Open source plasmids: just add bacteria and reproduce at will


#1

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#2

won’t be long now before custom proteins are the cover project of Make…


#3

ABOUT TIME!!!

Quite an enabler for lower-than-insane cost of biotech, including for DIYbio/biopunk.


#4

Molecular biology researcher here with experience in protein purification:

This is a great idea, but it’s important to note that going from bacteria overexpressing a protein product is a far step from ending up with a purified protein product. Keeping enzymes in their correct conformation throughout the purification process is difficult, and the machinery and experience needed to go through the purification protocol is not inconsiderable (depends greatly on method of purification - GST/His tag, size separation).


#5

This is great!
Addgene.org has long had cheap vectors, but they only ship to verified academic/non-profit institutions. BioBricks and their Foundation have attempted to get genetic parts into the hands of non-scientists, but I think they too use Universities as the gatekeepers, so I, as a scientist, welcome a new program to disseminate the knowledge.

On other hand, for pros, time is often as much a limiting factor as money. Sure, you can make your own Taq, but unless you have undergrads to exploit help, it’s a hassle make, purify and validate on your own. That said, I’m always looking for a way to stick it to Fisher Scientific, who is among the worst offenders in over-charging for reagents.

I hope this takes off.


#6

Can you please elaborate? Many pieces of equipment can be designed and built for pennies on a dollar, and a list of requirements can be pretty helpful for such design process.


#7

The important question is: will this turn us all into Splicers?


#8

Sure thing. To preface, this is coming from my experience working in an academic lab. I don’t see why the preparation of protein in a biotech setting would really be different, but it might be. All I can say is that, assuming that lab practices for getting clean protein purification are standard, this is a short (and likely incomplete) list of the stuff you’ll need:

  • Specialized competent cells for the overexpression of protein (we used BL21’s from invitrogen). I haven’t gotten a chance to look over the protocol, but usually you induce overexpression of protein with IPTG. You can also buy more expensive autoinduction media that begins induction of overespression based on quorum molecule concentrations, I believe). If you want to optimize growth, a way to measure the density of the bacteria you’re growing (spectrophotometer) is important.
  • All of the equipment required to transform bacteria (water bath, incubator, LB ampicillin plates, access to an autoclave)
  • A bacterial culture shaker to grow bacteria (including other components such as large glassware flasks - ones with baffles are the best to optimize bacterial cell growth)
  • A floor centrifuge to spin down the cells and lysate after growth and lysis
  • A protein purification column - size can vary
  • GST/his beads (or other antibody beads as necessary - again, not sure about what the protocol calls for in terms of the tag on the recombinant protein). These are generally where you get pretty expensive, but they can be re-used a few times.
  • A peristaltic pump to load fluid onto your column
  • metalware and a set up to hold your column and all of the associated tubing
  • To get ideal purification, a gradient tower to allow a gradient of eluent to run through the column
  • all of the buffers and chemicals that make up those buffers to load, wash, and elute the protein without denaturing it
  • A fractionater set up to distribute your eluate as it comes off of the tube
  • a set up for SDS-PAGE to analyze the fractions in which your desired protein is purest and at its highest concentration
  • Specialized tubes of an appropriate filter size to concentrate your desired fractions into working volumes.
  • storage buffer for your concentrated, purified protein.

#9

Very good list, thank you.
Some of the gadgets/materials seem to be easy(ish) to achieve for low-cost DIYbio or low-budget school labs.

Spectrophotometer, water bath, incubator, gloveboxes, autoclave. Shaker. All these are simple(ish) electronics and mechanics. Centrifuge, that’s a bit more involved mechanics if we talk about higher g. Peristaltic pump and fractionator, also fairly easy. Metalware too. Electrophoresis (PAGE) takes a bit of effort but should be doable, at least the mechanical assembly and the power supply; the gels and chemicals are another question.

At hackaday.com there are quite some pieces of otherwise expensive lab automation equipment made from LEGO. They are fairly concentrated under the “lego” tag.

The buffers and media may be a bit tricky to do at low cost. The main problems I see will be the purity of the available materials.

The special cells and the purification columns and concentrating filters, and the antigen coated beads, are where my brain so far fires blanks…

Thoughts, comments?


#10

The power of free-market capitalism (meaning the nonsecretive, noncrony kind) would finally be unleashed to tear down the barriers to biotechnology-based scientific wealth, as it has done with so many barriers before it

What? You knew someone was gonna make that reference here


#11

It’s pretty standard practice in underfunded labs to DIY competent cells from “borrowed” E.Coli strains, reducing the costs somewhat. It’s also easy enough to isolate your own E.Coli from a swab. I’ve never put the two processes together, but given time, patience and a little luck, it might be doable, providing you’re not too fussy about efficiency. If I still had access to the facilities, I’d give it a shot just for kicks.

I’ve also heard of home-brewed purification columns, too, but I always managed to bludge free ones from the Invitrogen rep. Not much help for the backyard bio-hacker there. But IME there’s usually a cheap way to do things and I’ve seen some great results from shoestring budgets and elderly equipment.


#12

There is also some talk about using Lactobacillus strains. These have the advantage of being about as easy to work with as making a yoghurt, unlike the somewhat fussy E.coli. No idea how well would the overexpression work with those.

I’ll have to look more at the homebrew columns… I am more electronics and somewhat chemistry than “pure” biotech, but the real fun is where the areas overlap.


#13

wanders off to read protocols…
Lactobacillus is a really nice idea. From a quick glance, it’s definitely worth further investigation. And you can keep it in your fridge without running the risk of poisoning the rest of the household.

I went the other way from electronics to bioscience. There’s far more of an overlap than many people think and some of the skills came in very handy when wrangling recalcitrant lab equipment and troubleshooting experiments. Good times. :smile:


#14

And with GFP as the hello-world app you can have a nice glowy radioactive-slime yoghurt.

The compartmentalization of sci/tech is pretty much artificial and arbitrary. A lot of things can be reduced to a combination of physics and engineering.

(And I just won a small battle with a bootloader for an experimental curve tracer/potentiostat. And possibly more toys, perhaps even a power supply for an electrophoresis rig; the thing is intended for easy modifications.)


#15

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