TABLE 1.

E. coli strains, plasmids, and primers used in this study

Strain, plasmid, or oligonucleotide	Descriptiona	Reference, source, or site
Strains
Novablue	endA1 hsdR17 (rK12− mK12+) supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proA+B+ lacIqZΔM15::Tn10]	Novagen
EB68	MG1655 (F− λ−)	George Church, Harvard Medical School (24)
EB356	MG1655 (F− λ−) araBAD::ispF(optimal RBS) kan	This work
EB370	MG1655 (F− λ−) araBAD::ispF(optimal RBS) kan ΔispF	This work
Plasmids
pKO3	E. coli gene replacement plasmid described by Link and coworkers	24
pKO3-ispFflank	pKO3 with ispF flanks from EB68	This work
pKD46	Red recombinase expression plasmid for transformation of linear DNA in E. coli	11
pBluescript SKII+	Cloning vector	Stratagene (La Jolla, Calif.)
pBS-araBADflank	pBluescript with araBAD flanks from EB68	This work
pBS-araBADflankkan	pBluescript with Kanr cassette inserted between araBAD flanking sequence	This work
pBS-araBADflankispFkan	pBluescript with ispF (optimal RBS) and Kanr cassette inserted between araBAD flanking sequence	This work
Oligonucleotides
BAD-a	5′-AAGGAAAAAAGCGGCCGCACCGCGAATGGTGAGATTGAGAATATA-3′	NotI
BAD-bb	5′-CACGCAATAACCTTCACACTCCGCCCGGGCAACCAACGGTATGGAGAAACAGT-3′	SrfI
BAD-cb	5′-GTTGCCCGGGCGGAGTGTGAAGGTTATTGCGTGCTGTATAAAACCACAGCCAA-3′	SrfI
BAD-d	5′-CGCACGCATGTCGACTTCAGACGGGCATTAACGATAGTG-3′	SalI
BAD-e	5′-ATGCAGGATTTTTGCCCAGA-3′
kan-F	5′-TGTGTTTAAACGTATGTGGAAGGTGGAAAGCCACGTTGTGTCTC-3′	PmeI
kan-R	5′-TAACCAATTCTGATTAGAAA-3′
ispF-Fc	5′-GGGGTACCAATAAGGAGGAAAAAAAAATGCGAATTGGACACGGT-3′	KpnI
ispF-R	5′-GCTCTAGATCATTTTGTTGCCTTAAT-3′	XbaI
ispF-a	5′-AAGGAAAAAAGCGGCCGCGAATCATCCGCAAATCACCGT-3′	NotI
ispF-bd	5′-ACGCAATAACCTTCACACTCCAAATTTATAACCATTATGTATTCTCCTGATGGATGGTTC-3′
ispF-cd	5′-GTTATAAATTTGGAGTGTGAAGGTTATTGCGTGAAATGATTGAGTTTGATAATCTCACTTACT-3′
ispF-d	5′-CGCACGCATGTCGACCCCAAAACGTTGGGCACC-3′	SalI

^aUnderlined sequences indicate restriction enzyme sites.

^bBoldface sequence indicates complementarity between BAD-b and BAD-c.

^cBoldface sequence indicates optimal ribosome binding site (RBS).

^dBoldface sequence indicates complementarity between ispF-b and ispF-c.