Team:Evry/Notebook/Sensing/PCBs/08-21-2014

From 2014.igem.org

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<u>Sensor construction hbpR/PhbpC and hbpR2/PhbpR1</u>
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<u>Sensor construction bphR2/PbpR1</u>
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<br/>The part BBa_J23114 was located on 2014 Distribution kit plates and resuspended with 10 µL sterile water. That permits to obtain a DNA concentration around 0.2 ng/µl (according to the registry. Solution was transferred into 1 ml eppendorf tubes and stored at -20°C.
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<br/><a href="parts.igem.org/Part:BBa_J23114">BBa_J23114</a> was resuspended with 10 µL sterile water in order to obtain a DNA concentration around 0.2 ng/µl (according to the registry). Solution was transferred into one 1 ml eppendorf tube and stored at -20°C.
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<br/>Transformations of registry parts (BBa_B0015, E1010 and J23114) and vector PSB1C3G3 were performed to obtain colonies with plasmid containing parts for future amplifications. <br/> The transformation was performed on DH5 alpha ''E. coli'', as followed: </p>
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<br/>Transformations of constitutive promoter <a href="parts.igem.org/Part:BBa_J23114">BBa_J23114</a>, RFP <a href="parts.igem.org/Part:BBa_E1010">BBa_E1010</a> and terminator <a href="parts.igem.org/Part:BBa_B0015">BBa_B0015</a> and vector pSB1C3G3 was done on DH5alpha as followed: </p>
<ol>
<ol>
<li> Remove E. coli competent tubes from -80°C and keep it on ice
<li> Remove E. coli competent tubes from -80°C and keep it on ice
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<li> Incubate 2 minutes on ice
<li> Incubate 2 minutes on ice
<li> Add 2 ml of LB medium and incubate 60 minutes at 37°C with an agitation at 200 rpm
<li> Add 2 ml of LB medium and incubate 60 minutes at 37°C with an agitation at 200 rpm
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<li> Plate 200 µl of PSB1C3G3, BBa_B0015 and E1010 on a chloramphenicol LB agar plate or ampiciline LB agar plate for BBa_J23114
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<li> Plate 200 µl of pSB1C3G3, BBa_B0015 and E1010 on a chloramphenicol LB agar plate and ampicilline LB agar plate for BBa_J23114
<li> Incubate plate overnight at 37°C
<li> Incubate plate overnight at 37°C
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</ol>
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<br/>pSB1C3 was digested with EcoRI and pstI and a ligation with bphR2 was done before the transformation in DH5a.
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<br/> 50 µL were plated on Cam Lb plate and incubated at 37°C overnight
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<br/><br/>
<span class="cd-date">Aug 21</span>  
<span class="cd-date">Aug 21</span>  

Latest revision as of 00:15, 13 October 2014

Picture

Sensor construction bphR2/PbpR1
BBa_J23114 was resuspended with 10 µL sterile water in order to obtain a DNA concentration around 0.2 ng/µl (according to the registry). Solution was transferred into one 1 ml eppendorf tube and stored at -20°C.
Transformations of constitutive promoter BBa_J23114, RFP BBa_E1010 and terminator BBa_B0015 and vector pSB1C3G3 was done on DH5alpha as followed:

  1. Remove E. coli competent tubes from -80°C and keep it on ice
  2. Add 3 µl of template (here solubilized plasmids from the registry distribution kit) and mix gently
  3. Incubate 10 minutes on ice
  4. Perform an heat shock 30 seconds at 42°C
  5. Incubate 2 minutes on ice
  6. Add 2 ml of LB medium and incubate 60 minutes at 37°C with an agitation at 200 rpm
  7. Plate 200 µl of pSB1C3G3, BBa_B0015 and E1010 on a chloramphenicol LB agar plate and ampicilline LB agar plate for BBa_J23114
  8. Incubate plate overnight at 37°C

We received primers.
IMAGE
Symbol
Table 4: Received primers with numbers and sequences for PCB sensing constructions



pSB1C3 was digested with EcoRI and pstI and a ligation with bphR2 was done before the transformation in DH5a.
50 µL were plated on Cam Lb plate and incubated at 37°C overnight

Aug 21

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