E.coli in-frame gene deletion projects

Latest update: 19-Mar-1999 by GMC, ymasarova@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