Re: Possible Kickstarter projects

Enzyme rescue can be achieved either through transformation and
selection, or PCR.

The former works simply enough; using high-efficiency transforming cells
(you might have to invest in some prepared Top10s if you're not good at
prepping high-efficiency cells, or just try repeatedly with simpler
methods), transform E.coli with the enzyme* and select on a variety of
commonly-used antibiotics. If there's enough plasmid in the enzyme to
transform the cells, you should see colonies on at least one plate. If
you see transformants on more than one plate, the strain might contain
more than one plasmid, which hints at a more complex expression system;
you might need both..

The latter works simply too but makes some big assumptions: look up the
sequence of the wild-type enzyme-coding-DNA, and make primers for it.
Then run PCR with these primers and a little of the (heat-inactivated)
enzyme prep as template. If the enzyme is being produced with
mostly-original DNA, you should get a band corresponding to the size of
the gene.

Of course, a kickstarter could probably fund synthesis, which would be
nice for a few reasons: The enzymes are usually prepared industrially
with chromatography (HPLC), which is hard to reproduce. You'd waste a
lot of time cloning the gene into a form that's easy to isolate
(ice-purification is nice, but I like the look of elastin-like-polymers
for a DIY-friendly enzyme prep), and spend a lot of money on appropriate
enzymes, primers and buffers. I'm a big believer that for those without
a pre-existing lab, it's cheaper to get it synth'd.

I'd be delighted to get on board such an effort; it's on my list of
things to do already. I want a suitcase full of strains that make
everything you need for a new biolab; enzymes, dyes, polymers, sugars,
antibiotics...

*If you fail with raw enzyme, you *could* try purifying plasmid from the
enzyme with a gel extraction spin column kit. Miniprep probably won't
work, as it doesn't "expect" the input to be buffered as strongly as
enzymes generally are..

On 15/11/11 06:37, Robert O'Callahan wrote:
> The Ice Nucleation Protein is part of an iGem distribution, so that part
> would not need to be synthesized. Most of the genes of interest could be
> cheaply cloned out of their species of origin, which could be had either
> through DSMZ or another culture collection or just asking authors of papers
> working with the bugs.
>
> A basic bootstrapping set of enzymes might be the enzymes required for
> basic iGem standard assembly: thermostable pol, EcoRI, PstI, XbaI, SpeI, T4
> ligase. Adding T4 DNA pol would be useful for SLIC cloning.
>
> Re salvaging proprietary DNA from polymerase extracts, I had wondered if
> anyone had tried that one. How I would love to know the secrets behind
> Kapa's HiFi pol... I oughta try that trick out this week.
>
> -Rob

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