E. coli GFP transformation

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Revision as of 00:56, 9 November 2012 by Mycoplasma (talk | contribs)

Genetic modification

We're interested in doing genetic modification on micro-organisms at the London Hackspace. Needed for GFP experiment (that we don't already have):

Fridge? Incubator. Build or buy. Cleaning: Bleach, gloves, paper towels, hand cleaning gel. Bunsen burner + fuel. Glassware, pipettes, tools. Ethanol. Lots.

To apply for a license we will probably first need: Written protocols for stuff, especially cleaning up.

Disposal. Chemical. Sterilisation. Do we need incineration? Find a waste disposal company that will take biohazard material?

Restriction enzymes. At least two. Pricey? GFP plasmid. Antibiotics. Ampicillin. Initial e. coli culture. Growth medium/ broth. LB. Plates. Standard 10cm plates. Agarose. Ice. 40C bath.

Proper storage.

Centrifuge - bigger : hold

Miniprep kit

Maxiprep kit

Growing competent E. coli

Here is the protocol we used with UCL to grow competent cells during the making of the public biobrick. So we need to get E. coli (cannot find how to buy this yet), LB - £66, 0.1M CaCl2 - £32.50, M9 salts - £57.60, MgSO4 - £20, bacteriological agar solution - £42.90, thiamine - ~£20, D glucose - ~£20, and plates. All links are only the first ones I found, maybe cheaper options available.

Self-cloning

Here's a protocol to do a 'self-cloning' transformation, which we would not need a licence for, and so could do now to practise.

  • plate spreaders
  • petri dishes
  • LB
  • Agar
  • Howard has a good protocol for transformation

Storage when materials arrive

  • Some must be stored at -20. E. coli should be refrigerated. (Can we freeze it instead?)
  • Once E. coli is opened needs to be used in a few days. Implies all needs to be used at once.
  • Shipped first class post mon - thurs

Legal

There are a number of things we have to do in order to set up a GM lab.

The relevant regulations are The Genetically Modified Organisms (Contained Use) Regulations 2000 (PDF)

Here is a summary of those regulations: GMO Regulations 2000 summary

New lab

Financial feasibility

Income:

  • Current membership fee is £8 a month.
  • At the moment I would say maximum paying members we could get is 10.
  • So we'd make £80 a month from membership fees

Costs:

  • Rent, lets say £500 a month
  • Other space costs. Utility bills, business rates/council tax.
  • Reagents. We should work out cost per reaction, assuming an extraction, PCR and electrophoresis. I estimate this will be between £1 and £5 per reaction.

So we have a big funding shortfall.

Options for new space

  • Talk to Russ about accomodating us in the new space.
  • Investigate funding from other sources as cannot fund an independent space ourselves.

Funding sources:

Requirements for lab

  • Tiling / washable surfaces
  • Sink
  • Lockable
  • Enough room for 10-15 people to work
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