Methods and reagents: Preparing ultra-competent Escherichia coli
 
Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet.  This month's column discusses how to get the most out of your
transformation procedure and how to prepare your own ultra-competent
Escherichia coli cells.  For details on how to partake in the newsgroup, see
the accompanying box.

Do you lack consistency with your transformation protocol or find your
preparation time is far too long? Then, maybe it's about time you switched
protocols.  Procedures based on a simple CaCl2-wash method provide a fair
number of transformants when you only need to change bacterial host strains or
if you receive plasmid DNA from someone else and need to propagate it.

The hare
********
The simplest methods [1-3] are quick and dirty, but rarely yield more than 1 x
10^4 to 1 x 10^6 transformants per ug of purified plasmid DNA, which could
amount to a substantial loss of recombinants when efficiency is of the utmost
importance, for example when constructing a random library.

The most widely known procedure used for commercial preparations of
ultra-competent E. coli uses techniques based on the work of Douglas Hanahan
[4,5], in which he optimized many different parameters. By washing the cells in
many different divalent cations and by adding reducing agents, chemical
transformation can be quite efficient, and because the Hanahan procedure
usually yields from 1 x 10^5 to 1 x 10^8 transformants per ug of DNA, some
people are convinced that this is still the best protocol available.

However, not only have others tried to reproduce much of Hanahan's work with
mixed results [6], but they have addressed some other factors that have not
been previously tested and that have since been shown to increase competency.
Methods other than those above can be faster and simpler, with minimal loss of
competency. For example, the use of a single solution method can provide
competent cells in less than 20 min.

Chung et al. [7] showed that JM109 cells prepared in a single transformation
and storage solution composed of LB media with 10% PEG, 5% DMSO, and 50 mM
Mg2+, pH 6.5 (TSS) were able to produce 5.12 x 10^7 transformants per ug of
pUC19 DNA when made fresh, and after being frozen at -70 degrees C for 1-18
weeks, gave 6.17 x 10^7 transformants per ug of DNA.  Another study [8]
extended this, showing that 36% glycerin and 12% PEG added to the media allowed
1.07 x 10^8 JM103, 1.17 x 10^8 DH1, and 1.29 x 10^8 HB101 transformants per ug
of pBR322 DNA.

Epicentre Technologies now prefers the use of this method for preparing their
frozen competent cells and claim the standard TSS gives an efficiency of 2.4 x
10^6, while their own modified TSS protocol gives 7.5 x 10^6 HB101
transformants per ug of pGEX-2T DNA using 5 ng.  In addition, one netter said
that he found an increase of about 50- to 100-fold in efficiency when he
switched from the standard CaCl2 method to the single-step TSS protocol.

The tortoise
************
By far, the most preferred method of netters for producing ultra-competent E.
coli cells by a chemical method is that of Inoue et al. [9] Interestingly, a
very important step included in this protocol seems to be the growth of cells
at 18 degrees C.  It was reported that 1.44 x 10^9 JM109, 1.84 x 10^9 DH5alpha,
and 1.00 x 10^9 HB101 transformants per ug of pBR322 DNA were obtained with
this method.  In practice, however, one netter said that an efficiency of 2 x
10^8 to 3 x 10^8 was more typical and that only occasionally might one expect
to see upwards of 2 x 10^9 XL1-blue cells transformed per ug of pBluescript
DNA.  On the other hand, some netters have reported that 1 x 10^8 to 1 x 10^9
for DH5alpha and XL1-blue strains is common, and some have reported obtaining
efficiencies as high as 1 x 10^10 on occasion.

Although the efficiencies using this method are among the highest, the down
side is the requirement for a cooled incubator, and owing to the lower
temperature of incubation, the growth of the bacterial culture takes much
longer to arrive at the desired cell density, which makes this a much more
lengthy protocol. Also, several washing steps must be performed adding to the
overall prep time.

Netters feel that the tradeoff in time is worth it and that preparing a large
batch of cells and freezing aliquots of them at -70 degrees C can limit the
number of preparations done in a year.  Also, the long wait can be avoided by
determining the doubling time (usually 3 to 4 hours for DH5alpha and XL1-blue)
and by inoculating late in the evening, then harvesting the cells early the
next day.  Netters also agree that the culture can be grown at room temperature
(20-22 degrees C) without significant loss of efficiency, thereby avoiding the
use of the cooling equipment. In addition, the longer doubling time might help
alleviate the difficulty in catching the cells at the more optimal cell density
of an OD600 of 0.94 [Ref. 10] as discussed in a previous Methods and reagents
column (TIBS 19, 426-427), although this has not yet been thoroughly
investigated.

As nobody knows why this lower temperature growth works so well, there are some
gaps in the knowledge of exactly how the cells become competent.  One idea put
forth by Mikhail Alexeyev (malexeyev@biost1.thi.tmc.edu) is that E. coli might
be most susceptible to chemical treatment that induces competency only at a
certain stage of its cell cycle. The slower growth of the cells at lower
temperatures could allow more cells to be captured at this stage and maybe this
temperature somehow synchronizes them and they can be caught at the correct
stage more easily.  This idea could be tested quite easily by synchronizing
cells at different stages and then testing their competency.

Although some might think that the development of bacterial transformation
protocols has been through its heyday long ago, the ability to consistently
produce highly competent E. coli cells still remains somewhat elusive.
Variability in transformation efficiencies seen by netters suggests that many
parameters still need to be explored.

Overall, a single method can be chosen from the many methods available, but a
combination of various different methods is most likely the best bet for
increasing efficiency beyond 1 x 10^9 transformants per ug on a regular basis.
For example, one of the parameters not addressed in the work of Inoue et al.
[9] is the inclusion of reducing agents in the transformation reactions, as
noted in earlier work.

Zophonias O. Jonsson (zjons@vetbio.unizh.ch) wrote that he did a comparison of
BL21(DE3)plysS transformed with a pET derivative using cells grown at 23
degrees C that were prepared by the Inoue method. When treated with reducing
agents as per the Hanahan procedure, beta-mercaptoethanol increased efficiency
five-fold while dithiothreitol did so by 20-fold.

Increasingly, commercial suppliers are offering frozen ultra-competent cells
that are claimed to provide the best efficiency for transformation. It appears
that the companies have tweaked the process for increased transformants and,
having created their own slight modifications, refuse to reveal exactly how
they have pushed the limits of competent cells in order to remain competitive.
Netters are not only distraught by this type of action, but think that it is
time a new protocol be made publicly available that can push the competency
level one step closer to that obtained by electroporation.  Most feel that many
different parameters still need to be explored, and that it is probably worth
some effort to develop a new and better protocol.  They are convinced that the
high transformation efficiencies of using electroporation might some day be
replaced with an ultra-competent-producing chemical method pushed through the 1
x 10^10 efficiency envelope (for a discussion on electroporation, see TIBS 20,
248-249).

Breaking the cell competency barrier could all but eliminate the need for
expensive equipment and cut the high cost of frozen electro-competent cells and
special cuvettes now sold by the suppliers.  Netters feel that one day soon
ultra-competent cells will be able to be prepared in-house by chemical means
and that this capability may be just around the corner.

References
**********

[1]  Cohen, S. N., Chang, A. C. Y. and Hsu, L. (1972)
     Proc. Natl. Acad. Sci. USA 69, 2110-2114
 
[2]  Dagert, M. and Ehrlich, S. D. (1979)
     Gene 6, 23-28

[3]  Takahashi, R., Valeika, S. R. and Glass, K. W. (1992)
     BioTechniques 13, 711-715

[4]  Hanahan, D. (1983)
     J. Mol. Biol. 166, 557-580

[5]  Hanahan, D. (1985)
     in DNA cloning: a practical approach (Glover, D. M., ed.),
     pp. 109-135, Oxford

[6]  Liu, H. and Rashidbaigi, A. (1990)
     BioTechniques 8, 21-25

[7]  Chung, C. T., Niemela, S. L. and Miller, R. H. (1989)
     Proc. Natl. Acad. Sci. USA 86, 2172-2175

[8]  Nishimura, A. et al. (1990)
     Nucleic Acids Res. 18, 6169

[9]  Inoue, H., Nojima, H. and Okayama, H. (1990)
     Gene 96, 23-28

[10] Tang, X. et al. (1994)
     Nucleic Acids Res. 22, 2857-2858

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Any statements made by the author are not meant to advocate the use of a
particular commercial product or endorse any company. All opinions are
those of the author and do not reflect the opinion of the National Cancer
Institute or the National Institutes of Health.

Copyright: This manuscript is not copyrighted by Elsevier Publishing Company.
However, you may not reproduce any portion for resale or edit the text for
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You found this at the World Wide Web (WWW) Uniform Resource Locator (URL)
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/feb96.txt

Any reference to this column must be cited as the following published article:
Hengen, P. N. 1996. Methods and reagents - Preparing ultra-competent
Escherichia coli. Trends in Biochemical Sciences 21(2):75-76.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh@ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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