E. coli GFP transformation

From London Hackspace Wiki

Genetic modification

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

  • The 'hello world' GFP experiment
  • Get some ideas from iGem teams

Needed for GFP experiment (that we don't already have):

Fridge? Incubator. Build or buy. Or could use hackspace environmental chamber. Can be programmed with right temp but non-sterile. 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 (one possibility), 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.

Kit:

  • Kit has enough materials "for 16 students, working in pairs" - so 8.

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:

  • 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.
  • Need an incubator (that is dark inside)

Storage when materials arrive:

  • Some reagents must be stored at -20. E. coli should be refrigerated.
  • Once E. coli pack is opened needs to be used in a few days.
  • Shipped first class post mon - thurs

Legal

There are a number of things we have to do in order to comply with the 'contained use' regulation and set up a GM lab. We will need to carry out a risk assessment, submit it to the HSE (with a fee, currently seems to be £465. There may be provision for us to apply for a discount), and create a 'Genetic modification safety committee'.

A good summary of the process is available from NCBE. We have also been in touch with Cathal Garvey in Ireland who has successfully gone through the process.

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 of our own space

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. Therefore we need to stay with the hackspace.

Funding sources:

Requirements for lab + HSE risk assessment

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