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Team:KULeuven/Lab/Blue Light Receptor
From 2009.igem.org
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| - | Purifying the promoter region of the blue light receptor from E. Coli. This region needs to | + | Purifying the promoter region of the blue light receptor from E. Coli. This region needs to be ‘cleaned’ and possible restriction sites mutated out. After a biobrick can be made. |
==required== | ==required== | ||
Revision as of 12:23, 24 July 2009
Contents |
Planning
Goal
Purifying the promoter region of the blue light receptor from E. Coli. This region needs to be ‘cleaned’ and possible restriction sites mutated out. After a biobrick can be made.
required
- e coli stam ( MC4100)
- Primers (1) for PCR: already ordered (nummers: 2171 (FP) - 2172 (RP))
- 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
