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Fig. 3 | Biotechnology for Biofuels

Fig. 3

From: Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955

Fig. 3

Screening of single and double-crossover mutant at tryptophan synthase beta locus. a A schematic representation of the two possible single crossover (SC) integrants, LC and RC. The former is where integration occurs at the 500 bp, left homology arm (LHA), while the latter is where integration is at the 500 bp, right homology arm (RHA). Illustrated genes are trpB tryptophan synthase beta, BCV53_15585 indole-3-glycerol phosphate synthase, BCV53_15595 tryptophan synthase alpha. The position and orientation of the four primers (1 to 4) used for PCR screening are shown above and below each represented region. b PCR screening of twelve single crossover integrants which have either integrated at the LHA (lanes 2–7) or the RHA (lanes 8–13). With the former, the amplified DNA fragment using primer 1 (FC_TRP) and primer 2 (RC_Plasmid) was 1125 bp, as opposed to 3005 bp if integration at the RHA had occurred. In the case of SC integrants where integration occurred at the RHA (lanes 8–13) the size of the DNA fragment amplified using primers 3 (FC_Plasmid) and 4 (RC_TRP) was 1171 bp as opposed to 2993 bp if integration had been at the LHA. Lane 1 is the wildtype (WT) strain, which generates no DNA fragment with any primer combination. c Screening of putative double crossover (DC) mutants using primers 1 and 4. The WT (lane 1) generates a 2202 bp DNA fragment in PCR whereas DNA template from a ΔtrpB mutant (lanes 2–7) generate a 1089 bp fragment. MW 2-log DNA marker (NEB) molecular weight marker

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