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E. coli GFP transformation: Difference between revisions

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E. coli GFP transformation (view source)

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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]
* 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]


=== Still need ===
* GFP + antibiotic resistance plasmid.
* Ampicillin. - [http://www.sigmaaldrich.com/catalog/product/sial/a9518?lang=en&region=GB | Sigma]
* Initial e. coli culture.
=== Already have ===
* Plates.
* Agar
* LB Broth
* Temp controlled water bath


== Growing competent E. coli ==
== 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&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.
[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]]


[[Category:Biohacking]]
[[Category:Biohacking]]
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