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∆sdhA∆iclR
|
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∆sdhA∆iclR
|
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
|