Development of a novel type of cloning vector for suicide selection of recombinants.
Based on the primary structure of the rat corticostatin R4 (antimicrobial defensin RatNP-1), a synthetic gene was designed, synthesized, and inserted into the IPTG-inducible prokaryotic expression vector pFLAG, with an additional 13 codons between the FLAG sequence and the synthetic R4 sequence. This construct, N-p1.2, was further developed by inclusion of multiple cloning sites right after the FLAG sequence, forming a new plasmid pSCV-1. Escherichia coli transformants containing pSCV-1 or N-p1.2 could only be propagated on agar plates in the absence of IPTG due to the detrimental expression of R4 fusion peptide to the growth of bacteria upon IPTG induction. A 214-bp bovine IGF-II cDNA and a 700-bp Ly-6C.2 cDNA fragment were subcloned into pSCV-1 and N-p1.2 respectively. Only the E. coli cells transformed with recombinant plasmids grew on IPTG agar plates. This "suicide" selection against nonrecombinants was further tested in cDNA library construction using pSCV-1. Analysis of plasmid DNA prepared from randomly picked colonies growing on ampicillin agar plates containing IPTG showed all plasmids contained cDNA inserts. The lambda Hind III fragments were used for comparing the cloning efficiency of pSCV-1 to pBluescript. Four of the 60 (6.6%) analyzed white colonies transformed with pBluescript were false positives. All of the analyzed pSCV-1-transformed colonies growing on IPTG plates contained recombinant forms of plasmid. The percentage recovery of each ligatable lambda Hind III fragment was similar in both pBluescript and pSCV-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Chan
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RY
Endocrine Laboratory, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Palfree
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RG
Congote
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LF
Solomon
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S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004926:Escherichia coli; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000302:isolation & purification; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011995:Recombination, Genetic; D015183:Restriction Mapping
D017931:DNA Primers; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; C054598:corticostatin; D007335:Insulin-Like Growth Factor II