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Molecular Cloning
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| Line 43: | Line 43: | ||
SRRZ-NcoI-for | SRRZ-NcoI-for | ||
| + | | ||
<span style="color: Green">5- ATCCATGGATGAAGATGCCAGAAAAACATGACCTGTTG-3</span> | <span style="color: Green">5- ATCCATGGATGAAGATGCCAGAAAAACATGACCTGTTG-3</span> | ||
SRRZ-rev-in | SRRZ-rev-in | ||
| - | 5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Orange">5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3</span> | ||
SRRZ-BamHI-rev-out | SRRZ-BamHI-rev-out | ||
| - | 5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: red">5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3</span> | ||
'''(2) Pp Operon primers''' | '''(2) Pp Operon primers''' | ||
| Line 57: | Line 64: | ||
PP-PhaP-NotI-for | PP-PhaP-NotI-for | ||
| - | 5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Brown">5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3</span> | ||
PP-PhaP-HindIII-rev | PP-PhaP-HindIII-rev | ||
| - | 5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Purple">5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3</span> | ||
LacILVA-HindIII-for | LacILVA-HindIII-for | ||
| - | 5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Yellow">5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3</span> | ||
LacILVA-rev-in | LacILVA-rev-in | ||
| - | 5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Green">5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3</span> | ||
LacILVA-rev-PstI-out | LacILVA-rev-PstI-out | ||
| - | 5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Brown">5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3</span> | ||
IGEM-ZouYLP-CreT7ter—PstI-for | IGEM-ZouYLP-CreT7ter—PstI-for | ||
| - | 5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Red">5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3</span> | ||
IGEM-ZouYLP-CreT7ter-rev-in | IGEM-ZouYLP-CreT7ter-rev-in | ||
| - | 5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Brown">5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3</span> | ||
IGEM-ZouYLP-CreT7ter-EcoRI-rev-out | IGEM-ZouYLP-CreT7ter-EcoRI-rev-out | ||
| - | 5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Yellow">5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3</span> | ||
'''(3) Pr operon primers''' | '''(3) Pr operon primers''' | ||
| Line 91: | Line 122: | ||
PR-PhaR-NotI-for | PR-PhaR-NotI-for | ||
| - | 5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Blue">5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3</span> | ||
PR-PhaR-NdeI-rev | PR-PhaR-NdeI-rev | ||
| - | 5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: red">5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3</span> | ||
LRLacILP-for-in | LRLacILP-for-in | ||
| - | 5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Yellow">5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3</span> | ||
LRLacILP-NdeI-for-out | LRLacILP-NdeI-for-out | ||
| - | 5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Green">5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3</span> | ||
LRLacILP-rev-in | LRLacILP-rev-in | ||
| - | 5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Orange">5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3</span> | ||
LRLacILP-KpnI-rev-out | LRLacILP-KpnI-rev-out | ||
| - | 5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Brown">5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3</span> | ||
'''(4) EGFP Primers''' | '''(4) EGFP Primers''' | ||
| Line 117: | Line 166: | ||
EGFP-for-KoZg-for | EGFP-for-KoZg-for | ||
| - | 5-ccatgggcagcaagggcgaggagctgttc-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: red">5-ccatgggcagcaagggcgaggagctgttc-3</span> | ||
EGFP-rev-in | EGFP-rev-in | ||
| - | 5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Green">5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3</span> | ||
EGFP-rev-out | EGFP-rev-out | ||
| - | 5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3 | + | |
| + | | ||
| + | | ||
| + | <span style="color: Purple">5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3 | ||
| + | </span> | ||
| - | == | + | == 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== | == 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 == | == Cut and Insert == | ||
| + | The restriction enzymes are chosen as following: | ||
| + | |||
| + | {| border="1" cellpadding="20" cellspacing="0" | ||
| + | !align="right" |Fragment | ||
| + | |Enzyme A | ||
| + | |Enzyme B | ||
| + | |- | ||
| + | |||
| + | !align="right" |SRRz/EGFP | ||
| + | |NcoI | ||
| + | |BamHI | ||
| + | |- | ||
| + | |||
| + | !align="right" |PpPhaP | ||
| + | |NotI | ||
| + | |HindIII | ||
| + | |- | ||
| + | |||
| + | !align="right" |LacI(LVA)Cre(T7ter) | ||
| + | |HindIII | ||
| + | |SacI | ||
| + | |- | ||
| + | |||
| + | !align="right" |PrPhaRLoxPLacILoxP | ||
| + | |NotI | ||
| + | |KpnI | ||
| + | |- | ||
| + | |} | ||
| + | |||
| + | |||
| + | |||
Latest revision as of 14:14, 29 October 2008
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Molecular Cloning
Contents |
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:
| Fragment | Enzyme A | Enzyme B |
|---|---|---|
| SRRz/EGFP | NcoI | BamHI |
| PpPhaP | NotI | HindIII |
| LacI(LVA)Cre(T7ter) | HindIII | SacI |
| PrPhaRLoxPLacILoxP | NotI | KpnI |
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