Molecular Cloning

From 2008.igem.org

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== Fragment Preparation ==
== Fragment Preparation ==
-
SRRz: PCR with primer SRRZ-NcoI-for and SRRZ-rev-in;
+
'''SRRz:''' PCR with primer SRRZ-NcoI-for and SRRZ-rev-in;
 
 
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Purify the product and run PCR with primer SRRZ-NcoI-for and primer SRRZ-BamHI-rev-out;
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;
+
'''EGFP:''' PCR with primer EGFP-for-KoZg-for and EGFP-rev-in;
 
 
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Purify the product and run PCR with primer EGFP-for-KoZg-for and primer EGFP-rev-out;
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;
+
'''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;
+
'''LacI(LVA):''' PCR with primer LacILVA-HindIII-for and LacILVA-rev-in;
 
 
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Purify the product and run PCR with primer LacILVA-HindIII-for and primer LacILVA-rev-PstI-out;
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
+
'''CRE(T7ter):''' PCR with primer CreT7ter—PstI-for and CreT7ter-rev-in
 
 
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Purify the product and run PCR with primer CreT7ter—PstI-for and  CreT7ter-EcoRI-rev-out;
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;
+
'''PrPhaR:''' PCR with primer PR-PhaR-NotI-for and PR-PhaR-NdeI-rev;
-
LoxPLacILoxP: PCR with primer LRLacILP-for-in and LRLacILP-rev-in
+
'''LoxPLacILoxP:''' PCR with primer LRLacILP-for-in and LRLacILP-rev-in
 
 
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== 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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