Increased Hydrogen Production by Genetic Engineering of Escherichia coli - Genetic Engineering: Recent Developments in Applications - page 11

and ppc genes, respectively. The entire coding sequences of the hybC, narL, and
ppc genes were deleted, while only 589 nucleotides at the 5 ′ terminal sequence
of the focA gene were deleted since the sequence downstream of it overlaps the
pfl operon. The gene disruptions were verified by PCR analyses of genomic DNA
using the primers k1 and k2 to the Km R gene, and the FW and RV primers to the
flanking sequences of the disrupted genes.
Table 5. E. coli strains and plasmids used in this study.
The plasmid pACYC177 (Invitrogen) was used for the multi-copy expression
of the E. coli genes fnr, arcAB, ihfAB, modE, and selC. The genes were amplified
by PCR, using Pfx50™ DNA polymerase (Invitrogen) to produce blunt ended
PCR products, from genomic DNA of E. coli strain W3110. For the construction
of pfnr, pmodE and pselC, the lacZ promoter sequence from the pGEM®-T easy
vector (Promega) was transferred into pAYCY177 by amplification using primers
Plac-BamHI-FW and Plac-PstI-RV, digestion with BamHI and PstI, and ligation
into the corresponding sites of pACYC177. The modified pACYC177 vector was
designated pACYC177L. The fnr, modE, and selC genes were amplified with
primer pairs: fnr-FW/fnr-RV, modE-FW/modE-RV and selC-FW/selC-RV, re-
spectively, digested with PstI and FspI, and ligated into the corresponding sites of
pACYC177L. For the construction of parcAB and pihfAB, primer Plac-ScaI-RV