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).


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 

yjgS 

lacI 

p & K 

glpG 

glpG 

p & K 

frvR 

frvR 

p & K 

yjhU 

sorC 

p & K 

yjbK 

fur 

p & K 

yfeC 

yfeD 

yfeD 

yfeD 

yebK 

rpiR 

yfhH 

rpiR 

p & K 

yehV 

merR 

yhdM 

merR 

yiaJ 

iclR 

p & K 

yjhI 

iclR 

ttk 

tetR 

p & K 

envR 

tetR 

ygaA 

ntrC 

ygeV 

ntrC 

p & K 

yfhA 

ntrC 

yhgB 

ntrC 

p & K 

ygbI 

deoR 

agaR 

deoR 

yihW 

deoR 

yjfQ 

deoR 

p & K 

yjhJ 

deoR 

p & K 

yhjB 

luxR 

p & K 

yjjR 

luxR 

yjjQ 

luxR 

p & K 

evgA 

luxR 

p & K 

yhiF 

luxR 

p & K 

y64_o6 

luxR 

p & K 

lldR 

gntR 

p & K 

yihL 

gntR 

yidP 

gntR 

p & K 

yhcK 

gntR 

p & K 

yhfR 

gntR 

farR 

gntR 

p & K 

phnF 

gntR 

p & K 

yjiR 

gntR 

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 

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

yabB 

BCR URF 

yabC 

SAM-methylase

yceC 

pseudouridylate synthase

ycfB 

GuaA (ATP)

ycfG 

thymidylate kinase

ycfH 

ACR URF (metals?)

ychF 

GTPase 

yebC 

ACR URF 

yfhD 

D-ribulose-5P-3epimerase

ygfA 

5,10-methenyl-THF synthase

yggH 

SAM-depend

ygiH 

permease 

yhbF-glmM

phosphoglucosamine mutase

yhbZ 

GTPase 

yibDp 

glycosyltransferase

yibK 

rRNA methylase

yibO 

phosphoglycerate mutase

yidA 

hydrolase 

yigL 

hydrolase 

yihA 

GTPase 

yjfH 

rRNA methylase

yraL 

BCR URF 

E.coli-specific Urfs

o261_238123

no family

ybfD 

transposonase:ydcC,yhhI 

yijP 

urfs: yjgX, yhbX, yhjW, yjdB

hdeA 

no family 

p & K