Molecular Cloning

Molecular Cloning

Plasmids
1 pUCPhbCAB 2 pACYC(lacI+) DuetSRRz 3 pACYC(lacI+) DuetEGFP 4 pACYC(lacI-) DuetSRRz 5 pACYC(lacI-) DuetEGFP 6 pACYC(LacI-)DuetSRRzPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP 7 pACYC(LacI-)DuetEGFPPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP

Template Resources
PhbCAB: PBHR68 Plasmid Lac I: PET28a CRE: PpPhaP: Ralstonia eutropha H16 genome PrPhaR: Ralstonia eutropha H16 genome SRRz: EGFP:

Primers
(1)	SRRz Primers

SRRZ-NcoI-for

5- ATCCATGGATGAAGATGCCAGAAAAACATGACCTGTTG-3

SRRZ-rev-in

5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3

SRRZ-BamHI-rev-out

5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3

(2) Pp Operon primers

PP-PhaP-NotI-for

5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3

PP-PhaP-HindIII-rev

5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3

LacILVA-HindIII-for

5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3

LacILVA-rev-in

5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3

LacILVA-rev-PstI-out

5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3

IGEM-ZouYLP-CreT7ter—PstI-for

5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3

IGEM-ZouYLP-CreT7ter-rev-in

5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3

IGEM-ZouYLP-CreT7ter-EcoRI-rev-out

5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3

(3) Pr operon primers

PR-PhaR-NotI-for

5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3

PR-PhaR-NdeI-rev

5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3

LRLacILP-for-in

5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3

LRLacILP-NdeI-for-out

5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3

LRLacILP-rev-in

5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3

LRLacILP-KpnI-rev-out

5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3

(4) EGFP Primers

EGFP-for-KoZg-for

5-ccatgggcagcaagggcgaggagctgttc-3

EGFP-rev-in

5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3

EGFP-rev-out

5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3

Fragment Preparation
SRRz: PCR with primer SRRZ-NcoI-for and SRRZ-rev-in;

Purify the product and run PCR with primer SRRZ-NcoI-for and primer SRRZ-BamHI-rev-out;

EGFP: PCR with primer EGFP-for-KoZg-for and EGFP-rev-in;

Purify the product and run PCR with primer EGFP-for-KoZg-for and primer EGFP-rev-out;

PpPhaP: PCR with primer PP-PhaP-NotI-for and PP-PhaP-HindIII-rev; LacI(LVA): PCR with primer LacILVA-HindIII-for and LacILVA-rev-in;

Purify the product and run PCR with primer LacILVA-HindIII-for and primer LacILVA-rev-PstI-out;

CRE(T7ter): PCR with primer CreT7ter—PstI-for and CreT7ter-rev-in

Purify the product and run PCR with primer CreT7ter—PstI-for and CreT7ter-EcoRI-rev-out;

PrPhaR: PCR with primer PR-PhaR-NotI-for and PR-PhaR-NdeI-rev;

LoxPLacILoxP: PCR with primer LRLacILP-for-in and LRLacILP-rev-in

Purify the product and run PCR with primer LRLacILP-NdeI-for-out and primer LRLacILP-KpnI-rev-out.

Fusion PCR
(1) Fuse LacI(LVA) and CRE(T7ter) together and amplify with the primer LacILVA-HindIII-for and CreT7ter-EcoRI-rev-out;

(2) Fuse PrPhaR and LoxPLacILoxP together and amplify with the Primer PR-PhaR-NotI-for and LRLacILP-KpnI-rev-out. We’ve tried to fuse more by PCR, but failed.

Vector preparation
We choose pACYCDuet-1 as our second vector while PhbCAB operon is cloned into PUC18.

Since lacI is one of the elements we also used in our own system, we knocked out the lacI coding region in pACYCDuet-1 successfully.

Cut and Insert
The restriction enzymes are chosen as following: