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| | <div style="width: 80%; margin-left: 10%;"> | | <div style="width: 80%; margin-left: 10%;"> |
| | <p class="detail_h1">Purpose:</br></p> | | <p class="detail_h1">Purpose:</br></p> |
| − | <p class="detail_p">make connection of promoter and rbs</br></p> | + | <p class="detail_p">Ligation of promoter and RBS_B0032</br></p> |
| | <p class="detail_h1">Steps:</br></p> | | <p class="detail_h1">Steps:</br></p> |
| | <p class="detail_p"> | | <p class="detail_p"> |
| − | 1. Double enzyme digestion of LacI and rbs_B0032</br> | + | 1. Double enzyme digestion of P<sub>lac</sub> and RBS_B0032</br> |
| | 2. Electrophoresis analysis of double digested result of plasmid</br></p> | | 2. Electrophoresis analysis of double digested result of plasmid</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" /> | | <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" /> |
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:
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 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:
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 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:
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
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:
Ligation of the final circuit with RBS_B0032
Steps:
1. Double enzyme digestion of circuits with RBS_B0032_gene_ldh and RBS_B0034_gene_fdh.
2. Electrophoresis analysis of double digested result.
3. Extract double digested product
4. Ligate 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:
Ligation of the final circuit with RBS_B0034
Steps:
1. Double enzyme digestion of circuit with RBS_B0034_gene_ldh and RBS_B0034_gene_fdh.
2. Electrophoresis analysis of double digested result
3. Extract double digested product
4. Ligate 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_B0034
Purpose:
Ligation of the final circuit with RBS_B0030
Steps:
1. Double enzyme digestion of circuit with RBS_B0030_gene_ldh and RBS_B0034_gene_fdh.
2. Electrophoresis analysis of double digested result
3. Extract double digested product
4. Ligate 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_B0030
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
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.
