E.coli in-frame gene deletion projects

Latest update: 19-Mar-1999 by GMC, ymasar@genetics.med.harvard.edu , Up to: Church Lab page: http://arep.med.harvard.edu/

So far, our in-frame deletion efforts have focused on URFs (reading frames of unknown function) which contain DNA-binding Helix-Turn-Helix (HTH) motifs. There are also a few known genes, conserved URFs, and URFs without motifs on our short list.

**NEW** E. coli knockout and amplification primers

See our related web pages for details on homologous recombination general mutation substitution strategy (pKO3) and multiplex knockout strain pool competition readout methods. See also: Link, A.J., Phillips, D. and Church, G.M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: Application to open reading frame characterization. J. Bacteriol. 179: 6228-6237.

The 33 bp tag oligomers that we and others have used for the in frame deletions are:

5' gtt ata aat ttg gag tgt gaa ggt tat tgc gtg 3' :c22plex tag forward

5' cac gca ata acc ttc aca ctc caa att tat aac 3' :c22plex tag reverse

 Constructs using these 33-mers should be compatible with our growth rate quantitation methods and should all make the same (innocuous) peptide.

The yabC nomenclature was setup by Kenn Rudd and Amos Bairoch and is analogous to the zabC type transposon Demerec-like designators where the "a" represents the map-minutes tens place and the b the ones place (a=0, b=1, ... j=9, k=1 ...). The "C" is in rough order within the minute position set by "ab". We intend to include gene name synonyms and sequences in later versions of the table.

Note that the list below is to help coordinate comprehensive knockout projects. It is not intended to inhibit other labs from making similar constructs nor to offer strains (at present).

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E.coli genes with in-frame deletions currently being tested:

In addition to the table below there are 24 in-frame deletions using the same 33-bp tag have been constructed for E.coli orfs with homologs in Mycoplasma kindly provided by Hannes Loferer. See Nature Biotech. 16: Sept. 1998: yibO, yhaA, yjcW, yagC, ygaO, yejF, ytfR, ybbA, yehX, yhdZ, yabJ, yjfH, yebC, yeiP, yhiG, yrbF, yadG, ybhA, yabB, yfiF, yggH, yheG, ytfL, yciO. Plus 6 conditional lethals (yhbZ, ygjD, ycfB, yfiI, yihA, and yjeQ).

 Another 10 are in early construction phases: sdhA, rpe, talA, sucD, mgs, gpmA, nuoA, cyoA, gcd, mdh.

The p & K indicators below mean that we have putative plasmid and/or knockout constructs made but not neccessarily proven/characterized. The absence of K does not imply that the knockout is lethal.

Gene	Family	plasmid/KO
Unknown Function
yidL_3858583	araC	p & K
yijO	araC	p & K
yhiW	araC	p & K
yhiX	araC	p & K
yjeB	yjeB	p & K
yijC	yijC	p & K
yiaG	yiaG	p & K
yheO	yheO	p
yjgS	lacI	p & K
glpG	glpG	p & K
frvR	frvR	p & K
yjhU	sorC	p & K
yjbK	fur	p & K
yfeC	yfeD
yfeD	yfeD	p
yebK	rpiR	p
yfhH	rpiR	p & K
yehV	merR
yhdM	merR
yiaJ	iclR	p & K
yjhI	iclR
ttk	tetR	p & K
envR	tetR	p
ygaA	ntrC	p
ygeV	ntrC	p & K
yfhA	ntrC	p
yhgB	ntrC	p & K
ygbI	deoR	p
agaR	deoR	p
yihW	deoR	p
yjfQ	deoR	p & K
yjhJ	deoR	p & K
yhjB	luxR	p & K
yjjR	luxR	p
yjjQ	luxR	p & K
evgA	luxR	p & K
yhiF	luxR	p & K
y64_o6	luxR	p & K
lldR	gntR	p & K
yihL	gntR	p
yidP	gntR	p & K
yhcK	gntR	p & K
yhfR	gntR
farR	gntR	p & K
phnF	gntR	p & K
yjiR	gntR	p
pssR	lysR	p & K
yoxA	lysR	p & K
ydhB	lysR	p & K
yfiE	lysR	p & K
leuO	lysR	p & K
yafC	lysR	p & K
ybeF	lysR	p & K
yeiE	lysR	p & K
yhcS	lysR	p & K
yiaU	lysR	p & K
yidZ	lysR
yjiE	lysR	p
y65_19	lysR	p & K
yhjC	lysR	p & K
ygiP	lysR	p & K
perR	lysR
cbl	lysR
lrhA	lysR
ybhD	lysR
yhaJ	lysR
Known DNA-binding
nhaR	lysR	p & K
gcvA	lysR	p & K
oxyR	lysR
crp		p & K
ihfA		p & K
hupB		p & K
hupA		p & K
iciA	lysR
cynR	lysR
cysB	lysR
ilvy	lysR
lysR	lysR
metR	lysR
xapR	lysR
Conserved URFs
yjbJ	BCR urf	p & K
yaaC	FAD synthase	p
yabB	BCR URF	p
yabC	SAM-methylase	p
yceC	pseudouridylate synthase	p
ycfB	GuaA (ATP)
ycfG	thymidylate kinase
ycfH	ACR URF (metals?)
ychF	GTPase
yebC	ACR URF	p
yfhD	D-ribulose-5P-3epimerase	p
ygfA	5,10-methenyl-THF synthase	p
yggH	SAM-depend	p
ygiH	permease	p
yhbF-glmM	phosphoglucosamine mutase	p
yhbZ	GTPase	p
yibDp	glycosyltransferase
yibK	rRNA methylase	p
yibO	phosphoglycerate mutase
yidA	hydrolase
yigL	hydrolase
yihA	GTPase	p
yjfH	rRNA methylase	p
yraL	BCR URF	p
E.coli-specific Urfs
o261_238123	no family
ybfD	transposonase:ydcC,yhhI
yijP	urfs: yjgX, yhbX, yhjW, yjdB
hdeA	no family	p & K