Regulatory elements involved in the expression of competence genes in naturally transformable Vibrio cholerae

Mirella Lo Scrudato; Sandrine Borgeaud; Melanie Blokesch

doi:10.1186/s12866-014-0327-y

BMC Microbiology

Table 2 Primers used in this study

From: Regulatory elements involved in the expression of competence genes in naturally transformable Vibrio cholerae

Primer name	Sequence	Description
Primer name	(in 5′ to 3′ direction)	Description
Rev[VC1917]-NotI	GCGGCCGCGAGCTCTAGAGGTTTCTTAG	For inverse PCR leading to plasmids:
		pBR-[−700]comEA,
		pBR-[−500]comEA,
		pBR-[−300]comEA,
		pBR-[−100]comEA,
		pBR-[−40]comEA
Fwd[VC1917]-700	AGAGCTCGCGGCCGCAGGTGTTAACCACTCCTGCGGTAC	Inverse PCR to generate
Fwd[VC1917]-700	AGAGCTCGCGGCCGCAGGTGTTAACCACTCCTGCGGTAC	pBR-[−700]comEA
Fwd[VC1917]-500	AGAGCTCGCGGCCGCCAACAAGCACTTGAACTGGGTAAC	Inverse PCR to generate
Fwd[VC1917]-500	AGAGCTCGCGGCCGCCAACAAGCACTTGAACTGGGTAAC	pBR-[−500]comEA
Fwd[VC1917]-300	AGAGCTCGCGGCCGCTATCGTTGTGATTGAGTTGAGC	Inverse PCR to generate
Fwd[VC1917]-300	AGAGCTCGCGGCCGCTATCGTTGTGATTGAGTTGAGC	pBR-[−300]comEA
VC1917-134-NotI	GCGGCGGCCGCATTCTTAGTGTAATTGATATG	PCR to generate
pBR-TET_MCS after	ATCATGCGCACCCGTGGCCAGGACCC	pBR[−134]comEA,
Fwd[VC1917]-100	AGAGCTCGCGGCCGCGGGCTACAGCAGTAGCCCGTTC	Inverse PCR to generate
Fwd[VC1917]-100	AGAGCTCGCGGCCGCGGGCTACAGCAGTAGCCCGTTC	pBR[−100]comEA
Fwd[VC1917]-40	AGAGCTCGCGGCCGCCGCTATCATAAGCCCTCAACAAC	Inverse PCR to generate
Fwd[VC1917]-40	AGAGCTCGCGGCCGCCGCTATCATAAGCCCTCAACAAC	pBR[−40]comEA
gyrA-157-fwd	AATGTGCTGGGCAACGACTG	qRT-PCR for gyrA transcription [10]
gyrA_332_bwd	GAGCCAAAGTTACCTTGGCC	qRT-PCR for gyrA transcription [10]
comEA_50_fwd	CGACATTACCGTTACTGGCC	qRT-PCR for comEA transcription [10]
comEA_224_bwd	CCGTTGGCTTCTCGATAATCG	qRT-PCR for comEA transcription [10]
comEC_1029_fwd	GGTCGCGATTGTTCTCTACC	qRT-PCR for comEC transcription [10]
comEC_1186_bwd	CCAAATTGTACAGAACTGCCG	qRT-PCR for comEC transcription [10]
VC0396_188_fwd	GCCTGATTCGCCAGCAATTG	qRT-PCR for qstR transcription [22]
VC0396_356_bwd	CCAAGACCGTGGGCAATAAAG	qRT-PCR for qstR transcription [22]
hapR-230-fwd	CCAACTTCTTGACCGATCAC	qRT-PCR for hapR transcription [22]
hapR-399-bwd	GGTGGAAACAAACAGTGGCC	qRT-PCR for hapR transcription [22]
hapA_175_fwd	ACGGTACAGTTGCCGAATGG	qRT-PCR for hapA transcription [22]
hapA_358_bwd	GCTGGCTTTCAATGTCAGGG	qRT-PCR for hapA transcription [22]
comEA_284_rev	CGCACTGTCGCTTCACCAATCC	5′RACE: synthesis of first strand cDNA of comEA
comEA_217_rev	CTTCTCGATAATCGACAATGGCCTGAGC	5′RACE: PCR amplification of Poly(A) cDNA comEA
oligo dT-Anchor primer (Roche)	GACCACGCGTATCGATGTCGAC	5′RACE: PCR amplification of Poly(A) cDNA comEA
F-EcoRI_Anchor_P	CCAAGAATTCGACCACGCGTATCGATGTCGAC	5′RACE: PCR fragment of Poly(A) cDNA comEA cloned into plasmid pBR-Tet_MCSI
R-BamHI_comEA_217	CCAAGGATCCCTTCTCGATAATCGACAATGGCCTGAGC
T7RNA-pol-750-down	GCTGAGGCTATCGCAACCCGTGC	DNA uptake assay: amplification of donor DNA; primer specific for E. coli BL21(DE3); [9],[13]
T7 RNAP-end-bw	TTACGCGAACGCGAAGTCCGACTCTAAG
lacZ-missing-fw	GCCGACTTTCCAATGATCCACAATGGG	DNA uptake assay: amplification of acceptor DNA; primer specific for V. cholerae A1552 and lacZ⁺ derivatives of it; [9],[13]
lacZ-missing-bw	CCCTCGCTATCCCATTTGGAAATGCC

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