Team:IISER-Mohali-INDIA/Experiments

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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 :

Flow chart

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 :

Flow chart

B. Strategy for cloning construct 2 (module 1) by SOE PCR :

Flow chart

C. Strategy for cloning construct 1 (module 2) by SOE PCR :

Flow chart

D. Strategy for cloning construct 2 (module 2) by SOE PCR :

Flow chart

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
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