Craig Venter synthesis, possible with pcr?

[Koeng]

I was checkin out this map of how Craig Venter created a synthetic bacteria cell

http://www.jcvi.org/cms/fileadmin/site/research/projects/first-self-replicating-bact-cell/handouts/image5-lettersize.pdf

Could've this been done with just a hell lot of PCR reactions, PCRing clusters of genes and leaving out the ones that they thought were not needed? Considering all the costs, would it have been cheaper? Also, at the cheapest how much would a PCR reaction be (with and without polymerase)? Any thoughts?

Koeng

[Dakota Hamill]

I'm guessing mainly because if everything wasn't synthesized in an
oligo machine, and was selectively PCR'ed out of a living organism
instead, it wouldn't be a truly "synthetic" organism.

[Koeng]

True. The company probably "spares no expense" since they have a buttload of money from big oil

[Dakota Hamill]

Yeah you asking that question made me re-read some things about
Venter. He has done a lot of grandiose things and seems like a
badass.

I'm still trying to figure out how he became a multimillionaire and
got so many huge projects funded. Wikipedia says he graduated with a
PhD, was a professor for a little, then went to the NIH. But then I
don't know what happened in between and all I remember is hearing
about him years ago out on a giant sail boat sequencing things in the
ocean, then doing the human genome project, then doing synthetic
genomic things.

[Koeng]

I think he convinced an oil company that he would make them awesome biofuels. Too bad they are spending millions on a project that could have been done with thousands.  

[Mega]

Of course it would have been possible by PCR, and yes, probably much cheaper. 

They just took the genes of one bacterium, left some away. So basically you only'd need one template (+ Resistance marker template & optical marker)

But, as Dakota pointed out, that would ba a semi-natural oranism, or rather a truely natural organism, as its genome was created by biological reactions (in vitro)...

[Eugen Leitl]

It's basically a modern iteration on http://en.wikipedia.org/wiki/W%C3%B6hler_synthesis

[Bryan Bishop]

On Sat, Apr 6, 2013 at 7:42 PM, Dakota Hamill <dko...@gmail.com> wrote:

I'm still trying to figure out how he became a multimillionaire and
got so many huge projects funded.  Wikipedia says he graduated with a

You have missed all the Celera things and his plans for world domination by patenting the human genome.

- Bryan
http://heybryan.org/
1 512 203 0507

[Cathal Garvey]

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To be fair (though I'd rather be harsh, because Venter's a jerk and
owns patents over parts of me), you can't realistically PCR/clone
whole chromosomes. At such scales, not only does the error rate
propagate wildly, but the simple fragility of large, unpacked DNA
molecules comes seriously into play.

What smart-people-employed-by-Venter developed were powerful ways to
incrementally assemble large DNA molecules from smaller (but still
pretty big) subunits while avoiding these issues; some of these
methods involve in-vivo assembly, so co-transformation of lots of
manageable chunks leads to the cell doing the hard work of assembling
the full chromosome.

Gibson assembly, if I'm not mistaken, was one of the innovations to
emerge from this work, but even Gibson assembly is unlikely to work
for very large molecules. Another neat trick is co-transformation of
large linear DNA with homologous ends into B.subtilis (think this
works with Yeast, too), whereupon the cell does the end-joining for
you. This doesn't work in species lacking efficient recombination
machinery; B.subtilis is nice because it features active linear DNA
uptake coupled with a deliberate recombination system, so efficiency
is apparently pretty good.

I'm interested to see developments in serial assembly, so you
transform cells with your minimum genome, and it contains cassettes
and target regions that await further input in the form of additional
linear segments, and assemble them as-given in order. That way, you
could do the *really* hard part once (getting your minimal genome in
place) and then do serial transformations with every additional piece
to assemble a more complex genome on top of it. I imagine this working
like a ratchet, where each new segment contains a selection cassette
like ampicillin, and after each successful selection the cells are
induced to excise and destroy this selection cassette, allowing
selection with the same cassette again. A bit of DNA logic and
recombinases, and there you go..

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[Ashley Heath]

I suggest that you all calm down a little and read Ventner's autobiography. That might put his efforts and successes into a more reasonable perspective!

[Cathal Garvey]

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His effort to patent the whole human genome wasn't termed the "Human
Genome Project": That label generally applies to the efforts of the
Human Genome Consortium that spared at least a large fraction of our
genetic heritage from Venter's grasp.

Although, it's highly debatable whether Venter could even have
sequenced the genome successfully using the methods Celera were
attempting to push at the time; his successes with Bacterial
chromosomes didn't translate well over to redundant, repeat-rich,
massive human genomes.

Suffice to say, if you're willing to leverage potential ownership over
something critical to scientific advancement in human biology and
medicine, you can get plenty of business-before-ethics people on board
to pay for your big projects. And, likewise for the concept of
"minimal cells", which will no doubt be very broadly patented by
Venter & co, too as soon as they succeed in making a reference
implementation. Given the amount of big oil money being lavished on
their work, I don't doubt they'll beat the better intentioned labs
around the world to that goal, as there's no "minimal cell consortium"
to save the day this time.

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[Mega]

Brought me of a nice idea: A talen-coding gene, induced by tetracycline. The Talen destroys the Ampicillin gene. Thus you could insert a cassette with a amp marker, select on amp, put the transformants onto tet-agar, and do another integration of a casette.

[Koeng]

Ampicillin induction? Is there any operators for that?

Also with the organisms that are minimal I believe are resistant to ampicillin :(

-Koeng

[Cathal Garvey]

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To answer that, you'd have to dig into the origins of the most
commonly used Ampicillin cassette. I believe that's "bla", for "Beta
lactamase", from a Shewanella species, although in the literature it
might have been called "Escherichia" at the time as I believe
Shewanella is a fairly recent reclassification.

One easy route is to take an "old" version of Amp from an early
cloning plasmid and run it through BLAST, selecting bacterial genomes
to filter out cloning plasmids and constructs. First few natural hits
should contain wild species with homologous genes, and you can analyse
their promoters to see if they are dumb/constitutive or if they are
induced by beta-lactam antibiotics like Amp.

Even if they are though, it might be (probably is) an indirect
induction mechanism, something like:
Amp -> Reduced Indicator of Health -> Stress Response Genes -> bla

..so it mightn't be particularly useful, given how many ways there are
to accidentally activate stress response genes during normal culture
conditions.

I'd say that's a shortcut that would require a lot of work to attempt
(characterising Amp-inducible promoters), and may not be worth it,
considering how many well-characterised and effective promoters there
are available in the literature. Use something lazy like light
induction, chemical induction, etc.

On 04/09/2013 02:18 PM, Koeng wrote:


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[SC]

Most of the finances for Venter's version of the human genome project came from sales and royalties of "Big Dye", which is a component of the first automated sequencing method.  He also got some revenue from the NIH, although that partnership turned sour according to his autobiography. 

The sequencing business has changed a lot since the Big Dye method, and he recently downsized his institute and laid off nearly half his staff. We interviewed many of them (they were all looking for jobs in the area at the same time), and they had horror stories.

 

[Bryan Bishop]

On Tue, May 7, 2013 at 11:50 AM, SC <stac...@yahoo.com> wrote:

The sequencing business has changed a lot since the Big Dye method, and he recently downsized his institute and laid off nearly half his staff. We interviewed many of them (they were all looking for jobs in the area at the same time), and they had horror stories.

Feel free to share the horror stories. I am sure many of us are interested. From talking with some of the JCVI programmers, their shop is not exactly focused on quality software or long-term maintenance.

[SC]

JCVI management knew about the layoffs months before they happened (private grant was not renewed), but didn't make an announcement to the employees.  Of course, rumors got out, they were denied by management, and all of a sudden everyone had two weeks notice.  I think this shows a general lack of respect for the employees.

.

When they broke ground in California to build another institute, the remaining people were told it would be in addition to the Rockville facility, and only found out the Rockville site was closing after seeing a press release. 

.

With all due respect for Venter's contributions to the field, he seems like a dick to work for.

.

His loss is our gain though, we hired some of his very talented former employees.