Cloning Strategies
Cloning of Module 1 in pZS21MCS:
Construct 1 --> Ptet - RBS1 - T7 RNA polymerase - terminator 1 - terminator 2.
Construct 2 --> PT7 - RBS2 - Chromoprotein II - RBS3 - tetR - terminator3.
Different parts like Ptet, PT7, RBS, T7 RNA polymerase, Chromoprotein II, tetR and terminator parts are taken from BioBricks library and are amplified by PCR. Different amplified PCR products are fused together through splice overlap extension PCR.
Cloning of Module 2 in pACYC177 :
Construct 1 --> Pmar - RBS4 - ToxR - terminator 4.
Construct 2 --> Pctx - RBS5 - chromoprotein I - RBS6 - TetR - terminator5.
Different parts like ToxR and chromoprotein I are PCR amplified from BioBricks library. Other parts like - Pmar is amplified from E. coli genome (BW25113), and terminator 4 is cloned from plasmid (modified pAH125). Parts are amplified by PCR and different parts are fused together by splice overlap extension PCR.
List of part numbers and primers are given in Table 1 and 2
General Cloning strategy :
Splice Overlap Extension PCR (SOE PCR):
It is a variant of PCR and used to join the PCR fragments and insert mutations. To join fragments, special primers are to be used at the ends to be joined. Primers are designed in such a way that it has 5’ overhang complementary to the end of the other molecule. Therefore,after annealing, during the replication the DNA is extended by a new sequence that is complementary to the molecule to be joined. Once both DNA molecules are extended in such manner, they are mixed and PCR is carried out with only the primers from ends.
A. Strategy for cloning construct 1 (module 1) by SOE PCR :
B. Strategy for cloning construct 2 (module 1) by SOE PCR :
C. Strategy for cloning construct 1 (module 2) by SOE PCR :
D. Strategy for cloning construct 2 (module 2) by SOE PCR :
Table 1: List of parts used from BioBricks library :
| Sr. No. | Name | Part no. |
|---|---|---|
| 1. | Ptet | R0040 |
| 2. | RBS1 | K1450004 |
| 3. | T7 RNA polymerase | K1450004 |
| 4. | Terminator 1 | K1450004 |
| 5. | Terminator 2 | K1450004 |
| 6. | PT7 | K1343022 |
| 7. | RBS2 | K1343022 |
| 8. | Chromoprotein II | K1033910 |
| 9. | RBS3 | K1343022 |
| 10. | tetR | K1343022 |
| 11. | Terminator 3 | K1343022 |
| 12. | ToxR | K641009 |
| 13. | RBS5 | K1343022 |
| 14. | RBS6 | K1343022 |
| 15. | Chromoprotein I | K1343022 |
| 16. | TetR | K1343022 |
| 17. | Terminator 4 | K1343022 |
Table 2. List of primers used for cloning circuit parts :
| Sr. No. | Primer ID | Sequence (5'-3') |
|---|---|---|
| 1. | P1 | ACCTGACGTCTCCCTATCAGTGATAGAGATTGAC |
| 2. | P2 | GTATTTCTCCTCTTTCTCTAGTAGTGCTCAGTATCTCTATCA |
| 3. | P3 | TGATAGAGATACTGAGCACTACTAGAGAAAGAGGAGAAATAC |
| 4. | P4 | ACCTGGGCCCTATAAACGCAGAAAGGCCCACCCG |
| 5. | P5 | ACCTGTCGACTAATACGACTCACTATAGGGAGACTCGAGGAGATCTGTTTAAAC |
| 6. | P6 | GCGCCTTCAGTCAGTGCCGTCATTGGTGGTTTCTCCTCTTTAATGG |
| 7. | P7 | CCATTAAAGAGGAGAAACCACCAATGACGGCACTGACTGAAGGCGC |
| 8. | P8 | CCTTCAAATTAACAGGAATCGGTTATTAACGGTACGTCTCCAGGTC |
| 9. | P9 | GACCTGGAGACGTACCGTTAATAACCGATTCCTGTTAATTTGAAGG |
| 10. | P10 | ACCTGGATCCCAAAAAACCCCTCAAGACCCGTTTA |
| 11. | P11 | ATGCGCTCGAGATCGATCATCCGAAAACCGATAAACGCGACG |
| 12. | P12 | GAGTTGTGTCCTAATCCGAACACATTAGTTGCCCTGGCAAGTAATTAG |
| 13. | P13 | CTAATTACTTGCCAGGGCAACTAATGTGTTCGGATTAGGACACAACTC |
| 14. | P14 | CAAGCTCAGCTAATTAAGCTTTTAAGCTACTAAAGCGTAGTTTTCGTCG |
| 15. | P15 | CGACGAAAACTACGCTTTAGTAGCTTAAAAGCTTAATTAGCTGAGCTTG |
| 16. | P16 | K1343022GTCGGCCCGGGGTAACAGAGCATTAGCGCAAGGTGATTTTTGTCTTC |
| 17. | P17 | AGTCGGGATCCAACAGAAAATGATAAAAAAGGAC |
| 18. | P18 | GGGTTTAAACAGATCTCCTCGAGAGAAAAATAATTGATCAAAAC |
| 19. | P19 | GTTTTGATCAATTATTTTTCTCTCGAGGAGATCTGTTTAAACCC |
| 20. | P20 | ATGCGGACGTCCAAAAAACCCCTCAAGACCCG |
