Difference between revisions of "Part:BBa K4604016:Design"
 
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Correct overhangs had to be designed for Golden Gate Cloning. The start codon of the bluB was changed to ATG to act as a start for translation in E. coli.
 
Correct overhangs had to be designed for Golden Gate Cloning. The start codon of the bluB was changed to ATG to act as a start for translation in E. coli.
  
 +
===Cloning of piG_01a===
  
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The tet promoter and rrnB terminator were given to us from iGEM Freiburg 2022. We used one of their plasmids as a template for a PCR but did site directed mutagenesis on the tet promoter to fit the iGEM parts standards. The <i>bluB</i> was synthesized by IDT, the sequence for that was taken from the National Library of Medicine (gene ID:61603312) out of <i>sinorhizobium meliloti 2011</i>. For the PCR we used the general protocol for the Q5 polymerase with varying parameters (elongation time and annealing temperature):
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<html>
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<table border=3 style="margin-left:auto;margin-right:auto;">
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    <caption><b>Fig. 1:</b> table showing parameters varying from PCR protocol for piG_01a</caption>
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  <tr>
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    <th>fragment</th>
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    <th>Annealing temp.</th>
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    <th>Elongation time</th>
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    <th>Fragment size (in bp)</th>
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  </tr>
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  <tr>
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    <th>Tet promoter</th>
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    <th>61°C</th>
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    <th>1 min</th>
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    <th>630</th>
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  </tr>
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  <tr>
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    <th><i>bluB</i></th>
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    <th>60°C</th>
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    <th>1 min</th>
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    <th>700</th>
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</tr>
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<tr>
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    <th>rrnB terminator</th>
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    <th>61°C</th>
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    <th>30 s</th>
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    <th>450</th>
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  </tr>
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</table></figure></html>
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The resulting PCR was loaded onto an agarose gel with a DNA ladder, the correct bands were cut out and extracted. The parts were assembled using the Golden Gate cloning method according to the general protocol. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from  overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to check for correct insertion and no mutation.
  
 
===Source===
 
===Source===
  
−
Assebled via Golden Gate Cloning. See more about exact sources on the pages of the BioBricks it is made up off.  
+
See more about exact sources on the pages of the BioBricks it is made up off.  
  
 
===References===
 
===References===

Latest revision as of 00:55, 12 October 2023


piG_01a (leaky_tetR_bluB)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 727
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1647


Design Notes

Correct overhangs had to be designed for Golden Gate Cloning. The start codon of the bluB was changed to ATG to act as a start for translation in E. coli.

Cloning of piG_01a

The tet promoter and rrnB terminator were given to us from iGEM Freiburg 2022. We used one of their plasmids as a template for a PCR but did site directed mutagenesis on the tet promoter to fit the iGEM parts standards. The bluB was synthesized by IDT, the sequence for that was taken from the National Library of Medicine (gene ID:61603312) out of sinorhizobium meliloti 2011. For the PCR we used the general protocol for the Q5 polymerase with varying parameters (elongation time and annealing temperature):


Fig. 1: table showing parameters varying from PCR protocol for piG_01a
fragment Annealing temp. Elongation time Fragment size (in bp)
Tet promoter 61°C 1 min 630
bluB 60°C 1 min 700
rrnB terminator 61°C 30 s 450


The resulting PCR was loaded onto an agarose gel with a DNA ladder, the correct bands were cut out and extracted. The parts were assembled using the Golden Gate cloning method according to the general protocol. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to check for correct insertion and no mutation.

Source

See more about exact sources on the pages of the BioBricks it is made up off.

References