Highly transformable bacterial cells and methods for...

Details Highly transformable bacterial cells and methods for... Highly transformable bacterial cells and methods for...

1997-05-01

2004-03-16

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S252330, C435S252100

Reexamination Certificate

active

06706525

ABSTRACT:

1.0. TECHNICAL FIELD OF THE INVENTION
The present invention generally relates to the field of recombinant DNA technology. More specifically, the invention describes a novel bacterial mutation characterized by an ability to confer a high efficiency of transformation phenotype, and methods for producing highly competent cells using bacteria comprising the novel mutation.
2.0. BACKGROUND OF THE INVENTION
The process of introducing DNA (and other similar polynucleotides) into host cells is a key aspect of recombinant DNA technology The process by which polynucleotides are introduced into host cells is called transformation. Bacterial cells generally remain the preferred hosts for the majority of recombinant DNA experiments and genetic engineering manipulations. Of particular interest for genetic engineering experiments is the bacteria
Escherichia coli
. Given that “competence” (the ability to efficiently uptake exogenous DNA) is not a natural feature of the
E. coli
growth cycle, artificial procedures must be used to introduce exogenous polynucleotides into
E. coli
. Of particular interest, are a variety of competency inducing procedures that render bacteria, including
E. coli
, more permeable to exogenous nucleic acid. Bacterial cells that have been treated to enhance their permeability to polynucleotides are generally referred to as competent cells.
There are many established procedures for making competent cells. These procedures include the CaCl
2
incubation methods of Mandel and Higa,
J. of Mol. Biol
. 53:159 (1970), as well as numerous well-known variants thereof. Hanahan has made a detailed study of factors that effect the efficiency of transformation of
E. coli
cells (
J. Mol. Biol
. 166:557-580 (1983)) where he describes a method of producing highly competent
E. coli
cells comprising the step of washing
E. coli
cells in a buffer comprising potassium acetate, KCl, MnCl
2
, CaCl
2
, and hexamine cobalt chloride, which is generally regarded as the best available method of producing highly competent
E. coli
. Another method of producing competent
E. coli
cells is described by Jessee et al., U.S. Pat. No. 4,981,797. Jessee et al. shows that high levels of competency may be induced by growing
E. coli
cells in a temperature range of 18° C. to 32° C. as part of the competency inducing procedure.
The various techniques for rendering
E. coli
cells competent produce competent
E. coli
cells having varying of transformation efficiencies. The precise mechanism by which DNA enters competent
E. coli
is not completely understood. Nor is it completely understood why one composition of competent
E. coli
cells differs in transformation efficiency from that of another composition of competent
E. coli
cells. Hanahan, in
Escherichia Coli and Salmonella Typhimurium: Cellular and Molecular Biology
, editor F. C. Neidhardt, American Society for Microbiology, Washington, D.C. (1987).
The above methods have been further optimized to achieve efficiencies of approximately 1×10
9
cfu/&mgr;g supercoiled plasmid DNA. Although this number appears high, the theoretical efficiency for the test plasmid (pUC) is 3×10
11
cfu/&mgr;g. Furthermore, when applied to practical laboratory conditions, such as the transformation of DNA substrates that were ligated rather than supercoiled, the actual number of colony forming units observed was many orders of magnitude lower than that achieved when supercoiled pUC was used as a test substrate.
Even with past developments, only a minute fraction of the cells in a preparation of “competent”
E. coli
cells, are actually competent for DNA uptake. Thus, the methods and cells presently used to generate compositions of competent
E. coli
cells may yet be significantly improved.
Alternatively, other methods of producing competent cells may result in the formation of competent
E. coli
cells that each have an enhanced ability to replicate and expresses exogenously added DNA. Hanahan, in
J. Mol. Bio
. 166:557-580 (1983) has speculated that competent
E. coli
cells contain channels for transport of DNA across the cell envelope, and that the limiting step in determining the competency for transformation of
E. coli
cells are events that occur in the cell after the cell has taken up the DNA of interest, i.e., the establishment step. Another factor affecting the transformation efficiency of a composition of competent
E. coli
cells is the genotype of the cells. Some strains of
E. coli
are known to produce more highly transformable competent cell compositions than other strains of
E. coli
that have been subjected to the same competency inducing procedure.
Subsequent to the initial discovery that
E. coli
could be rendered competent for DNA uptake, studies have been undertaken to increase transformation efficiency. The maximum level of transformation efficiency obtained using the method of Hanahan described in
J. Mol. Bio
. 166:557-580 (1987), which employs the step of washing cells in a buffer comprising potassium acetate, KCl, MnCl
2
, CaCl
2
, glycerol, and hexamine cobalt chloride, is approximately 1×10
9
transformants per microgram of supercoiled pUC18 plasmid DNA. On a per cell basis, this translates to approximately 1 cell out of 300 in the population actually becoming transformed. However, the above number generally only applies to small supercoiled plasmids. When large plasmids, or ligated molecules are involved, as is the case with many recombinant DNA experiments, the number of “competant” cells that actually become transformed is dramatically reduced. As such, a need continues to exist for new and improved methods for producing competent
E. coli
of superior transformability, as well as new strains of
E. coli
that demonstrate superior transformability. Such methods and strains would be of wide interest to most researchers in the field of genetic engineering in that the number of transformations required to obtain the desired result would be minimized. Thus, for example, larger genetic libraries could be built more easily as well as the construction of complex recombinant molecules achieved more readily.
3.0. SUMMARY OF THE INVENTION
The invention described herein provides a method of producing novel strains of highly transformable gram negative bacterial cells such as
E. coli
that may be used in a wide variety of competency inducing procedures. The methods of the subject invention involve methods of mutagenizing bacterial cells, selecting the mutagenized cells for a high efficiency transformation phenotype, and using the mutated genetic material responsible for (or associated with) high efficiency transformation to construct novel compositions of highly competent bacteria.
One embodiment of the present invention is a biologically pure strain of
E. coli
which is characterized as comprising an Hte mutation that confers a high efficiency of transformation (of foreign plasmids) phenotype relative to
E. coli
that lack an Hte mutation.
Another embodiment of the subject invention is the novel strain of
E. coli
XL10-GOLD, having the genotype &Dgr;(mcrA)183&Dgr; (mcrCB-hsdSMR-mrr)173endA1 supE44 thi-1 recA1 gyrA96 relA1 lac tet
R
Hte [F′ proAB lacI
q
Z&Dgr;M15 Tn10 (Tet
R
) Amy Cam
R
]. The invention further relates to frozen compositions of such cells, and methods for making the cells competent.
An additional embodiment of the present invention is the use of cells harboring an Hte mutation to clone or subclone heterologous genetic material of interest.
4.0. DETAILED DESCRIPTION OF THE INVENTION
The presently described invention includes gram negative bacterial cells, such as
E. coli
, that have been genetically modified to have enhanced transformation efficiency after being processed by an appropriate competency inducing procedure when compared to
E. coli
cells lacking the genetic modification. It has previously been shown that microorganisms bearing the proper genotype can display drastically enhanced transformation efficiency. In particular, U.S. Pat. No. 5,512,468, herein incorporated by

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