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

Team:KULeuven/Notebook/Blue Light Receptor

From 2009.igem.org

Contents

Planning

Goal

necessary

Where from

Steps