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Fig. 2 | Microbial Cell Factories

Fig. 2

From: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering

Fig. 2

Deletion of upp gene from Rhodobacter sphaeroides genome. a Number of colonies obtained on RÄ agar plates plated with 10−3 dilutions of R. sphaeroides conjugation mixtures with the homologous recombination (HR) control vectors pBBR_Cas9_Δupp500HR_NT and pBBR_Cas9_Δupp1000HR_NT, and HR editing vectors pBBR_Cas9_Δupp500HR_sp2 and pBBR_Cas9_Δupp1000HR_sp2. The error bars represent standard deviations from three replicate experiments. b Restreaks of randomly selected colonies from the above mentioned conjugations on RÄ agar plates supplemented with 5-FU. Only Δupp mutants can grow on 5-FU plates. c Genome specific colony PCR amplification of the upp locus in cells conjugated with the pBBR_Cas9_Δupp500HR_NT, pBBR_Cas9_Δupp1000HR_NT, pBBR_Cas9_Δupp500HR_sp2 and pBBR_Cas9_Δupp1000HR_sp2 vectors. Amplification yields a 2992 bp product for the wild type upp gene and a 2374 bp product for the deleted upp gene. d Sequence verification of the desired upp deletion by Sanger sequencing

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