
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/8/87/Amoy-Notebook_Node41_figure1.jpg" />
 <p class="detail_p"></br>
-. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol</br>
+. 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.</br>
 . Extract the plasmids</br>
 . Electrophoresis analysis of plasmids</br></p>
@@ Line 691: / Line 691: @@
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/c/c9/Amoy-Notebook_Node32_figure3.jpg" />
 <p class="detail_h1">Product:</br></p>
-<p class="detail_p">final circuit of RBS_B0032</br></p>
+<p class="detail_p">final circuit of RBS_B0034</br></br></br></p>
 </div>
 </div>
@@ Line 700: / Line 700: @@
 <div style="width: 80%; margin-left: 10%;">
 <p class="detail_h1">Purpose:</br></p>
-<p class="detail_p">make connection of the final circuit with RBS_B0032</br></p>
+<p class="detail_p">make connection of the final circuit with RBS_B0030</br></p>
 <p class="detail_h1">Steps:</br></p>
 <p class="detail_p">
-. Double enzyme digestion of circuit with RBS_B0032 and gene_ldh and gene_fdh circuit.</br>
+. Double enzyme digestion of circuit with RBS_B0030_gene_<i>ldh</i> and RBS_B0034_gene_<i>fdh</i> circuit.</br>
 . Electrophoresis analysis of double digested result</br></p>
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/1/1f/Amoy-Notebook_Node32_figure1.jpg" />
@@ Line 712: / Line 712: @@
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/8/87/Amoy-Notebook_Node41_figure1.jpg" />
 <p class="detail_p"></br>
-. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol</br>
+. 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.</br>
 . Extract the plasmids</br>
 . Electrophoresis analysis of plasmids</br></p>
@@ Line 720: / Line 720: @@
 <img style="width: 30%; margin-right: 70%; margin-top: 10px; margin-bottom: 0px;" src="https://static.igem.org/mediawiki/2015/c/c9/Amoy-Notebook_Node32_figure3.jpg" />
 <p class="detail_h1">Product:</br></p>
-<p class="detail_p">final circuit of RBS_B0032</br></p>
+<p class="detail_p">final circuit of RBS_B0030</br></br></br></p>
 </div>
 </div>

Revision as of 10:01, 13 September 2015

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

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 promote_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

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

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

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

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

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 LacI_RBS_B0034

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

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_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

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

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 ldh and double enzyme digestion

Steps:

1. pcr amplification system for gene ldh

Components	Volume
ddH20
LDH-F
LDH-R
Pub18-LDH
primestar

2. electrophoresis of PCR products of gene ldh
3. Double enzyme digestion of gene ldh

item	volume

ddH2O
buffer
ErcoI
SpeI

4. Cycle purity of digested gene ldh

Product:

Purpose:

pcr amplification for gene fdh and double enzyme digestion

Steps:

1. pcr amplification system for gene fdh

Components	Volume
ddH20
FDH-F
FDH-R
Pub18-FDH
primestar

2. electrophoresis of PCR products of gene fdh
3. Double enzyme digestion of gene fdh

item	volume

ddH2O
buffer
ErcoI
SpeI

4. Cycle purity of digested gene fdh

Product:

Purpose:

Make connection of gene and terminator

Steps:

1. Double enzyme digestion of gene and terminator_B0015
2. Electrophoresis analysis of double digested result of plasmid
3. Extract double digested gene
4. Cycle purity of digested terminator
5. Link gene with terminator under 16℃ for 8 hours
6. Transformation

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

plasmid of gene ldh and terminator

Purpose:

make connection of gene and terminator

Steps:

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

plasmid of gene fdh and terminator

Purpose:

make connection of gene and terminator

Steps:

1. Double enzyme digestion of gene and terminator_B1006
2. Electrophoresis analysis of double digested result of plasmid
3. Extract double digested gene
4. Cycle purity of digested terminator
5. Link gene with terminator under 16℃ for 8 hours
6. Transformation

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

plasmid of gene ldh and terminator

Purpose:

make connection of gene and terminator

Steps:

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

plasmid of gene and terminator

Purpose:

make connection of isolated circuit with RBS_B0032 and gene_ldh

Steps:

1. Double enzyme digestion of LacI_B0032 and LeuDH_T
2. Electrophoresis analysis of double digested result

3. Extract double digested product
4. Link gene with terminator under 16℃ for 8 hours
5. Link gene with terminator under 16℃ for 8 hours
6. Transformation

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

isolated circuit with RBS_B0032 and gene_ldh

Purpose:

make connection of isolated circuit with RBS_B0032 and gene_ldh

Steps:

1. Double enzyme digestion of LacI_B0032 and LeuDH_T
2. Electrophoresis analysis of double digested result

3. Extract double digested product
4. Link gene with terminator under 16℃ for 8 hours
5. Link gene with terminator under 16℃ for 8 hours
6. Transformation

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

isolated circuit with RBS_B0032 and gene_ldh

Purpose:

make connection of isolated circuit with RBS_B0032 and gene_ldh

Steps:

1. Double enzyme digestion of LacI_B0032 and LeuDH_T
2. Electrophoresis analysis of double digested result

3. Extract double digested product
4. Link gene with terminator under 16℃ for 8 hours
5. Link gene with terminator under 16℃ for 8 hours
6. Transformation

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

isolated circuit with RBS_B0032 and gene_ldh

Purpose:

make connection of isolated circuit with RBS_B0032 and gene_ldh

Steps:

1. Double enzyme digestion of LacI_B0032 and LeuDH_T
2. Electrophoresis analysis of double digested result

3. Extract double digested product
4. Link gene with terminator under 16℃ for 8 hours
5. Link gene with terminator under 16℃ for 8 hours
6. Transformation

7. Pick 8 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of chloramphenicol
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

isolated circuit with RBS_B0032 and gene_ldh

Purpose:

make connection of promoter and rbs

Steps:

1. Double enzyme digestion of LacI and rbs_B0032
2. Electrophoresis analysis of double digested result of plasmid

3. Extract double digested promoter LacI
4. Cycle purity of digested rbs_B0032
5. Link promoter with rbs under 16℃ for 8 hours
6. transformation

7. Pick 10 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of amp
8. Extract the plasmids
9. Electrophoresis analysis of plasmids

Product:

Plasmid of LacI linked with RBS_B0032

Purpose:

make connection of the final circuit with RBS_B0032

Steps:

1. Double enzyme digestion of circuit with RBS_B0032_gene_ldh and RBS_B0034_gene_fdh circuit.
2. Electrophoresis analysis of double digested result

3. Extract double digested product
4. Ligation under 16℃ for 8 hours
5. Transformation

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

Product:

final circuit of RBS_B0032

Purpose:

make connection of the final circuit with RBS_B0034

Steps:

1. Double enzyme digestion of circuit with RBS_B0034_gene_ldh and RBS_B0034_gene_fdh circuit.
2. Electrophoresis analysis of double digested result

3. Extract double digested product
4. Ligation under 16℃ for 8 hours
5. Transformation

9. Verify the results by double enzyme digestion

Product:

final circuit of RBS_B0034

Purpose:

make connection of the final circuit with RBS_B0030

Steps:

1. Double enzyme digestion of circuit with RBS_B0030_gene_ldh and RBS_B0034_gene_fdh circuit.
2. Electrophoresis analysis of double digested result

3. Extract double digested product
4. Ligation under 16℃ for 8 hours
5. Transformation

9. Verify the results by double enzyme digestion

Product:

final circuit of RBS_B0030

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20ul ddH20 to solve the dry powder
2. Suck 10ul of plasmid into 50ul of competent cell for transformation

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h
5. Plasmid minipre

Product:

Purpose:

Extract plasmid from dry powder

Steps:

1. Add 20ul ddH20 to solve the dry powde
2. Suck 10ul of plasmid into 50ul of competent cell for transformation

3. Pick a single colony from the agar plate using a sterile pipette tip.
4. Culture at 10ml LB of chloramphenicol for 12h
5. Plasmid minipre

Product:

Purpose:

Make connection of promoter and rbs

Steps:

1. Double enzyme digestion of promoter_J23100 and RBS_B0034
2. Cycle purity of digested products
3. Link promoter with rbs under 16℃ for 8 hours
4. Transformation

5. Pick 10 single colonies from the agar plate using sterile pipette tips. Put it into 10ml LB of amp
6. Extract the plasmids
7. Electrophoresis analysis of plasmids

Product:

Plasmid of promoter J23100 linked with RBS_B0034

Team
- Member
- Amoy
- Attributions
Project
- Description
- Background
- Methods
- Results
- Discussion
- Future Work
Notebook
- Notebook
- Protocols
- Parts
- Gallery
Interlab
Newsletter
Practices
Judging
- Medal Criteria
- Acknowledgement
Safety

Notebook

Notebook
Protocols
Parts
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.

Email: igemxmu@gmail.com

Website: 2015.igem.org/Team:Amoy

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