Construction of a novel zero background prokaryotic expression vector: potential advantages.
A novel DNA sequence, derived from the antisense strand of the DNA gyrase inhibitor protein, CcdB, was toxic to E. coli. This protein (approximately 6 kDa) decreased the growth rate of E. coli K12 by three orders of magnitude upon induction. The expressed toxic protein in E. coli K12 was soluble while it was insoluble in induced E. coli BL21. A high efficiency prokaryotic cloning/expression vector was constructed using this toxic gene sequence and gave zero background with approximately 100% cloning efficiency requiring no dephosphorylation. The toxic gene product also affected the survival of a ccdB resistant cell line, thus indicating a different mechanism of toxicity, other than DNA gyrase inhibition, as compared to the ccdB toxicity.
Mandi
Naganath
N
Lupin Limited, Biotechnology R & D, Gat # 1156, Ghotawade Village, Mulshi Taluka, Pune, 411042, India.
Kotwal
Prakash
P
Padmanabhan
Sriram
S
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D003001:Cloning, Molecular; D016373:DNA, Antisense / Q000235:genetics; D048168:Escherichia coli K12 / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D026901:Membrane Transport Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000633:toxicity*; D012641:Selection, Genetic; D014169:Transformation, Bacterial
D001425:Bacterial Outer Membrane Proteins; D016373:DNA, Antisense; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C075818:codB protein, E coli