E. coli Strains

ABLE C
E. coli C lac (lacZ -) [Kanr, mcrA-, mcrCB-, mcrF-, mrr-, hsdR(rK-mK-)] [F'proAB, lacIq,ZD M15, Tn10(Tetr)]. Reduces plasmid cop y number of ColE1-derived plasmids 4-fold; helps in cloning toxic gene products (Stratagene).

ABLE K
E. coli C lac(lacZ -) [Kanr, mcrA-, mcrCB-, mcrF-, mrr-, hsdR(rK-mK-)] [F'proAB, lacIq,ZD M15< /I>, Tn10(Tetr)]. Reduces plasmid copy number of ColE1-derived plasmids 10-fold; helps in cloning toxic gene products (Stratagene).
AG1
recA1, endA1, gyrA96, thi-1, hsdR17(rK- mK+), supE44, relA1
BL21
E. coli B, F-, < I>dcm, ompT, hsdS(rB-mB-), gal.

BL21(DE3)
E. coli B, F-, dcm, ompT, hsdS(rB-mB-< /SUP>), gall(DE3). Contains the T7 RNA polymerase gene under control of the lacUV5 promoter. The polymerase gene is integrated into the bacterial chromosome from l(DE3). Induce expression with IPTG. Use for non-toxic protein expression (Stratagene).
BL21(DE3) pLysE
E. coli B, F-, dcm, ompT, hsdSB(rB-mB-), gal(DE3) [pLysE Camr]. Have same DE3 lysogen as BL21(DE3)pLysS containing T7 lysozyme, a T7 RNA polymerase inhibitor to prevent leaky expression in uninduced cells, and contain pLysE p lasmid with T7 lysozyme (Invitrogen). Useful for expression of toxic proteins.
BL21(DE3) pLysS
E. coli B, F-, dcm, ompT, hsdS(rB-mB-), gall(DE3) [pLysS Camr]. These cells have the pLysS plasmid added to them containing T7 lysozyme, a T7 RNA polymerase inhibitor to prevent leaky expression in uninduced cells. Useful for expression of toxic proteins (Stratagene).
C600
Use with lgt10 growth & titer - parental & recombinant l grow; hsdR-, hsdM+, supE44, thr-1, leuB6, thi-1, lacY1, tonA21, mcrA-, mcrB+

C600 Hfl-
Use with lgt10; recombinant plaques are lytic & produce clear plaques vs lysogenic, turbid parental plaques

DH1
F-, endA1, gyrA96, thi-1, hsdR17(rK-,mK+), supE44, relA1. Used for plasmid and cosmid transformation.

< FONT SIZE=+1>DH5a
DH1 genotype and F-,F 80D lacZD M15, D (lacZYA-argF),U169. Used for plasmid and cosmid transformation. Blue-white selection.

HB101
Good for pBR plasmids and other non- a-complementing plasmids. hsd20(rB-, mB-), recA13, rpsL20(streptomycinR), leu, proA2.

INVaF'
F' endA1, recA1, hsdR17(rk-,mk+), supE44, thi-1, gyrA96, relA1, F80lacZD M15, D (lacZYA-argF)U169. Can produce ssDNA from vectors with f1 ori. Blue/white selection.

INV110
F' tra, D36proAB, lacIqZDM15/rpsL, StrR, thr, leu, endA1, thi-1, lacY, galK, galT, ara, tonA, tsx, dam, dcm, dupE44, D(lac-proAB), D(mrcC-mrr)102::Tn10 (tetR). Dereived from JM110 to grwo plasmids that are dam or dcm methylation sensitive. Can produce ssDNA from vectors with f1 ori. Blue/white selection.

JM101
supE, thi-1, D (lac-proAB), [F' traD36, proAB, lacIqD(la cZ) M15]

JM103
D (lac pro), thi-1, strA, supE, endA, sbcBC, hsdR-, F'(traD36, proAB, lacIq, D M15).

JM109
F' episome for filamentous phage infections; a-complementation; lacIq repressor, recA-. Good for M13 & pGEM. e14-(mcrA-), recA1, endA1, gyrA96, thi-1, hsdR17(rK-mK-), supE44, relA1, l-, D (lac-proAB), [F'traD36, proAB, lacIqZD M15].

JM110
rps, (Strr), thr, leu, thi-1, lacY, galK, galT, ara, tonA, tsx, dam, dcm, supE44, D (lac-proAB) , [F'traD36, proAB, lacIqZD M15]. For subcloning methylated DNA. EndA+ so poor plasmid preps. See SCS110 for endA-strain (Stratagene).

K802
hsdR2-(rK-, mK+), h sdM+, gal-, metB1, supE44, mcrA-, mcrB-; use with EMBL-3

LE392
Use with various l vectors with cDNA & genomic inserts. No blue/white selection. lon+, F-, hsdR514 (rK-, mK+), supE44, supF58, lacY1 or D (lacIZY)6, galk2, galT22, metB1, trpR55, l-.

LMG194
F-, DlacX74, galE, thi, rpsL, DphoA, (PvuII), Dara714, leu::Tn10.

M15
kanR

NM522
supE, thi-1, D(lac-proAB), D(mcrB-hsdSM)5,(rK-mK-), [F' proAB lacIq,D (lacZ)M15]

NM538
hsdR(rK-, mK+), supE, F80R

NM539
from NM538 for selection of Spi- phage recombinants

RR1
F-, mcrB, hsd20(rB-,mB-), leuB1, ara-14, proA2, lacY1, galK2, xyl-5, mtl-1, rpsL20(smR), supE44, l-.

SCS1
recA1, endA1, gyrA96, thi-1, hsdR17(rK-mK+),supE44, relA1

SCS110
rpsL, (Strr), thr, leu, endA, thi-1, lacY, galK, galT, ara, tonA, tsx, dam, dcm, supE44, d(lac-proAB), [F' traD36, proAB, lacIqD M15]. Derived from the JM110 strain with an endA- mutation for plasmid preps. For subcloning methylated DNA (Stratagene).


The fol lowing three strains were obtained from Susan Gottesman, NCI, NIH. They are described in Meth. Enz. 185:119-129, 1990.

SG1611
der of JM101: lon-. Can be used with M13 or pUC vectors

SG21173
der of MC4100: lon-, clp-, htpR kanR, tS

SG12044
der of C600: kanR lon-, clp-


SURE
e14-(mcrA-), D (mcrCB-hsdSMR-mrr)171, endA1, supE44, thi-1, gyrA96, relA1, lac, recB, recJ, sbcC, umuC::Tn5(Kanr), uvrC, [F' proAB, lacIqDM15, Tn10(Tetr)]. This strain is good for subcloning genes with long inverted repeats (Stratagene).

SURE 2< /A>
e14-(mcrA-), D (mcrCB-hsdSMR-mrr)171, endA1, supE44, thi-1, gyrA96, relA1, lac, recB, recJ, sbcC, umuC::Tn5(Kanr), uvrC, [F' proAB, lacIqDM15, Tn10(Tetr), Amy, Camr]. Camr @<40 µg/ml, Cams @ 100 µg/ml. This strain is good for subcloning genes with Z-DNA secondary structures (Stratagene).
TB1
F-, ara,D(lac-proAB), rpsL, F80lacZD M15, hsdR17(rK-,mK-).< /P>
TG1
supE, thi-1, D(lac-proAB), D(mcrB-hsdSM)5, (rK-mK-), [F' traD36, proAB, lacIq D M15]

TKB1
E. coli B, F- dcm, ompT, hsdS(rB-mB-),gall(DE3), [pTK, Tetr]. Derived from BL21 cells with a chromosomal copy of T7 RNA polymerase gene. Also

TKX1
D (mcrA)183, D (mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac [F' proAB, lacIqD M15, Tn5(Kanr)], [pTK Tetr]. Derived from XL-1 Blue cells and containing an elk tyrosine kinase gene controlled by a trp promoter, leading to induced phosphorylation of some proteins (Stratagene).

TOP10
F-, mcrA, D(mrr-hsdRMS-mcrBC), f80lacZDM15 DlacX74, deoR, recA1, araD139 D(ara-leu)7697, galK, rpsL(StrR), endA1, nupG . High efficient cloning (Invitrogen)

TOP10F'
[F' [lacIq, Tn10(TetR)], mcrA, D(mrr-hsdRMS-mcrBC), f80lacZDM15, DlacX74, deoR, recA1, araD139 D(ara-leu)7697, galK, rpsL(StrR), endA1, nupG. High efficient cloning with TetR gene on F' episome (Invitrogen).

TOP10/P3
F-, mcrA, D(mrr-hsdRMS-mcrBC), f80lacZDM15, DlacX74, deoR, recA1, araD139 D(ara-leu)7697, galU, galK, rpsL(StrR), endA1, nupG, [P3:kanR, ampR (amber), tetR (amber)]. Allows production of supF containing p lasmids such as pCDM8, and pcDNA1.1 (Invitrogen). KanR on own, but obtain ampRand TetR from supression of amber mutations on supF plasmids.

TOPP 1 & 2
Rifr, [F' proAB lacIqZD M15, Tn10(Tetr)]. Non-K12 E. coli that may enhance hard to express proteins (Stratagene).

TOPP 3
< font size=3>Rifr, [F' proAB lacIqZD M15, Tn10(Tetr) (Kanr)]. Non-K12 E. coli that may enhance hard to express proteins (Stratagene).
VCS257
Use with lLong-C, Stratagene

XL-1 Blue
recA1, endA1, gyrA96, thi-1, hsdR17(rK-,mK+), supE44, relA1, l-, lac-, [F' proAB, lacIqZD M15, Tn10(Tetr)].

XL-1 Blue MR
D (mcrA)183, D (mcrCB-hsdSMR-mrr)173, recA1, endA1, gyrA96, thi-1, supE44, relA1, l-, lac-. No blue-white selection, good for methylated DNA (Strata gene).

XL-1 Blue MRF'
D (mcrA)183, D (mcrCB-hsdSMR-mrr)173, recA1, endA1, gyrA96, thi-1, supE44, relA1, l-, lac-, [F' proAB, l acIqZD M15, Tn10(Tetr)]. Restriction minus strain of XL-1 Blue (Stratagene).

XL-1 Blue MRF' Kan
D (mcrA)183, D (mcrCB-hsdSMR-mrr)173, recA1, endA1, gyrA96, thi-1, supE44, relA1, l-, lac-, [F' proAB, lacIqZD M15, Tn5(Kanr)]. Good for Tetr plasmid subclones (Stratagene).

XL2 Blue
recA1, endA1, gyrA96, thi-1, hsdR17(rK-,mK+), supE44, relA1, lac-, [F' proAB, lacIqZD M15, Tn10(Tetr), Amy, Camr]. Camr @<40 µg/ml, Cams @ 100 µg/ml (Stratagene).

XL2 Blue MRF'
D (mcrA)183, D (mcrCB-hsdSMR-mrr)173, recA1, endA1, gyrA96, thi-1, supE44, relA1, l-, lac-, [F' proAB, lacIqZD M15, Tn10(Tetr), Amy, Camr]. C amr @<40 µg/ml, Cams @ 100 µg/ml (Stratagene).

XL1-Red
endA1, gyrA96, thi-1, hsdR17(rK-,mK+), supE44, relA1, lac-, mutD5, mutS, mutT, Tn10(Tetr). Deficient in three mut DNA repair pat hways, thus increasing chance of cloning mutated genes (Stratagene). Retransform into XL1 Blue once mutants are screened.

XLmutS Kans
D(mcrA)183, D (mcrCB-hsdSMR-mrr)173, endA1, gyrA96, thi-1, supE44, relA1, lac-, mutS::Tn10(Tetr), [F' proAB, lacIqZD M15, Tn5]. Do not perform mismatch repair from in vitro mutagenized constructs, and have less degraded plasmids in minipreps with endA- mutation (Stratagene).

XLmutS Kanr
D(mcrA< /I>)183, D (mcrCB-hsdSMR-mrr)173, endA1, gyrA96, thi-1, supE44, relA1, lac-, mutS::Tn10(Tetr), [F' proAB, lacIqZD M15, Tn5 (Kanr)]. Do not perform mismatch repair from in vitro mutagenized constructs, and have less degraded plasmids in minipreps with endA- mutation (Stratagene).

Y1088
F' DlacU169, supE, supF, hsdR(rk-,mk+), trpR, fhuA21, proC::Tn5 (pMC9;tetRAmpR).

Y1089
Use for preparative production of ß-gal fus ion proteins with lgt11. D lacU169, proA+, D lon, araD139, strA, hflA150, [chr::tn10], pMC9.

Y1090
blue/white color selection; use for antibody screening of expression libraries. D lacU169, proA+, D lon, araD139, strA, supF, [trpC22::Tn10], pMC9.

E. coli Mutations and Brief Descriptions

ara-14
Blocks arabinose catabolism

argF
Ornithine carbamoyltransferase mutati on blocks ability to use arginine

dam
DNA adenine methylase mutant. Blocks methylation of adenine at GmATC; Dam+ cells methylate A of GATC at N6 to produce N5-me-A. Low transformation efficiency with Dam-modified vectors.

dcm
DNA cytosine methylase mutation. Blocks methylation of cytosine at Cm CAGG or CmCTGG; Dcm+ cells methylate the 5 position of the 3' C of CC(A/T)GG to produce 5me-C.

deoR
Regulatory mutant for constitutive deoxyribose synthesis to allow uptake of large plasmids. Some reports indicate the transformation efficiency of 40-60 kB plasmids is increased by an order of magnitude in deoR strains.

dut
With ung allows uracil incorporation into DNA via dUTPase mutation. UTPase deficient.

dnaJ
Certain chaparonins are inactivated.

e14
Carries mcrA and other genes making the strain mcrA-

endA,A1
Mutation in endonuclease I increases quality of plasmids. These strains are useful for generating ss-DNA of M13, since the endonuclease digests ds-DNA, but not packaged ss-DNA.

F-
Host lacks the F' episome

F'
Host contains the F' episome containing a particular g enotype from the E. coli chromosome.

galK
Galactokinase mutation blocks catabolism of galactose

galU
Glucose-1-phosphate uridylyltransferase mutation blocks ability to use galactose

gyrA96
DNA gyrase mutant p roduces resistance to nalidixic acid

hflA,A150(1)
Increases frequency of lysogeny in l phage containing cI repressor gene.

hfl
Inhibited from lytic cycle, increases frequency of lysogeny.

hsdR(rK-,mK+)
A restriction endonuclease. Restriction minus (via EcoK restriction system mutant, so no restriction at unmethylated EcoK I sites), modification plus; no cleavage of DNA by endogenous restriction enzymes from non-E. coli sources. EcoK I sites are CC(N6)GTGC or GCAC(N6)GTT.

hsdRMS(rK-,mK-)
Restriction minus (via EcoK restriction system mutant, so no restriction at unmethylated EcoK I sites), modification minus (no methylation at EcoK I sites). EcoK I s ites are CC(N6)GTGC or GCAC(N6)GTT.

hsd S
Eliminates restriction of DNA by hsdS. Restriction minus, modification minus; mutated EcoB restriction and methylation; no cleavage of DNA by endogenous restriction ezymes; no protective methylation. EcoB sites is TGA(N8)TGCT.

lacIq
High levels of lac repressor protein produced, inhibits transcription from lac promoter. Overcome this repression by binding to IPTG.

lacY
Blocks use of lactose via lactose permease mutant

lacY1
Blocks use of lacto se via lactose permease mutant

lacZ
ß-D-galactosidase gene. Mutations yield colorless (vs. blue) colonies in presence of X-gal.

lacZ D M15
A partial
deletion of the NH2-terminal region of ß-D-galactosidase to permit a-complementatio n with certain vectors that encode this region (pUC, M13) thus producing blue/white color selection. D U169, X111, & X74 all delete entire lac operon. X111 also deletes proAB so needs proline unless F'lac proAB is present. Often present on f-80 or F'

l-
Nonlysogenic (phage won't inc orporate into chromosome.

l(DE3)
l phage containing a T7 RNA polymerase gene is integrated into host chromosome.

leuB
requires leucine for growth on minimal media via ß-isopropyl malate dehydrogenase mutation.< /FONT>

D lon
Reduces proteolysis via lon protease mutant; increases stability of fusion proteins.

D malB
Deletion of malEFG, malK, lamB, & malM. Prevents expression of maltose binding prote in.

mcrA
Blocks restriction of DNA methylated at G(m)CGC.

mcrB
Blocks restriction of DNA methylated AG(mC)T. Same as mcrCB (?).

metB
Requires methionine for growth on minimal media via cy stathionine g-synthetase mutant.

mrr
Blocks adenine methylation; prevents cleavage of C(m)AG and G(mA)C.

mtl-1
Blocks catabolism of mannitol.

mtl
Blocks ability to use mannitol.

Mud
Defective Mu prophage.

P1

Cell contains P1 prophage carrying the P1 restrictionsystem.

P2 (2)
Cell contains P2 prophage. l phages with Red+ and Gam+ genes are growth inhibited (Spi- selection) by P2 lysogens in these hosts.

P3

Used to screen for supF plasmids. Cell contains P3 prophage or plasmid with AmpR and TcR genes with amber mutations, and KanR gene.

f80
Cell carries F80 prophage. Some E. coli strains carry defective lac M15.

proAB
Requires proline for growth on minimal media

recA, A1, A13
Involved in DNA repair and recombination. Mutations reduce homologous recombination of vector with ho st DNA giving more stable inserts; gives UV-light sensitivity.

recB, C
Reduces recombination & repair of radiation damaged DNA via exonuclease V mutant. Sometimes aberrant plasmid replication. recB and recJ together produce the recA phenotype.

recD
Recombination is present but exonuclease V a ctivity is absent. Aberrant plasmid replication.

recF
Prevents plasmid-plasmid recombination.

recJ
Exonuclease involved in recombination; alternate to recA pathway. prevents plasmid-plasmid recombination. With recB, confers recA- phenotype. With sbcC, reduces Z-DNA rearrangements.

relA
RNA is synthesized in absence of protein synthesis (relaxed phenotype).

rpoH; htpR
Lacks a heat shock transcription factor thus reducing certain proteases other than lon.
rpsL< br>
StreptomycinR via S12 mutant in 30S ribosome.
sbcBC
Permits recombination in recBC hosts via exonuclease I mutant. With recJ, reduces rearrangements of Z-DNA.
supB,C,G,L,M,N,O
tRNA tyrosine suppressors of ochre (UAA) and amber (UAG) to glutamine (supE).
supE,F
tRNA glutamine suppressor of amber (supE)(UAG) or tyrosine (supF). supF is needed for growth of some phage vectors.
thi-1
Requires thiamine for growth on minimal media.
thr
Requires threonine for growth on m inimal media.
Tn3
AmpicilinR via a transposon.
Tn5
KanamycinR via a transposon.
Tn10
TetracyclineR via a transposon.
tonA
Bacteriophage T1R via mutation in outer membrane protein.
traD,D36
Prevents transfer of F' episome via transfer factor mutation.
tprR
Requires tryptophan for growth on minimal media.
tsp
Deletion of a periplasmi c protease.
tsx
Confers resistance to phage T6 and colicin K.
umuC
Component of SOS repair pathway. Reduces rearrangements of inverted repeats.
ung
Uracyl-N-glycolase deficiency; normally removes uracil from DNA.
uvrC
Component of UV repair pathway. Reduces rearrangements of inverted repeats.
xyl-5
Blocks catabolism of xylose.


VECTORS

Lambda Vectors

lgt·WES

lgt10
7.6 kb insert - good for cDNA & smaller genomic DNA fragments; EcoRI site; 32.71 kb left arm, 10.63 right arm.

lgt11
6 kb inserts at end of lacZ gene - can produce f usion protein; left arm 19.6 kb, right arm 24.1 kb.

EMBL-3 sp6/T7
9-23kb inserts - good for genomic libraries; BamHI, XbaI, SacI, SfiI sites; grows on K802 & NM538, Spi recombinants grow on NM539; 20 kb left arm, 14 kb stuffer, 8 kb right arm; .

lLong-C
0-11 kb insert, EcoRI, SacI, & XbaI sites, use with VCS257, Stratagene, arms are 21.2 & 18.3 kb

Plasmid Vectors

pBR322
4361bp ampR, TcR

pBR325
6.0kb? ampR, TcR, CmR

pUC18 & 19
2.69kb ampR

pGEM
3.05kb ampR

pSP64
3.0 kb ampR

pSL1180
3.42kb ampR, Pharmacia, super polylinker, use with NM522 or D H5a, host needs F' factor, can make ssDNA (M13 ori), no blue/white selection. ref: Brosius, J. DNA 8:759-777, 1989.

Sequencing Vectors

M13mp18 & 19
7250 bp

Prokaryotic Expression Plasmids

pQE9,11
3.44 kb BamHI site, Quiagen

pGEX-2T,-3X
GST fusion protein, has thrombin or factor X site, available from Pharmacia

pMAL
7.0 kb; fusion protein with E. coli malE gene which produces the maltose binding protein; inducible P-tac promotor via IPTG induction.

The followin g plasmids were obtained from Dr. Masayori Inouye, Biochem, Robert Wood Johnson Med School, Piscataway NJ.

pIN-III-B1,B2,B3
Inducible promotor; use in any host; in W620recA host
pIN-III-ompA1,A2,A3
Secretion vector to periplasmic space; in W620recA host; EcoRI, BamHI, HindIII sites.

    Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Bacterial Hosts and Vectors - Genetics. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/hostvect.html). 1997. Oklahoma City. Revision Date: January 13, 2000."