The plasmid pBL was constructed by insertion of a 70-bp chemically synthesized multiple cloning site into the 2.5-kb PCR-generated plasmid backbone of pBluescript II KS(+) (Stratagene) and deletion of the LacZα ORF by conventional cloning. The plasmid pBL-DL was constructed by insertion of a 1-kb PCR fragment from pGEM®-luc (Promega) into the NotI/SalI sites of pBL by SLiCE. The suicide plasmid pGT1 was constructed by SLiCE-mediated insertion of a 830-bp PCR-amplified fragment spanning the 3′ region of the E. coli DH10B cynX gene and an araC-pBAD-redα/EM7-redβ/Tn5-gam expression cassette isolated from plasmid pBAD24 (8 (link)) and lambda phage DNA (NEB) into the SmaI site of plasmid pEL04 (9 (link)). pGT1 also contains a temperature-sensitive replicon and a chloramphenicol selection marker.
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Versatile Plasmid Construction via SLiCE
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Arac
Backbone
Chloramphenicol
Deletion
E coli
Gene
Ks 5 2
Lambda phage
Pgem
Plasmid
Promega
Replicon
Smai
Corresponding Organization :
Other organizations : Albert Einstein College of Medicine
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Variable analysis
independent variables
- Insertion of a 70-bp chemically synthesized multiple cloning site into the 2.5-kb PCR-generated plasmid backbone of pBluescript II KS(+)
- Deletion of the LacZα ORF from pBluescript II KS(+)
- Insertion of a 1-kb PCR fragment from pGEM®-luc into the NotI/SalI sites of pBL
- SLiCE-mediated insertion of a 830-bp PCR-amplified fragment spanning the 3′ region of the E. coli DH10B cynX gene and an araC-pBAD-redα/EM7-redβ/Tn5-gam expression cassette isolated from plasmid pBAD24 and lambda phage DNA into the SmaI site of plasmid pEL04
- Construction of plasmids pBL, pBL-DL, and pGT1
- Temperature-sensitive replicon in pGT1
- Chloramphenicol selection marker in pGT1
- No positive or negative controls were explicitly mentioned.
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