Name Description, relevant genotype or primer/oligo sequence (5’ 3’) Source
E. coli host strains
DH5α F−, endA1, glnV44, thi-1, recA1, relA1, gyrA96, deoR, nupG φ80d lacZ∆acZd ladlacZYA – argF) U169, hsdR17(rK-mK +), λ- Lab stock
BW25113 F-, ∆(araD-araB)567, ∆lacZ4787(::rrnB-3), λ-, rph-1, ∆(rhaD-rhaB)568, hsdR514 (Datsenko and Wanner 2000)
BW∆ldhA BW25113 ldhA null mutant (Srirangan et al. 2014)
DMH BW∆ldhA/pK-hemA This study
DMH∆sdhA DMH∆sdhAiclR sdhA null mutant of DMH sdhA and iclR mutants of DMH This study This study
DMH-CT DMH/pK-hemA/pgRNA-bacteria/pdcas9-bacteria This study
DMH-L1 DMH/pK-hemA/pgRNA-L1/pdcas9-bacteria This study
DMH-L2 DMH/pK-hemA/pgRNA-L2/pdcas9-bacteria This study
DMH-L3 DMH/pK-hemA/pgRNA-L3/pdcas9-bacteria This study
DMH-L4 DMH/pK-hemA/pgRNA-L4/pdcas9-bacteria This study
DMH-L4∆sdhA sdhA null mutant of DMH-L4 This study
DMH-L4∆iclR iclR null mutant of DMH-L4 This study
DMH-L4∆sdhAiclR sdhA and iclR null mutants of DMH-L4 This study
Plasmids
pCP20 pK184 Flp+, λ cI857+, λ pR Rep(pSC101 ori)ts, ApR, CmR p15A ori, KmR, Plac::lacZ’ (Cherepanov and Wackernagel 1995) (Jobling and Holmes 1990)
pdcas9-bacteria p15A ori, PTet-dCas9 (Qi et al. 2013)
pgRNA-bacteria ColE1 origin, PJ23119-gRNA (Qi et al. 2013)
pgRNA-L1 Derived from pgRNA-bacteria, PspeI::hemB-gRNA-L1 This study
pgRNA-L2 Derived from pgRNA-bacteria, PspeI::hemB-gRNA-L2 This study
pgRNA-L3 Derived from pgRNA-bacteria, PspeI::hemB-gRNA-L3 This study
pgRNA-L4 Derived from pgRNA-bacteria, PspeI::hemB-gRNA-L4 This study
pK-hemA Derived from pTrc99a, Ptrc::phaAB This study
Primers/oligo sequences
v-ldhA
GATAACGGAGATCGGGAATGATTAA; GGTTTAAAAGCGTCGATGTCCAGTA
(Akawi et al. 2015)
v-sdhA
CTCTGCGTTCACCAAAGTGT; ACACACCTTCACGGCAGGAG
This study
v-iclR
GGTGGAATGAGATCTTGCGA; CCGACACGCTCAACCCAGAT
This study
c-frt
AGATTGCAGCATTACACGTCTTGAG; CCAGCTGCATTAATGAATCGGGCCATGGTCCATATGAATATCCTCC
(Srirangan et al. 2014)
c-ptrc cf-gRNA q-hemB q-rrsA g-hemA g-pK-hemA g-pgRNA hemA-gRNA-L1 hemA-gRNA-L2 hemA-gRNA-L3 hemA-gRNA-L4 CCGATTCATTAATGCAGCTGG; GGTCTGTTTCCTGTGTGAAATTGTTA ATCTTTGACAGCTAGCTCAGTCC; CAAGCTTCAAAAAAAGCACCGA GCGCGCTATGTTTGAAGAGA; TCGGTATGGTGAGAGATGCC TCCAGGTGTAGCGGTGAAAT; TGAGTTTTAACCTTGCGGCC CACAGGAAACAGCTATGACCATGGACTACAATCTGGCACTCGA; GAGCTCGAATTCGTAATCATTCAGGCAACGACCTCGGC GCGCCGAGGTCGTTGCCTGAATGATTACGAATTCGAGCTCGGTAC; AGTGCCAGATTGTAGTCCATGGTCATAGCTGTTTCCTGTGTG GTTTTAGAGCTAGAAATAGCAAGTT; ACTAGTATTATACCTAGGACTGAGC GTCCTAGGTATAATACTAGTCCCTCGATTCCACAAACATCGTTTTAGAGCTAGAAATAGC; GCTATTTCTAGCTCTAAAACGATGTTTGTGGAATCGAGGGACTAGTATTATACCTAGGAC GTCCTAGGTATAATACTAGTAAAGCCGTTGAAGCCATGCCGTTTTAGAGCTAGAAATAGC; GCTATTTCTAGCTCTAAAACGGCATGGCTTCAACGGCTTTACTAGTATTATACCTAGGAC GTCCTAGGTATAATACTAGTGCCTGATGTTTGTGGAATCGGTTTTAGAGCTAGAAATAGC; GCTATTTCTAGCTCTAAAACCGATTCCACAAACATCAGGCACTAGTATTATACCTAGGAC GTCCTAGGTATAATACTAGTATGCGCTTCCGGCAGCTTCAGTTTTAGAGCTAGAAATAGC GCTATTTCTAGCTCTAAAACTGAAGCTGCCGGAAGCGCATACTAGTATTATACCTAGGAC (Srirangan et al. 2014) This study This study This study This study This study This study This study This study This study This study