According to Essential Science Indicators
from
Thomson
Reuters, the paper ranked at #2 in the field of
Microbiology is "One-step inactivation of chromosomal
genes in Escherichia coli K-12 using PCR
products," (Datsenko KA, Wanner BL, Proc. Nat.
Acad. Sci. 97[12]: 6640-5, 6 June 2000), with 2,023
citations up to December 31, 2008.
The paper's authors are Dr. Barry Wanner and Dr. Kirill Datsenko, both
of whom hail from the Department of Biological Sciences at Purdue
University in West Lafayette, Indiana. Dr. Wanner is a full professor in
the department, and Dr. Datsenko is a research associate there. Dr. Wanner
has published 100 manuscripts and book chapters, including 31 papers
recorded in Essential Science Indicators as cited a total of 2,932
times, and Dr. Datsenko's record includes 9 papers cited a total of 2,118
times.
In the interview below,
ScienceWatch.com correspondent Gary Taubes talks with both
authors about this paper and its impact on the research
community.
In 2000, you published a methods paper in
PNAS that now has over 2,000 citations: "One-step inactivation
of chromosomal genes in Escherichia coli K-12 using PCR
products." Why is this method of gene inactivation so special? What
exactly does it do?
Wanner: The method itself is actually a very rapid and efficient method for
making mutations. As geneticists who study physiology, one of the key ways
in which we do our work is basically to break a pathway—break
something in a cell—and see what’s broken and how that affects
the physiology or growth of the cell. This method was a way to target
particular genes for interruption. Thirty years ago, when I started in this
field, we would look for random mutations and then identify the gene that
was mutated, that was responsible, and try to understand the process. Now
we have 4,000 or 5,000 genes, or DNA sequences that we believe are genes,
and we have a way of targeting each of them with high precision.
That’s what this method is all about. It’s a way to do reverse
genetics very efficiently. It makes making mutations easy so you can focus
on trying to determine the function of the genes, on the biology rather
than the labor. It’s a technology, one that’s quite simple to
use and can be used in different ways, not just knocking out genes but
modifying them.
Wanner: Prior to this work it would take maybe two to three weeks to make a
single mutation from start to end. Using this method, as we are now, with
robots involved, we can make hundreds of mutations in a single day.
What were you working on in the 1990s that led up to
this work?
Wanner: I’ve been studying signal transduction using E. coli
as a model cell for a long time. This cell was the focus of both my Ph.D.
work and postdoctoral research—in particular signal transduction and
the question of how a signal gets across a membrane. I’ve continued
to study that at a more and more detailed level ever since.
Datsenko: I was trained as a classical microbiologist. I started my career
studying the phosphotransferase system in the phytopathogenic bacteria
Erwinia, and when I moved to Purdue, I shifted my research to E.
coli because that was the focus of the laboratory. That was in early
1999.
What gave you the idea for your gene-inactivation method
in E. coli?
Wanner: What prompted this particular study was actually a paper published
in 1993, reporting the development of a similar method for a different
organism, a eukaryote—Saccharomyces cerevisiae. What it did
was make gene disruption extremely simple in yeast, but nothing like it
existed for other organisms at the time. That method was actually used
later to make the first knockout organisms, which were also done in
Saccharomyces cerevisiae. So I started work on this as a part-time
project, along with other members of my group who had left the lab by the
time Kirill arrived. I had actually done some of the last experiments on
this technique with my own hands. Once Kirill arrived, a new person in the
group, it became his project as an introduction to the lab.
Why Kirill? Was there something unique about his
expertise?
Wanner: It was more a matter of timing. He had just gotten his Ph.D. in
Moscow before joining my group, and he was interested in learning bacterial
genetics. The last person other than myself to work on this was a post-doc
who had left the laboratory more than a year earlier. The other researchers
in my lab were grad students with other projects to work on. So Kirill got
it.
Were you aware at the time how significant this could
be?
Datsenko: I have to admit that before this paper was published, I
didn’t think it was such a big deal. For me it started like a side
project: I was supposed to develop this technique as a tool to isolate a
certain mutant; at that time, it was not the main focus of my research. It
wasn’t until November 1999, when Dr. Wanner came into the lab and
congratulated me because the paper had been accepted in PNAS that
I realized it was something special.
What was the major obstacle or challenge that had to be
overcome to make this method work?
Wanner: We always knew the method would work—based on earlier results
we had obtained. The problem was that it was extremely inefficient. Until
Kirill persevered with it, getting it to the point that it was 100%
reliable, it was never efficient enough to use.
Was there one particular breakthrough that made it more
efficient or just a series of minor fixes and improvements?
Wanner: The method was working all along but the problem was that we would
isolate maybe 1,000 cells on a petri dish and only a few of those would be
correct. We had a way of identifying those one or two that were correct,
and what Kirill started doing was looking at the other 998 or 999 and
trying to figure out what was wrong with them.
So his breakthrough was to find out what was wrong with the
majority—this high background—and then how to eliminate the
background so we’d end up with just the one or two that were correct.
Well, maybe there were 10,000 cells on a plate and 10 and or 20 that were
correct. Either way, what we did was just eliminate the
background—the incorrect cells. So Kirill was tinkering to find out
what was wrong with them and that was the real technological breakthrough.
Other people had always focused on the correct cells; Kirill figured out
what was wrong with the incorrect ones and how to eliminate them.
How do you eliminate the incorrect ones?
Wanner: The method is based on synthesizing DNA using PCR. In order to do
that, you use a template, and the problem was that the template plasmid was
undergoing other types of recombination events—the template itself
was actually the source of the background. We worked out a couple of ways
to prevent the template plasmid from entering the cells. Simple chemical
purification of the PCR product was insufficient. We always had some
template plasmid present.
One approach was to use a template plasmid that would be unable to
replicate; another way was to use a certain type of restriction enzyme that
restricted or digested only the template plasmid after amplification. Using
the two together we completely eliminated this background problem.
Was it immediately clear from the response of the
community that you had achieved something particularly significant in
the field?
"It’s a way to do reverse
genetics very
efficiently."
Datsenko: Actually, the opposite. I remember when Dr. Wanner first reported
about our procedure at a meeting, before the paper was published in
PNAS, no one got excited. Nobody asked any questions. They treated
it as just another boring report. That was it.
Wanner: Every two or three months, a group of us from universities in the
Midwest would get together and discuss our research. I had given a
presentation describing this work at one of these meetings. I was quite
excited about it because we were in the business of making mutations and
that’s what this method allowed us to do. But it didn’t seem to
be well appreciated at that meeting. Maybe a couple of people came up to
talk to me about it afterward.
Malcolm Casadaban, a geneticist from the University of Chicago, said it was
the best thing since baked bread or something like that. He was a very
bright guy and he was very enthusiastic. But most people just didn’t
get it. Maybe my presentation wasn’t as clear as it could have been
and Dr. Casadaban understood anyway, because he was a geneticist.
Shortly after that, I presented it at another meeting of geneticists and
they were all quite excited. Ever since then, we’ve been getting
requests for this system from a wide range of people, even those who have
no expertise in genetics. In fact, half a dozen have visited the lab to
learn the technique, although we never thought that was necessary. Still,
we continue to get requests, usually one or two a day, for the actual
system and the necessary materials.
Why did you decide to publish it in PNAS rather
than more of a specialty journal?
Wanner: Previously I had published three or four papers in PNAS,
and I wanted a journal that is read widely. We might have published in
other excellent journals like Molecular Microbiology, although
this was a methods paper and it doesn’t take methods papers, or the
Journal of Bacteriology, which does take methods, but I thought
this deserved a broader audience.
Was there any element of serendipity in making this PCR
method work?
Wanner: I’m a great believer in serendipity in science, but that
wasn’t the case here. This one worked through perseverance and
because we tried a number of approaches until we hit on the right one.
Are there competing techniques for accomplishing
targeted gene disruption in E. Coli?
Wanner: Francis Stewart in Germany had published a method that was
analogous to ours, but much less efficient and not very practical. He had
published his paper before ours in Nature Biotechnology, but I
didn’t actually become aware of it until I was writing up our
manuscript.
During the summer of 1999 I did speak to Don Court at NIH, who had
developed a method very similar to ours. We had exchanged strains and
things and I discussed with him specific problems we were having with our
background. In fact, I cited him in the acknowledgements for these
discussions, because he told me of a method he was using that reduced the
background in his case.
We ended up using both his approach as well as the digestion approach using
these restriction enzymes. He also published about the same time we
did—also in PNAS. And he has gone on to develop this
technology much farther. We were never interested in developing our
technology so much as using it for research.
How are you using the method in your research
now?
Wanner: We’re working in collaboration with Hirotada Mori’s
group at the Nara Institute of Science and Technology in Japan. The work is
largely done there, although both Kirill and I are co-authors. We’ve
employed this technology to make a complete knockout set of all
nonessential genes of E coli. We’ve recently gone farther in
a manuscript I’m now writing, making a second knockout set. The
reason for making two is we’re embarking on making double knockouts
to look at how one gene interacts with another gene, doing these genetic
interactions genome wide. We published the first two manuscripts on this
last summer in Nature Genetics, along with Carol Gross’s
group at UCSF, and Jack Greenblatt and Andrew Emili’s group at
University of Toronto. We developed the technology in collaboration with
Mori and have now published six or seven papers with him since around 2003.
What we’re gearing up to do is use these double knockouts with
genome-wide scans. We’re using double plates that have 1,536
wells—96 x 16—and that’s because even a simple organism
like E. coli has 4,000 nonessential genes. It has about 4,400
genes total. We want to make doubles of all 4,000. That’s 16 million
strains and it has to be done in quadruplicate for statistical reasons.
This is very, very high throughput. And the wealth of information
we’re getting out of this is just amazing. We’ve already been
discussing this work at meetings. Some of it is now reported, most is not.
Already we're getting requests for the system from other groups.
Did the 1999 PNAS paper change the way you saw
your career progressing?
Datsenko: It allowed me to establish many research relationships with
scientists around the world, which resulted in multiple successful
collaborations.
Wanner: I have actually become better known myself for this technology than
any of the actual science I’ve done, either before or
since.
Dr. Barry L. Wanner
Department of Biological Sciences
Purdue University
West Lafayette, IN, USA
Dr. Kirill A. Datsenko
Department of Biological Sciences
Purdue University
West Lafayette, IN, USA
Datsenko KA, Wanner BL, "One-step inactivation of
chromosomal genes in Escherichia coli K-12 using
PCR products," Proc. Nat. Acad. Sci. USA 97(12):
6640-5, 6 June 2000. Source:
Essential Science Indicators from
Thomson
Reuters.