|
|
| (14 intermediate revisions by 3 users not shown) |
| Line 1: |
Line 1: |
| = 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. Biorad do a [http://www.bio-rad.com/prd/en/UK/LSE/PDP/619b8f74-9d3f-4c2f-a795-8a27e67598b7/pGLO-Bacterial-Transformation-Kit kit]. Carolina do just the [http://www.carolina.com/biotechnology-plasmid-lambda-dna/pgreen-plasmid-1-ug-200-ul-0005-ugul/211449.pr?catId=&mCat=&sCat=&ssCat=&question=pgreen+plasmid plasmid]
| |
| * 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 ==
| |
|
| |
| [http://wiki.london.hackspace.org.uk/w/images/a/af/DIYbio_Collaboration_protocols.pdf 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 ([http://www.bio-rad.com/prd/en/US/LSE/SKU/166-0408EDU/E.-coli-Strain-HB101-K-12 one possibility]. Could also try NCBE. They sell e. coli as a transformer kit replacement part. Finally these are suggested for schools by NCBE: Sciento, Blades Biological and Philip Harris Education), [http://www.sigmaaldrich.com/catalog/product/fluka/l3152?lang=en®ion=GB LB] - £66, [http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=en®ion=GB 0.1M CaCl2] - £32.50, [http://www.sigmaaldrich.com/catalog/product/SIGMA/M6030?lang=en®ion=GB M9 salts] - £57.60, [http://www.sigmaaldrich.com/catalog/product/sigma/m3409?lang=en®ion=GB MgSO4] - £20, [http://www.sigmaaldrich.com/catalog/product/sigma/l2025?lang=en®ion=GB bacteriological agar solution] - £42.90, [http://www.sigmaaldrich.com/catalog/product/sial/t4625?lang=en®ion=GB thiamine] - ~£20, [http://www.sigmaaldrich.com/catalog/product/sigma/g8769?lang=en®ion=GB D glucose] - ~£20, and plates. All links are only the first ones I found, maybe cheaper options available.
| |
|
| |
| == Self-cloning ==
| |
|
| |
| [http://www.ncbe.reading.ac.uk/ncbe/protocols/transformer.html 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 (1 per 4 people), incubate at room temperature (18-25C) in the dark for 3-4 days (or incubate at 37C for 18-24 hours, but results from this method not as good).
| |
| * Before session - make agar plates and refrigerate (these can be left for up to 7 days). Make transformation buffer. Dispense plasmid DNA into tubes. Prepare recovery broth.
| |
| * Day 5 - Warm plates to 37C. Chill transformation buffer on ice for at least 5 mins. Take cells and mix with transformation buffer. Mix. Chill on ice for 10 mins. Add this tube to plasmid tube and mix. Heat shock at 40-42C for exactly 30s, then return to ice for 1-2 mins. Add recovery broth (at 37C) to tubes and mix. Add bacteria to (37C pre-warmed) plates.
| |
| * Incubate plates overnight at 37C.
| |
| * Day 6 - Examine results (or store plates in fridge until viewing time)
| |
| * Clean up - destroy cultures in autoclave. Disinfect all working materials.
| |
|
| |
| ''Storage when materials arrive:''
| |
|
| |
| * Plasmids must be stored at -20.
| |
| * E. coli should be stored at 4C
| |
| * Once E. coli pack is opened needs to be used in a few days.
| |
| * Shipped first class post mon - thurs
| |
|
| |
| ''Anything we need that we don't already have and doesn't come with the kit?''
| |
|
| |
| * 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.
| |
| * 37C incubator (investigate using environmental chamber)
| |
| * 42C water bath (look at using new hot plate, check size sufficient)
| |
| * Disinfectant (We currently have bleach and IPA)
| |
|
| |
| == 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 [http://www.ncbe.reading.ac.uk/ncbe/safety/dnasafety3.html NCBE]. We have also been in touch with Cathal Garvey in Ireland who has successfully gone through the process.
| |
|
| |
| The relevant regulations are [http://www.legislation.gov.uk/uksi/2000/2831/pdfs/uksi_20002831_en.pdf The Genetically Modified Organisms (Contained Use) Regulations 2000] (PDF)
| |
|
| |
| Here is a summary of those regulations: [[Project:Biohacking/Genetic_modification/Regulations_2000_summary|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:''
| |
|
| |
| * [http://www.wellcome.ac.uk/Funding/Public-engagement/index.htm Wellcome trust public engagement fund]
| |
| * Arts catalyst
| |
| * Synthetic aesthetic
| |
| * UCL. Imperial.
| |
| * Workshops
| |
| * Kickstarter
| |
| * Businesses to get free kit from
| |
| * BBC for new publicity.
| |
| * NESTA
| |
| * NCBE
| |
|
| |
| ==== Requirements for lab + HSE risk assessment ====
| |
|
| |
| * Tiling / washable surfaces
| |
| * Sink
| |
| * Lockable
| |
| * Enough room for 10-15 people to work
| |
| * Disposal procedures
| |
|
| |
| [[Category:Biohacking]]
| |
