Biologically pure escherichia coli cell line which is a deoR.sup.- mutant and which is more transformation efficient with foreign plasmids than deoR.sup.+ escherichia coli cell lines, processes for obtaining these cell lines, methods of use

A method for obtaining E. coli cell lines which carry the deoR mutation is described, as well as the cell lines themselves. These cell lines are useful in cell transfection and transformation, as they transfect transform at much higher frequencies than the previously available cell lines.

FIELD OF THE INVENTION 
This invention relates to novel cell lines (or strains) of Escherichia coli 
with enhanced properties as a transformation host for DNA, as well as 
methods for producing said cell lines. 
BACKGROUND AND PRIOR ART 
The ability to transfer plasmids into E. coli has come to be an integral 
part of the repertoire of tools used in molecular biology. Plasmid 
transformation of E. coli was first observed by Cohen, et al, Proc. Natl. 
Acad. Sci. 69: 2110 (1972), by applying the observation of Mandel, et al, 
J. Mol. Biol. 53: 159 (1970) that E. coli and bacteriophage .lambda., when 
combined in CaCl.sub.2 containing solutions at 0.degree. C. caused 
transfection of the E. coli with the phage. 
Much work has been undertaken since then, with the goals of improving 
transformation frequency, and characterizing the parameters involved. 
Examples of this work include Hanahan, J. Mol. Biol. 166: 557-580 (1983), 
which is incorporated by reference herein; Bergmans, et al, J. Bacteriol 
146: 564 (1981); Jones, et al, J. Bacteriol 146: 841 (1981); Norgard, et 
al, Gene 3: 279 (1978). 
What the art as a whole teaches, in summary, is that E. coli and DNA 
interact at low temperatures in an environment containing divalent 
cations. Many factors improve transformation frequency, including heat 
shock, inclusion of monovalent cations in the transforming buffer, the 
addition of hexamine cobalt chloride, treatment with solvents and 
sulfhydryl reagents, and growth in media containing elevated magnesium 
levels. These treatments have improved transformation from 1 plasmid in 
10.sup.5 cells, to up to 1 in 10.sup.2 cells. 
One cell line which has been of particular use in this regard is cell line 
DH-1, which is described in Hanahan, J. Mol. Biol. 166: 557-580 (1983). 
This cell line is an E. coli mutant (recAI), and has served as a host for, 
e.g., cDNA cloning, plasmid rescue, and cloning of large DNA fragments. 
Recently, it has been learned that a mutation, identified as deoR, can be 
induced in E. coli cells. When present, the cells which carry the mutant 
serve as unexpectedly better hosts for all of the techniques described 
supra, as compared to DH-1. The DH-1 cell line itself has been mutated to 
contain the deoR mutation, and the resulting cell line is known as DH-5. 
Cell line DH-5 has been deposited with the American Type Culture 
Collection, 12301 Parklawn Drive, Rockville Maryland and bears accession 
number 53868. This deposit affords permanency of cell line and provides 
ready access to the public. 
A method for obtaining deoR mutated E. coli cell lines, the mutated cell 
lines themselves, and methods of using these for, e.g., cell 
transformation, are the subject of this invention. 
While the deoR mutations might occur in nature, substantially pure cultures 
of these have never before been available. The inventions described herein 
provides a method to one skilled in the art whereby a regular source of 
the mutant line now becomes available.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
General Protocol for Obtaining deoR Mutations 
The deoR mutation involves a gene which encodes a repressor protein. This 
repressor suppresses the expression of a set of genes involved in 
nucleotide utilization. In the absence of this protein, the regulated 
genes are expressed constitutively. When the repressor is absent, as it is 
in deoR mutations, colonies of the mutants grow more rapidly than 
non-mutants on inosine containing medium. Inosine is a nucleotide which 
does not induce derepression of the coordinated genes in question, as do 
other nucleotides. Munch-Peterson, et al, Eur. J. Biochem 27: 208-215 
(1972). 
To select for the deoR mutant, whether arising naturally or by, e.g., 
chemical radiation, or genetically induced mutation, the subject cell 
sample is inoculated (10.sup.4 to 10.sup.5 cells) into SynIn medum. SynIn 
medium consists of 10 mM NaCl, 10 mM NH.sub.4 Cl, 1 mM MgSO.sub.4. 
7H.sub.2 O, 2.5 mM K.sub.2 HPO.sub.4, 0.1 mg/ml thiamine, and 1.5 mg/ml 
Inosine. When used in Agar plates, 1.5% Bacto-Agar is added. For 
Syn-Adenosine Medium or plates, adenosine replaces inosine. 
5-50 ml of cell sample is used, depending upon the mutagenization protocol. 
When naturally occurring mutants are to be selected, larger amounts of 
sample should be used, while smaller amounts (5-10 ml) are appropriate for 
induced mutations. 
Cells are cultured at 37.degree. C. with agitation until confluency is 
reached (10.sup.9 cells/ml), and then 50 .mu.l of cells are taken into 5 
mls of fresh SynIn medium, and cultured at 37.degree. C. until confluency 
is reached. 
The cycle described supra is repeated 2-5 times, with confluency requiring 
less and less time, because normal cells will be overgrown by deoR 
mutations. 
Serial dilutions of the cells are then plated into SynIn agar plates, and 
these are incubated at 37.degree. C. to produce colonies. deoR colonies 
are noticeably larger. 
Candidate deoR cells are then streaked on fresh SynIn plates, together with 
parenteral deoR.sup.+ cells. Incubation is at 37.degree. C. deoR colonies 
appear much faster. 
The foregoing protocol was used on E. coli cell line DH1, which is 
described in Hanahan, J. Mol. Biol 166: 557-580 (1983) but can be used on 
any E. coli cell line. 
In the examples that follow, deoR mutant cell line DH-5 was used, although 
one skilled in the art will recognize the applicability of the protocols 
and experiments set forth herein to any E. coli cell line. 
EXAMPLE I 
Preparation And Isolation of Cell Line DH 5 
A sample of E. coli cell line DH 1 was mutagenized with nitrosoguanidine, 
following the protocol set forth in Miller, Experiments in Molecular 
Genetics (1972). These mutagenized cells were grown in a minimal essential 
medium which contained either uridine or inosine. These nucleosides were 
used as carbon sources because of the observation that cytR and deoR 
mutant cell lines could be discerned in mixed cell cultures due to the 
higher growth rate of the mutants on these media (cytR on uridine, deoR on 
inosine). 
After the cells were grown for a relatively long period of time, samples 
were either streaked on plates, or studied in liquid medium to determine 
their rate of growth on either uridine or inosine containing media. The 
results are set forth in following Table 1: 
TABLE 1 
______________________________________ 
cytR and deoR mutants 
Selection of Mutants 
Liquid 
Medium Assay of cloned cells 
Serial Plate by growth on plate 
Sample 
passages) 
Medium Uridine 
containing Inosine 
______________________________________ 
1 Ur Ur f f 
2 Ur In f f 
3 Ur In mf mf 
4 In Ur f m 
5 In Ur m m 
6 In Ur m m 
7 In In m f 
8 In In m m 
9 In In m m 
10 In In m m 
11 In In s vf 
12 In In m f 
DH 1 vs vs 
______________________________________ 
f = fast 
s = slow 
vf = very fast 
vs = very slow 
m = medium 
Cell sample 11, it will be seen, grows very fast on inosine containing 
medium, and slowly on uridine containing medium. This is indicative of the 
presence of a deoR mutation, and the absence of cytR. This colony was 
selected as cell line DH 5. Addtional cell lines, which are referred to 
infra, are DH-4 (cytR, but not deoR), and DH 6 (both cytR and deoR). 
EXAMPLE II 
Transfection With Plasmids 
Additional experiments were performed to determine the rate of transfection 
with various plasmids. In these experiments, plasmids pBR322, pXAD, and 
p66 which is a 66 kilobase plasmid composed of human B globin gene 
sequences inserted into pBR322, were used. These are derivative plasmids 
of pBR322, which are known to the skilled artisan. See, e.g., Hanahan, 
supra which also sets forth the protocols used for the transformation 
experiments. The results are set forth in terms of colony forming units, 
for two separate runs on each cell line. 
______________________________________ 
Plate 
Plasmid [%] Cell line 
CFU (Avg) 
______________________________________ 
10 pg pBR322 1% DH 1 31,41 
36 
DH 4 50,34 
42 
DH 5 80,90 
85 
DH 6 70,50 
60 
200 pg 
pXAD 2% DH 1 24,26 
25 
DH 4 40,34 
37 
DH 5 58,68 
63 
DH 6 58,70 
64 
500 pg 
p66 15% DH 1 2,0 1 
DH 4 0,0 0 
DH 5 3,3 3 
DH 6 5,4 4 
10 pg pBR322 + DMSO DH 5 85 
pBR322 (no DMSO) DH 5 31,37 
34 
pBR322 + DMSO DH 6 60 
pBR322 (no DMSO) DH 6 22,28 
25 
______________________________________ 
Clearly, DH 5, an example of a deoR mutant, is showing transfection far in 
excess of other mutants, and especially in excess of the preferred cell 
line for transformation, DH-1. DH6 transforms similarly, as it also 
carries the deoR mutation. 
EXAMPLE III 
Comparison of DH1 and deoR mutant (DH5) 
The cell cultures of DH1 and DH5, described supra, were used in comparative 
tests. pBR322 and p66 were used as well as a preparation of cDNA ligated 
into the vector pUC8 (in 20:1 mass ratio of cDNA to vector), at 5ng, 
2.5ng, 1.25ng, and 0.5ng concentrations, as well as pUC9 +cDNA at the same 
amounts. pUC8 + pUC 9 are described in the art. 
The cDNA used was RI-SalI DNA fragments from rat muscle. Additionally, as 
controls, 10pg pBR322 and 1ng p66 were used. The results of these 
experiments are set forth as follows: 
______________________________________ 
DH1 DH5 
1% 10% 1% 10% 
______________________________________ 
10 pg pBR322 20,20 -- 50,60 
-- 
1 ng p66 -- 2,4 -- 30,40 
Frequency of pBR322 2 .times. 10.sup.8 
5.5 .times. 10.sup.8 
transformation (as 
colonies formed per 
microgram of DNA) 
0.5 ng pUC8 7 51 17 160 
1.25 ng pUC8 16 70 32 207 
2.5 ng pUC8 7 82 32 284 
5.0 ng pUC8 18 145 64 550 
0.5 ng pUC9 2 21 5 92 
1.25 ng pUC9 3 40 23 120 
2.5 ng pUC9 2 48 24 180 
5.0 ng pUC9 10 90 35 340 
______________________________________ 
Yet again, it can be seen that DH5 is tranformed by all of the plasmids 
used at rates 3 and 4 times greater than those for DH1. As the only 
difference in the experimental parameters was the use of DH5 as compared 
to DH1, clearly the improvement resides in the deoR mutation. 
EXAMPLE IV 
Another set of experiments were performed using 30 mls of cultured cells, 
as described supra, in which the various plasmids described, supra, were 
used. These results were as follows: 
______________________________________ 
Plasmid CFUs AVG. 
______________________________________ 
DH1 (1%) 10,10 10 
10 pg pBR322 
DH5 (1%) 48,78 63 
200 pg pXAD 
DHl (10%) 11,13 12 
DH5 (10%) 205,185 195 
1 ng p66 DH1 (10%) 1,1 1 
DH5 (10%) 34,26 30 
1 ng pUC9 DHl (10%) 11,15 13 
+ cDNA DH5 (10%) 115,125 120 
1 ng pUC8 DH1 (10%) 24,24 24 
+ cDNA DH5 (10%) 240,260 250 
______________________________________ 
EXAMPLE V 
Tests were than performed to determine if the cells retained their enhanced 
tranformation properties after cold storage. 
5.times.2.5ml colonies of the cells (DH1 or DH5), were mixed with 40 mls of 
SOB medium and grown to OD.sub.550 of 0.6 DH1), and 0.48 (DH5). 
3.times.10 ml samples were then treated by centrifugation for 30 minutes on 
ice to obtain cell pellets. These pellets were then resuspended in one of 
the following media: 
(A) SF: 10% glycerol, 10 .mu.M KCH.sub.3 COO, 50 .mu.M CaCl.sub.2.2H.sub.2 
O, 100 .mu.M KCl at pH 6.1. 
(B) RFT: 15% glycerol; 30 .mu.M, KCH.sub.3 COO; 100 .mu.M RbCl; 10 .mu.M 
CaCl2.2H.sub.2 O; 50 .mu.M MnCl2.4H.sub.2 O (pH 5.8). 
(C) Frozen Storage Buffer (FSB) 
______________________________________ 
KCl 7.4 g 100 mM 
MnCl.sub.2.4H.sub.2 O 
8.9 g 45 mM 
CaCl.sub.2.2H.sub.2 O 
1.5 g 10 mM 
HACoCl.sub.3 0.8 g 3 mM 
Potassium acetate 
10 ml of a 1 M stock 
10 mM 
(pH 7.5) 
Redistilled glycerol 
100 g 10% (w/v) 
(final pH 6.20 .+-. 0.10) 
______________________________________ 
These were kept on ice for 50 minutes, and then resuspended in the same 
medium. DMSO was added to FSB, and each sample was aliquoted into 5 nunc 
tubes, and flash frozen at -70.degree. C. 
Twenty four hours later, samples were thawed until just liquid, and then 
were mixed with samples of pBR322 (100pg), and incubated, followed by 
plating. The following results were obtained. 
______________________________________ 
CFUs AVG. 
______________________________________ 
DH1 
SB 10,8 9 
RB 6,10 8 
FSB 8,2 5 
DH5 
SB 55,57 56 
RB 52,48 47 
FSB 34,40 37 
______________________________________ 
Not only does DH5 transform at much higher frequencies than DH1, it also 
appears to remain more stable following storage than the cell line of 
choice, DH1. 
As will be understood from the foregoing, especially Example I, the 
transformant used was an E. coli cell line which had been mutagenized by 
treatment with nitrosoguanadine and which contained the deoR mutant. These 
mutation appears to be key to the enhanced transformation frequency as DH1 
and DH5 do not differ in any respect other than this. Additionally, 
comparison to other E. coli mutants, including ctyR and cytR +deoR shows 
that this ctyR mutation does not effect the transformation frequency of 
the cell line. 
EXAMPLE VI 
One skilled in the art will note immediately that while a specific chemical 
agent was used to obtain the deoR mutant used herein, additional chemical 
mutagens may be used to create the deoR mutation. Presence of the mutation 
can be determined in a given cell sample following the method set forth in 
Example I, supra, wherein growth on an inosine containing medium is seen 
to indicate its presence. 
Additional methods of mutagenizing the E. coli cell line to obtain the 
desired deoR mutant cell line will be apparent to one skilled in the art. 
For example, both ultraviolet and X-irradiation may be used to produce 
mutations which may then be selected using the inosine containing medium 
described supra. Irradiation protocols may be found in, e.g., Miller, 
Experiments in Molecular Genetics (1972), (Cold Spring Harbor Laboratory, 
New York). Transposon insertional or deletional mutagenesis may be used as 
well. In this form of mutagenesis, transposons known to the art such as 
Tn5 and Tn10 can be inserted into the E. coli DNA, frequently with 
associated DNA deletions. Mutagenesis protocols may be found in, e.g., 
Davis et al., Advanced Bacterial Genetics (1980; Cold Spring harbor 
Laboratory, New York); Gilhavy et al., Experiments With Gene Fusions 
(1984; Cold Spring Harbor Laboratory, New York). 
Spontaneous mutations to deoR can be selected and isolated by use of the 
inosine synthetic medium. While a low rate of mutation is to be expected 
(.about.10.sup.-7 cell/generation), this can be increased by inoculating 
about 50 ml of a cell sample to the SynIn medium described supra. 
The terms and expressions which have been employed are used as terms of 
description and not of limitation, and there is no intention in the use of 
such terms and expressions of excluding any equivalents of the features 
shown and described or portions thereof, it being recognized that various 
modifications are possible within the scope of the invention.