E. coli GFP transformation: Difference between revisions

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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]]

Revision as of 17:17, 28 March 2015

Genetic modification

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

E coli transformed with PUC19 using ampicillin selection

28 mar 15

  • 2ml ecoli culture (bottle on shelf) incubated in 200ml LB (bottle in fridge)
  • -ve control is untransformed ecoli from same culture put on LBA
  • +ve control is transformed ecoli from same culture put on LB plates
  • Transform ecoli with PUC19 using electroporation
  • Plate onto LBA.

GFP transformation

  • The 'hello world' GFP experiment. Biorad do a kit. Carolina do just the plasmid


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. 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), 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.

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