The naturally competent strain Streptococcus thermophilus LMD-9 as a new tool to anchor heterologous proteins on the cell surface

Table 2 Primers and resulting fragments used in overlap extension PCR for construction of PrtH ⁺ and PrtH ⁺ WANS mutants

Fragment	Primer	Sequence (5′ – 3′)	Length (bp)	DNA matrix (strain)	Product size (kbp)	Hyb temp (°C)	Overlap product size (kbp)	Overlap hyb temp (°C)
PrtH⁺
F1	Style 1-1*	ACAAATTCATGCCGTTCATAAG	22	gDNA (LMD-9)	0.986	59.5	8.622	55
F1	PrtSssUp(H)_R	GCCTTAACTTGTTGTTC TGCAGCTACCGATGGTGC	35	gDNA (LMD-9)	0.986	59.5
F2	PrtHpp_F	GAACAACAAGTTAAGGCTAGTGTTGACAGCCAAACAAAAAC	41	gDNA (CNRZ32 CIRM-BIA 103)	5.752	62
F2	PrtHterm_R	AAAAGAGTAATGATCCTTCTCATTACTCTTTCATTATATGTAAATGATTATTTTAC	56	gDNA (CNRZ32 CIRM-BIA 103)	5.752	62
F3	EmpG9(H)_F	GAGAAGGATCATTACTCTTTT CAAACTTAAGAGTGTGTTGATAGTGC	47	pG+host 9	0.969	59.5
F3	EmpG9_R	GGACCTCTTTAGCTCCTTGG	20	pG+host 9	0.969	59.5
F4	PrtSDn(Em)_F	CCAAGGAGCTAAAGAGGTCC ATAATAAAACCGCTTAATCATTGTG	45	gDNA (LMD-9)	0.973	49
F4	PrtSDn_R	CGTCTATCAATCTTGTATTTTCTTG	25	gDNA (LMD-9)	0.973	49
PrtH⁺WANS
F5	UpH_F	CGGTATCAAGTGGGGTACTCG	21	gDNA (PrtH+)	1.395	69	4.186	55
F5	UpH(WANS)_R	CTTGCTTGGCTTGCAGA AACAGGAGCTGCAACTTGGTTATC	41	gDNA (PrtH+)	1.395	69
F6	WANS_F	TCTGCAAGCCAAGCAAG	17	gDNA (LMD-9)	0.473	58
F6	WANS(aphA3)_R	CTCAAATGGTTCGCTGG TTAGCAAACTTGTGATAAAGC	38	gDNA (LMD-9)	0.473	58
F7	aphA3_F**	CCAGCGAACCATTTGAG	17	gDNA (TIL1193)	1.4	54
F7	aphA3_R**	GTTGCGGATGTACTTCAG	18	gDNA (TIL1193)	1.4	54
F4	PrtSDn(aphA3)_F	CTGAAGTACATCCGCAAC ATAATAAAACCGCTTAATCATTGTG	43	gDNA (LMD-9)	0.971	61.7
	PrtSDn_R	CGTCTATCAATCTTGTATTTTCTTG	25

Primers Style 1-1, PrtSDn_R, and UpH_F correspond to those used for overlapping PCR reaction and italicised nucleotides correspond to homologous tails.
The name, sequence, length, DNA matrix, product size (including the added tails), hybridization temperature (hyb temp), overlap product size and overlap hybridisation temperature (hyb temp) are indicated.
bp (base pairs); kbp (kilobase pairs) and gDNA (genomic DNA) *[42], **[18].