Team:KULeuven/Notebook/Blue Light Receptor

From 2009.igem.org

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=Planning=
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==Goal==
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==13 July==
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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.
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*YCGF Gene, natural available in [http://ecoliwiki.net/colipedia/index.php/ycgF:Gene 3 E. Coli strains]. Can use the promotor of Ycgf gene for expression
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*Probable strain K12
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==necessary==
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*Promotor voor YCGF gene:
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e coli stam ( MC4100)
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**AACAATCCAGGGTAATGGGTGAGGCGAGAGTAAGACGGTAACAGACATATCTTCTTG TGTCTTTCTTTTAATACCAAAACATAACCGTTTCTTTACATTGATAAAAAATGGAAAAAG TTGAACACTAGTTGGCGAAAAATCTTGTATAGATTGTCAGTTAAATGATGCAATATGTT TTATCATAACACATTGTTTTATATGCATTAGCACTAATTGCAAAAAATTAATTTATCATT CTGTACACATATTTCGTACAAGTTTGCTATTGTTACTTCACTTAACATTGATTAACATTTTTAACAGAGGCGTAGC'''ATG''' (bron: [www.ncbi.nlm.nih.gov])
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Primers (1) for PCR: already ordered  nummer IGEM - 2172
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Primers (2) for ‘cleaning’ region
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Primers (3) for biobrick (nog te maken)
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==Where from==
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*Stam: from lab
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*Primers: self made and ordered
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  for PCR
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      Forward:  CATCAT GAATTCGCGGCCGCTTCTAGAG  TTT GAC AGG TTC GTC GTC
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      Reverse: CTGCAGCGGCCGCTACTAGTA  CCT CTG TTA AAA ATG TTA ATC AAT GTT AAG
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  for ‘cleaning’
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  for biobrick
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      only when actual promoter is known
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==Steps==
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1.PCR reaction to purify suspected promoter region.
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Probably has a promoter, RBS and SpeI restriction site.
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2.PCR fragment coupled to GFP
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Measuring reactivity of the promoter
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3.“cleaning”  region to only get promoter 
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Cutting in different pieces and measuring the GFP activity
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    a. Same reverse primer, shortening through forward primer.
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    b. Once there is no activity anymore with forward primer, keep it constant and shorten reverse inkorten
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    c. Once promoter found: updating those primers with a pre en suffix to make a biobrick out of the promoter
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4.Mutating  SpeI site  out ( 178 - 183) via PCR mutagenesis
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Revision as of 09:54, 24 July 2009

13 July

  • YCGF Gene, natural available in [http://ecoliwiki.net/colipedia/index.php/ycgF:Gene 3 E. Coli strains]. Can use the promotor of Ycgf gene for expression
  • Probable strain K12
  • Promotor voor YCGF gene:
    • AACAATCCAGGGTAATGGGTGAGGCGAGAGTAAGACGGTAACAGACATATCTTCTTG TGTCTTTCTTTTAATACCAAAACATAACCGTTTCTTTACATTGATAAAAAATGGAAAAAG TTGAACACTAGTTGGCGAAAAATCTTGTATAGATTGTCAGTTAAATGATGCAATATGTT TTATCATAACACATTGTTTTATATGCATTAGCACTAATTGCAAAAAATTAATTTATCATT CTGTACACATATTTCGTACAAGTTTGCTATTGTTACTTCACTTAACATTGATTAACATTTTTAACAGAGGCGTAGCATG (bron: [www.ncbi.nlm.nih.gov])
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