Difference between revisions of "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 '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]
-* 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.
-C 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&region=GB LB] - £66, [http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=en&region=GB 0.1M CaCl2] - £32.50, [http://www.sigmaaldrich.com/catalog/product/SIGMA/M6030?lang=en&region=GB M9 salts] - £57.60, [http://www.sigmaaldrich.com/catalog/product/sigma/m3409?lang=en&region=GB MgSO4] - £20, [http://www.sigmaaldrich.com/catalog/product/sigma/l2025?lang=en&region=GB bacteriological agar solution] - £42.90, [http://www.sigmaaldrich.com/catalog/product/sial/t4625?lang=en&region=GB thiamine] - ~£20, [http://www.sigmaaldrich.com/catalog/product/sigma/g8769?lang=en&region=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]]

Difference between revisions of "E. coli GFP transformation"

Latest revision as of 18:26, 2 April 2015

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