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:
plasmid of promoter J23100
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
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.
