Modified bacteria for the production of bioalcohol

Materials and Methods

Bacterial Strains, Plasmids, Primers, Enzymes and Methods:

[0120]List of bacterial strains, plasmids and primers used in the study has been provided in Table 1. E. coli DH5α strain (Invitrogen) was used for performing all the cloning work and E. coli B (Coli Genetic Stock Centre (CGSC), Yale University, USA) was used as parent strain for all the genomic manipulations. Restriction endonuclease and T4 DNA ligase were procured from New England Biolabs. Custom oligonucleotides (primers) were synthesized from Sigma-Aldrich for PCR amplifications. Taq DNA polymerase (Bangalore Genei) was used for performing verification PCR of the engineered strains. Plasmids pKD4, pKD46 and pCP20 (CGSC, USA) were used as the source of FRT-kan-FRT fragment, lambda Red recombinase and flippase, respectively, for performing genetic manipulation. Recombinant DNA techniques were performed according to standard procedures (Sambrook et al., 1989). DNA purification was performed using Qiagen kit