Team:Copenhagen/Project/Experimental

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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

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

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
CO measurements for the membrane preperations
Test the cytochromes ability to transform aminoacids to oximes with TLC

Comments or questions to the team? Please mail us at igemcopenhagen@gmail.com