[Hplusroadmap] Cheap bioreactor mostly for the construction of further bioreactors

[Bryan Bishop]

On Friday 30 May 2008, Frog wrote:
> Bryan,

Sorry for my delay in response. I was munching over your suggestions.

> Been watching from the sidlines for a while, but now something I can
> contribute.  I was at LTI (now know as the mud sucking dogs
> Invitrogen) for 15 years, making Taq and DNA polymerases and RT's and
> restriction enzymes, proteases, kinases, yada, yada, yada.  Also did
> quite a bit of Oligo synthesis

Neat. I think we'd all love to hear from you more frequently. I don't 
quite have an as-impressive resume built up quite yet.

> In order for your "aptamers" concept to work, our protein of interest
> would need to have some affinity for the oligonucleotide/peptide
> "backbone".  The main technical hurdle here would be the length of
> the aptamer molecule.  It would need to be pretty damn specific to
> the protein of interest.  Typically, this kind of chromatography
> would be used as a "polishing" step and really would be characterized
> as "affinity chromatography".  I believe it would likely be simpler,
> less technically challenging and less expensive to make the
> antibodies.

So I talked this over with a few buddies re: the aptamer specifity. It 
turns out that there's another conceptual problem to work off of too. 
If you're going to be making aptamers, you're probably going to be 
doing it from a pool of random RNA sequences. And then from there you 
can run your selection experiments once they are in aptamer form, and 
then clean everything else off, denature, transcription run, some gels, 
PCR, all of that fun stuff. Then you replicate the same sequences and 
get to make many many more aptamers. But if there's not a match in a 
random DNA pool of strands of length-N bases or whatever, it's going to 
take a DNA synthesizer in the process, which means length issues (both 
in the ability to build the molecule, but also its simulation and 
analytical constraints on the possibility space of the N units). Could 
be fun, if we already had all of those setups. How much easier are the 
antibody protocols?

> Ion exchange is much simpler, first step.  This would separate
> protein on the basis of their net charge (either positive or
> negative).  It can be done in  aggregate batch mode or in a simple
> fritted glass or plastic column.
>
> I am not sure why you think you'll need a "bioreactor" per se.  Most
> of the bugs you'd need to grow could be done in a mini-prep format:
> 1-2 Liters in any clean glass jar.  The proverbial "mayonnaise jar"
> approach.

Yeah, I'm all for the mayo jar approach.

> It will be important to have the purest protein prep achievable, as
> impurities will compound and amplify mistakes in any synthesis
> reactions.

On that note, how hard would it be to come up with sets of reactants and 
either the antibodies, aptamers, or chromatography setups to repeat the 
process on demand? The idea is to eventually have these same cells, or 
at least a subset of them in some strain/batch, produce the chemicals 
needed to do the transcription kits, etc.

> Then there is the necessity of a thermostable DNA polymerase for PCR
> and sources of pure nucleotides for the synthesis.

The pure nucloetide problem sounds like a hard one, so what about the 
oligo synthesis reaction routes for synthesizing individual 
nucleotides, or the possibilities of centrifuging cells and collecting 
nucleotides? Maybe even denaturing lots and lots of protein from 
acquirable sources, like grass or meat? 

As for thermostability in DNA polymerase. A good number of protocols 
that I have been looking at indicate that there's a few steps at 90 deg 
Celsius and then back down to stuff well below room temperature. I 
don't know if this is practical for do-it-yourself setups. There's 
certainly the possibility of using heat lamps coupled to a digital 
thermometer plus sensors in a box, but I don't know if this can be used 
accurately enough. Ideally we could just find something that works in 
something 'close to' room temperature. Is this what you mean by a 
thermostable DNA polymerase? 

> So, I have taken a slightly different approach.We have been
> successful scooping up gear (like fermentors, spectrophotometers,
> stir plates, heat blocks, microscopes, hoods etc.) and chemicals
> (including 25L of Sephadex resin and 25L of Protein G agarose, open
> market value @ ~ $1,000 per liter) for nearly free.  The catch is you

How? Networking? Dumpster diving? Foul play?

> need to find Biotechs that are going under (which mean you'll need a
> target rich environment) and have a means to haul the stuff out of
> their facility.  It's simple economics.  Most of these places have
> already expensed or capitalized their losses and are not looking to
> recuperate any revenue from their "junk".

Hm, so targeting the rich environment might really involve relocation 
from a 'non' biotech area. Kinda of an important step in the whole 
process. For those on the list up in Boston, I envy you. ;-)

- Bryan
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