Team:Amoy/Notebook
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 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
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 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.
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
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_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
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
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
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
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
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.
3. Extract double digested products. 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
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
3. Extract double digested products. 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.
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
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:
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
3. Extract double digested Plac 4. Cycle purity of digested RBS_B0032 5. Ligate 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. Culture at 37℃,200rpm for 12~14 hours. 8. Extract the plasmids. 9. Electrophoresis analysis of plasmids.
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.
3. Extract double digested products. 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_leudh and RBS_B0034_gene_fdh. 2. Electrophoresis analysis of double digested result.
3. Extract double digested products. 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_leudh and RBS_B0034_gene_fdh. 2. Electrophoresis analysis of double digested result
3. Extract double digested products. 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:
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 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.
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
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
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.
CONTACT US
Email: igemxmu@gmail.com
Website: 2015.igem.org/Team:Amoy
Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P.R.China 361005
