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| = Genetic modification =
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| We're interested in doing genetic modification on micro-organisms at the London Hackspace. Ideas for stuff to do:
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| == E coli, PUC19, ampicillin selection ==
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| '' 28 mar 15 ''
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| == GFP transformation ==
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| * 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]
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| === Still need ===
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| * GFP + antibiotic resistance plasmid.
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| * Ampicillin. - [http://www.sigmaaldrich.com/catalog/product/sial/a9518?lang=en®ion=GB | Sigma]
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| * Initial e. coli culture.
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| === Already have ===
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| * Plates.
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| * Agar
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| * LB Broth
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| * Temp controlled water bath
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| == Growing competent E. coli ==
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| [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.
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| == Self-cloning ==
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| [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.
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| ''Kit:''
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| * Kit has enough materials "for 16 students, working in pairs" - so 8.
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| ''Timescale:''
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| * 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).
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| * 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.
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| * 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.
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| * Incubate plates overnight at 37C.
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| * Day 6 - Examine results (or store plates in fridge until viewing time)
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| * Clean up - destroy cultures in autoclave. Disinfect all working materials.
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| ''Storage when materials arrive:''
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| * Plasmids must be stored at -20.
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| * E. coli should be stored at 4C
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| * Once E. coli pack is opened needs to be used in a few days.
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| * Shipped first class post mon - thurs
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| ''Anything we need that we don't already have and doesn't come with the kit?''
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| * 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.
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| * 37C incubator (investigate using environmental chamber)
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| * 42C water bath (look at using new hot plate, check size sufficient)
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| * Disinfectant (We currently have bleach and IPA)
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| == Legal ==
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| 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'.
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| 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.
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| 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)
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| Here is a summary of those regulations: [[Project:Biohacking/Genetic_modification/Regulations_2000_summary|GMO Regulations 2000 summary]]
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| [[Category:Biohacking]]
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