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Team:KULeuven/Notebook/Blue Light Receptor
From 2009.igem.org
Contents |
Planning
Goal
Purifying the promoter region of the blue light receptor from E. Coli. This region needs to bee ‘cleaned’ and possible restriction sites mutated out. After a biobrick can be made.
necessary
e coli stam ( MC4100) Primers (1) for PCR: already ordered nummer IGEM - 2172 Primers (2) for ‘cleaning’ region Primers (3) for biobrick (nog te maken)
Where from
Stam: from lab Primers: self made and ordered ===for PCR===
Forward: CATCAT GAATTCGCGGCCGCTTCTAGAG TTT GAC AGG TTC GTC GTC
Reverse: CTGCAGCGGCCGCTACTAGTA CCT CTG TTA AAA ATG TTA ATC AAT GTT AAG
===for ‘cleaning’===
===for biobrick=== only when actual promoter is known
Steps
1.PCR reaction to purify suspected promoter region. Probably has a promoter, RBS and SpeI restriction site. 2.PCR fragment coupled to GFP Measuring reactivity of the promoter 3.“cleaning” region to only get promoter Cutting in different pieces and measuring the GFP activity
a. Same reverse primer, shortening through forward primer. b. Once there is no activity anymore with forward primer, keep it constant and shorten reverse inkorten c. Once promoter found: updating those primers with a pre en suffix to make a biobrick out of the promoter
4.Mutating SpeI site out ( 178 - 183) via PCR mutagenesis
