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 be ‘cleaned’ and possible restriction sites need to be mutated out. Afterwards a biobrick can be made.
Purifying the promoter region of the blue light receptor from E. Coli. This region needs to be ‘cleaned’ and possible restriction sites need to be mutated out. Afterwards a biobrick can be made.
-
==required==
+
==Required==
*E.Coli strain ( MC4100)
*E.Coli strain ( MC4100)
*Primers (1) for PCR: already ordered (nummers: 2171 (FP) - 2172 (RP))
*Primers (1) for PCR: already ordered (nummers: 2171 (FP) - 2172 (RP))
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*Strain: from lab
*Strain: from lab
*Primers: self made and ordered
*Primers: self made and ordered
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**for PCR
+
**For PCR
***Forward:  CATCAT GAATTCGCGGCCGCTTCTAGAG  TTT GAC AGG TTC GTC GTC
***Forward:  CATCAT GAATTCGCGGCCGCTTCTAGAG  TTT GAC AGG TTC GTC GTC
***Reverse: CTGCAGCGGCCGCTACTAGTA  CCT CTG TTA AAA ATG TTA ATC AAT GTT AAG  
***Reverse: CTGCAGCGGCCGCTACTAGTA  CCT CTG TTA AAA ATG TTA ATC AAT GTT AAG  
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**for ‘cleaning’  
+
**For ‘cleaning’  
-
***under construction
+
***Under construction
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*the GFP ({{kulpart|BBa_E0240}})came from the freezer (-80)at the lab
+
*The GFP ({{kulpart|BBa_E0240}}) came from the freezer (-80) at the lab
==Steps==
==Steps==
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#PCR reaction to purify suspected promoter region.Probably has a promoter, RBS and SpeI restriction site.
+
#PCR reaction to purify suspected promoter region. Probably includes a promoter, RBS and SpeI restriction site.
-
#partial digestion with SpeI. After, run agarose gel to identify the correctly cut sequence and then perform a gel extraction. cut with EcoRI.   
+
#Partial digestion with SpeI. Afterwards, run an agarose gel to identify the correctly cut sequence and then perform a gel extraction. Cut with EcoRI.   
-
#cut GFP with EcoRI and XbaI.Couple PCR fragment to GFP. measure reactivity of the promoter.  
+
#Cut GFP with EcoRI and XbaI. Couple PCR fragment to GFP. Measure reactivity of the promoter.  
-
#cut vector with SpeI and use klenow to create blunt ends. then ligate together. use initial primers to recreate restriction sites and to select correctly ligated sequences. link to GFP to check the activity of the sequence.
+
#Cut vector with SpeI and use klenow to create blunt ends. Then ligate the fragments together. Use initial primers to recreate restriction sites and to select correctly ligated sequences. Link to GFP to check the activity of the sequence.
-
#“cleaning” region to strictly promoter. Cutting in different pieces and measuring the GFP activity:  
+
#“Cleaning” region to strictly promoter. 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.
-
     b. Once there is no activity anymore with forward primer, keep it constant and shorten reverse inkorten
+
     b. Once activity with forward primer ceased, keep it constant and shorten reverse  
-
     c. Once promoter found: updating those primers with a pre en suffix to make a biobrick out of the promoter
+
     c. Once promoter found: update those primers with a pre en suffix to make a biobrick out of the promoter
-
==important==
+
 
-
following conditions need to be kept in account:
+
[[Image:515px-Promoter_Instructions_Build.png‎|center|thumb|600px|standard promotor test]]
-
*for growth of the bacteria: 37°C
+
 
-
*for expression of the genes regulated by ycgF/E system: 16°C
+
==Important==
-
*at 16°C: expression will start after 50h and a very slow reversion to the ground state of ycgF
+
Following conditions need to be taken into account:
-
*working with a colony in the dark and one in light so that the effects of cold temperature on the gene expression pattern can be calculated out.  
+
*For growth of the bacteria: 37°C
-
*blue light does NOT induce stress and cell death in E. Coli
+
*For expression of the genes regulated by ycgF/E system: 16°C
-
*to monitor growth of the cells, measurement at OD 578 can be used
+
*At 16°C: expression will start after 50h and a very slow reversion to the ground state of ycgF
 +
*Working with a colony in the dark and one in light so that the effects of cold temperature on the gene expression pattern can be calculated out.  
 +
*Blue light does NOT induce stress and cell death in E. Coli
 +
*To monitor growth of the cells, measurement at OD 578 can be used

Latest revision as of 08:01, 11 August 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 need to be mutated out. Afterwards a biobrick can be made.

Required

  • E.Coli strain ( MC4100)
  • Primers (1) for PCR: already ordered (nummers: 2171 (FP) - 2172 (RP))
  • Primers (2) for ‘cleaning’ region (under construction)
  • GFP with RBS and Terminator sequence

Where from

  • Strain: 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’
      • Under construction
  • The GFP () came from the freezer (-80) at the lab

Steps

  1. PCR reaction to purify suspected promoter region. Probably includes a promoter, RBS and SpeI restriction site.
  2. Partial digestion with SpeI. Afterwards, run an agarose gel to identify the correctly cut sequence and then perform a gel extraction. Cut with EcoRI.
  3. Cut GFP with EcoRI and XbaI. Couple PCR fragment to GFP. Measure reactivity of the promoter.
  4. Cut vector with SpeI and use klenow to create blunt ends. Then ligate the fragments together. Use initial primers to recreate restriction sites and to select correctly ligated sequences. Link to GFP to check the activity of the sequence.
  5. “Cleaning” region to strictly promoter. Cutting in different pieces and measuring the GFP activity: 
   a.	Same reverse primer, shortening through forward primer.
   b.	Once activity with forward primer ceased, keep it constant and shorten reverse 
   c.	Once promoter found: update those primers with a pre en suffix to make a biobrick out of the promoter


standard promotor test

Important

Following conditions need to be taken into account:

  • For growth of the bacteria: 37°C
  • For expression of the genes regulated by ycgF/E system: 16°C
  • At 16°C: expression will start after 50h and a very slow reversion to the ground state of ycgF
  • Working with a colony in the dark and one in light so that the effects of cold temperature on the gene expression pattern can be calculated out.
  • Blue light does NOT induce stress and cell death in E. Coli
  • To monitor growth of the cells, measurement at OD 578 can be used
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