How to
Mutate
- Analysis have made you realise the need for mutations in the CYP in order to remove resctrictionsites
- You design primers that fits your template but contains the altered base
- You mix your primers with the template, dNTP, polymerase and HF buffer and run a non amplifying PCR
- Digest the parental template DNA (which is methylated) with Dpn1 (which cut methylated DNA only)
- Transform XL1-Blue competent cells with the mutated plasmid
- Once you have a colony on your plate you take it out and place it in and overnight culture
- You perform a Miniprep on your culture to purify your amplified plasmid
- Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site
- Run the digest of the plasmid and hope to see a number of bands corresponding to the number of restriction sites in the plasmid backbone. In the latter case your mutations failed
- Get it sequenced
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How to
make a BioBrick
- Add prefix and suffix containing primers for the CYP in question and amplify with PCR
- Purify with DNA Purification kit
- Cut with restrictionsenzymes EcoR1 and Pst1
- Heat shock to kill restrictionenzymes
- Cut the linarized plasmid backbone with Pst1 and EcoR1
- Ligate CYP and Plasmid
- Transform in XL1-Blue
- Overnight culture
- Mini Prep
- Get it sequenced
- Sent it to iGEM
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How to
make a CyperMan
- Cut the newly made Cyp BioBrick with restrictionsenzymes Xba and Pst1
- Heat Shock to kill the restriction enzymes
- Put it in the freezer
- Cut the purified BioBrick J004500 ekspression vector from the kit with Spe1 and Pst1
- Ligate CYP and Vector
- Transform in BL21
- Grow cells O/N while inducing ekspression of CYP with IPTG.
- Take tests (1h, 2h, 4h, next day) and prepare them for SDS page
- Run the SDS and hope to see increased amount of a band with the size of the CYP
- Make a membrane preparation to get micelles with cytochrome
- Test the cytochromes ability to transform aminoacids to oximes with TLC.
- Kill Fungus
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