High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties

Table 3 Primers, plasmids, and strains used in this work

Primer name	Sequence	Reference
SB122_F	CTCTAGATGAATTCTATATCGCCGACATCACC	This work
SB122_R	CTCTAGAAGCTGCAGGCGTTTAAGGGCACCAATAA	This work
SB123_F	GAATTCGCTTCTAGAGCGATATAGGCGCCAGCAAC	This work
SB123_R	CTGCAGAGCTACTAGTGGGGAATTGTTATC	This work
fadD_F	CATTTGGGGTTGCGATGACGACGAACACGCATTTTAGAGGTGAAGAATTGTGTAGGCTGGAGCTGCTTCG	This work
fadD_R	TAACCGGCGTCTGACGACTGACTTAACGCTCAGGCTTTATTGTCCACTTTATTCCGGGGATCCGTCGACC	This work
sbm_F	CGTAGGCGCAAATACCCTCATTTTGATTGCGTTTTACGGAGCAAATAATGATTCCGGGGATCCGTCGACC	This work
sbm_R	GGCGAATACTTTCTGCCAGCGTGGCTTCATTAATCATGATGCTGGCTTATTGTAGGCTGGAGCTGCTTCG	This work
fadE_check_F	CAAAAGCGAGAAGTACGGGCAGGTG	This work
fadE_check_R	CACCGTTTTACCATCAATCGAAAGC	This work
fadD_check_F	GTATAATCCCGGCCCCGCGAGAGTA	This work
fadD_check_R	ATGGAGAAATTTTGTGATGGACGGA	This work
sbm_check_F	GGTCACAAAGTCCTTCGTCAG	This work
sbm_check_R	GTCATTCATGCGGGTTTTATC	This work

Plasmid	Description	Reference
pET28a	Vector for expression of N-terminal His-tagged proteins under the T7 promoter; Km^R	Novagen
pKD13	Template plasmid for amplification of the FRT-flanked kanamycin cassette; Ap^R Km^R	[41]
pKD46	Temperature-sensitive replication plasmid for Red recombinase expression; Ap^R	[41]
pCP20	Temperature-sensitive replication plasmid for thermal induction of FLP synthesis; Cm^R Ap^R	[43]
pCR^®-BluntII-TOPO	Vector used for cloning of blunt PCR products; Km^R	Invitrogen
pBAD33	Vector for protein expression under P_BAD promoter; Cm^R	[44]
pCC05	pET28a derivative vector containing accA2, pccE and pccB genes; Km^R	[22]
pRT23	pET21 derivative vector containing mas gen; Ap^R	[24]
pMB03	pET28 derivative vector containing papA5 and fadD28; Km^R	[22]
pMB04	pBAD33 derivative vector containing papA5; Cm^R	[22]
pMB05	pBAD33 derivative vector containing fadD28; Cm^R	[22]
pMB06	pBAD33 derivative vector containing papA5 and fadD28; Cm^R	[22]
pMB07	pET28 derivative vector containing papA5, fadD28 and mas; Km^R	[22]
pSB122	Vector containing the p15A origin of replication from pBAD33; Cm^R	This work
pCA30	pSB122 derivative vector containing T7 promoter; Cm^R	This work
pMB20	pCA30 derivative vector containing accA2, pccE and pccB genes; Cm^R	This work
pMB21	pCA30 derivative vector containing E. coli fadR gene; Cm^R	This work

Strain	Description	Reference
DH5α	E. coli K12 F⁻ lacU169 (Φ80lacZΔM15) endA1 recA1 hsdR17 deoR supE44 thi-1 l2 gyrA96 relA1	[45]
BAP1	E. coli F⁻ ompT hsdSB (rB-mB-) gal dcm (DE3) prpRBCD:: T7prom-sfp-T7prom-prpE	[46]
RQ1	BAP1 ygfGH::T7prom-accA2-pccE-pccB-T7term	[22]
RQ3	RQ1 Δsbm	This work
RQ4	RQ1 ΔfadD	This work
RQ5	RQ1 ΔfadE	This work

ISSN: 2731-3654