Difference between revisions of "Part:BBa K4604015:Design"
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The start codon of the bluB was changed to ATG to act as a start for translation in E. coli.
 
The start codon of the bluB was changed to ATG to act as a start for translation in E. coli.
 
The region downstream of the tetA/R promoter was shortened (in comparison to piG_01a/BBa_K4604016) to counteract the leakiness.  
 
The region downstream of the tetA/R promoter was shortened (in comparison to piG_01a/BBa_K4604016) to counteract the leakiness.  
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===Cloning of piG_01b===
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Plasmid piG_01a (<a href="https://parts.igem.org/Part:BBa_K4604016">BBa_K4604016</a>) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 56°C and an elongation time of 5 minutes. A Dpn1 digest was done at 37°C for an hour, the DNA was loaded onto an 1% 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.
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Revision as of 19:12, 10 October 2023


piG_01b (tetR_bluB)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 710
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1618


Design Notes

The start codon of the bluB was changed to ATG to act as a start for translation in E. coli. The region downstream of the tetA/R promoter was shortened (in comparison to piG_01a/BBa_K4604016) to counteract the leakiness.

Cloning of piG_01b

Plasmid piG_01a (BBa_K4604016) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 56°C and an elongation time of 5 minutes. A Dpn1 digest was done at 37°C for an hour, the DNA was loaded onto an 1% 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

Modified piG_01a (BBa_K4604016) using Gibson Assembly.

References