Method capable of increasing competency of bacterial cell...

Details Method capable of increasing competency of bacterial cell... Method capable of increasing competency of bacterial cell...

2001-07-02

2004-03-23

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252800

Reexamination Certificate

active

06709854

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to improved bacteria, particularly
Escherichia coli
(
E. coli
) bacteria capable of high transformation efficiencies, methods for producing improved bacterial strains capable of high transformation efficiencies, and methods for obtaining high transformation efficiencies with bacteria, particularly
E. coli
bacteria. Specifically, it relates to methods of producing and using bacteria, particularly
E. coli
bacteria that contain F′ episome genetic material and are capable of exhibiting enhanced transformation efficiencies.
BACKGROUND OF THE INVENTION
High efficiency chemically competent
E. coli
bacteria (bacterial cells that can be transformed with DNA) are used extensively in the generation of cDNA libraries and the cloning of samples containing small amounts of target sequences. The ability to generate representative cDNA libraries, one in which each mRNA species present in the subject cell is represented in the library, relies on many factors. One of the major factors determining the quality of a cDNA library is the number of clones represented in the library. Using competent bacteria having a high transformation efficiency increases the probability of obtaining rare, under-represented clones in plasmid libraries. Also, when cloning samples containing small amounts of target DNA or cloning the DNA products of complex DNA manipulations such as the DNA products of single or multiple blunt ended ligations, the use of high efficiency bacteria is essential.
Early attempts to achieve transformation of
E. coli
were unsuccessful and it was generally believed that
E. coli
was refractory to transformation. However, Mandel and Higa (
J. Mol. Bio.
53: 159-162 (1970)) found that treatment with CaCl
2
allowed
E. coli
bacteria to take up DNA from bacteriophage &lgr;. In 1972, Cohen et al. showed CaCl
2
-treated
E. coli
bacteria were effective recipients for plasmid DNA (Cohen et al.,
Proc. Natl. Acad. Sci.,
69: 2110-2114 (1972)). Since transformation of
E. coli
is an essential step or cornerstone in many cloning experiments, it is desirable that it be as efficient as possible (Lui and Rashidbaigi,
BioTechniques
8: 21-25 (1990)). Several groups of workers have examined the factors affecting the efficiency of transformation.
Hanahan (
J. Mol. Biol.
166: 557-580 (1983), herein incorporated by reference) examined factors that affect the efficiency of transformation, and devised a set of conditions for optimal efficiency (expressed as transformants per &mgr;g of DNA added) applicable to most
E. coli
K12 strains. Typically, efficiencies of 10
7
to 10
9
transformants/&mgr;g can be achieved depending on the strain of
E. coli
and the method used (Liu & Rashidbaigi,
BioTechniques
8: 21-25 (1990), herein incorporated by reference).
Many methods for bacterial transformation are based on the observations of Mandel and Higa (
J. Mol. Bio.
53: 159-162 (1970)). Apparently, Mandel and Higa's treatment induces a transient state of “competence” in the recipient bacteria, during which they are able to take up DNAs derived from a variety of sources. Many variations of this basic technique have since been described, often directed toward optimizing the efficiency of transformation of different bacterial strains by plasmids. Bacteria treated according to the original protocol of Mandel and Higa yield 10
5
-10
6
transformed colonies/&mgr;g of supercoiled plasmid DNA. This efficiency can be enhanced 100- to 1000-fold by using improved strains of
E. coli
(Kushner, In:
Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering
, Elsevier, Amsterdam, pp. 17-23 (1978); Norgard et al.,
Gene
3:279-292 (1978); Hanahan,
J. Mol. Biol.
166: 557-580 (1983)) combinations of divalent cations ((Kushner, In:
Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering
, Elsevier, Amsterdam, pp. 17-23 (1978)) for longer periods of time (Dagert and Ehrlich,
Gene
6: 23-28 (1979)) and treating the bacteria with DMSO (Kushner, In:
Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering
, Elsevier, Amsterdam, pp. 17-23 (1978)), reducing agents, and hexamminecobalt chloride (Hanahan (J. Mol. Biol. 166: 557-580 (1983).
Incubation of
E. coli
. in solutions that contain multivalent cations is an important step in the transformation of
E. coli
. A number of multivalent cations are capable of affecting DNA transformation of
E. coli
. In addition to calcium cations, manganese, magnesium and barium cations can affect DNA transformation of
E. coli
and the use of manganese or barium cations rather than calcium cations has lead to higher transformation efficiencies with some strains of
E. coli
(Taketo, Z.
Naturforsch Sect. C
30: 520-522 (1975); Taketo, Z.
Naturforsch Sect. C
32: 429-433 (1975); Taketo & Kuno,
J. Biochem.
75: 895-904 (1975)). A variety of other compounds affect transformation efficiencies. Organic solvents and sulhydryl reagents can also influence transformation efficiencies (Hanahan (J. Mol. Biol. 166: 557-580 (1983); Kushner, In:
Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering
, Elsevier, Amsterdam, pp. 17-23 (1978); Jessee, J. A. and Bloom, F. R., U.S. Pat. No. 4,981,797 (1991)).
Incubation of
E. coli
at temperatures around 0° C., often on ice, in buffers containing multivalent cations is an important step in the production or generation of competent cells of
E. coli
. A rapid heat shock or temperature transition after incubation of the
E. coli
with target DNA further improves transformation efficiencies (Mandel and Higra, (
J. Mol. Bio.
53: 159-162 (1970)). Typically, the solutions containing
E. coli
and target DNA are transferred from 0° C. to temperatures between 37 and 42° C. for 30 to 120 seconds. The temperature at which
E. coli
bacteria are grown prior to incubation at 0° C. can also affect transformation efficiency. Growing
E. coli
bacteria at temperatures between 25 and 30° C. can improve the transformation efficiency of
E. coli
bacteria compared with
E. coli
bacteria grown at 37° C. (Jessee, J. A. and Bloom, F. R., U.S. Pat. No. 4,981,797 (1991)).
E. coli
bacteria that are grown at temperatures between 25 and 30° C., in contrast to 37° C., may require a heat shock at less than 37 to 42° C., or a heat shock of a shorter duration, for optimal results (Jesse and Bloom, U.S. Pat. No. 4,981,797 (1991); Inoue et al.
Gene
96:23-28 (1990)).
Transformation efficiency can be affected by the
E. coli
strain used. The selection of an
E. coli
strain that is capable of high transformation with the specific competence protocol adopted is an important step in the development of a procedure to produce
E. coli
bacteria capable of high transformation efficiencies. Different strains can exhibit different transformation efficiencies depending on the competence protocol used. Lui and Rashidbaigi,
BioTechniques
8: 21-25 (1990), compared the transformation efficiency of five
E. coli
strains, HB101, RR1, DH1, SCS1 and JV30 and showed that the transformation efficiencies of these strains varied according to the methodology adopted.
A number of procedures exist for the preparation of competent bacteria and the introduction of DNA into those bacteria. A very simple, moderately efficient transformation procedure for use with
E. coli
involves re-suspending log-phase bacteria in ice-cold 50 mM calcium chloride at about 10
10
bacteria/ml and keeping them ice-cold for about 30 min. Plasmid DNA (0.1 mg) is then added to a small aliquot (0.2 ml) of these now competent bacteria, and the incubation on ice continued for a further 30 min, followed by a heat shock of 2 min at 42° C. The bacteria are then usually transferred to nutrient medium and incubated for some time (30 min to 1 hour) to allow phenotypic properties conferred by the plasmid to be expressed, e.g. antibiotic resistance commonly used as a selectable marker for plasmid-containing cells. Protocols for the production of high ef

Affiliated with

Also associated with

Rating

Method capable of increasing competency of bacterial cell... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method capable of increasing competency of bacterial cell..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method capable of increasing competency of bacterial cell... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3271356