E. coli GFP transformation

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Genetic modification

We're interested in doing genetic modification on micro-organisms at the London Hackspace. Ideas for stuff to do:

  • The standard GFP experiment
  • Get some ideas from iGem teams

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.

Timescale:

  • Day 1 - prepare bacterial culture plates, incubate at room temperature in the dark
  • Before session - make agar plates and refrigerate
  • Day 5 - Class
  • Incubate plates overnight
  • Day 6 - Examine results

Requirements

  • Some steps state refrigeration is required (E. coli culture on arrival, . Need to check if freezer is ok
  • We'll need a fridge to store E. coli culture on arrival, LB plates once made, and culture plates if there's a delay between incubation and visualisation.

Storage when materials arrive

  • Some reagents must be stored at -20. E. coli should be refrigerated.
  • 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. We could increase membership fees for people who are very active.

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/Jonty/another trustee about accomodating us in the new hackspace. Ie. a bigger room that we could apply for a licence for. (We have no idea when the move will happen)
  • 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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