Team:KULeuven/Lab/Blue Light Receptor

From 2009.igem.org

(Difference between revisions)
(New page: =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 biobr...)
(→Steps)
Line 21: Line 21:
==Steps==
==Steps==
-
1.PCR reaction to purify suspected promoter region.
+
*1.PCR reaction to purify suspected promoter region.
Probably has a promoter, RBS and SpeI restriction site.   
Probably has a promoter, RBS and SpeI restriction site.   
-
2.PCR fragment coupled to GFP  
+
*2.PCR fragment coupled to GFP  
Measuring reactivity of the promoter  
Measuring reactivity of the promoter  
-
3.“cleaning”  region to only get promoter   
+
*3.“cleaning”  region to only get promoter   
Cutting in different pieces and measuring the GFP activity  
Cutting in different pieces and measuring the GFP activity  
     a. Same reverse primer, shortening through forward primer.
     a. Same reverse primer, shortening through forward primer.
Line 31: Line 31:
     c. Once promoter found: updating those primers with a pre en suffix to make a biobrick out of the promoter  
     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
+
*4.Mutating  SpeI site  out ( 178 - 183) via PCR mutagenesis

Revision as of 12:19, 24 July 2009

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
Retrieved from "http://2009.igem.org/Team:KULeuven/Lab/Blue_Light_Receptor"