Difference between revisions of "Part:BBa K4604018:Design"
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===Design Notes===
 
===Design Notes===
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Site directed mutagenesis was done between the tet promoter and RBS to remove a EcoRI restriction site. 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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Site directed mutagenesis was done between the tet promoter and RBS to remove a EcoRI restriction site. The region downstream of the tetA/R promoter was shortened (in comparison to piG_01a/<a href="https://parts.igem.org/Part:BBa_K4604016">BBa_K4604016</a>) to counteract the leakiness.  
  
  
 
===Cloning of piG_02b===
 
===Cloning of piG_02b===
 
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  Plasmid piG_02a (<a class="link" href="https://parts.igem.org/Part:BBa_K4604017">BBa_K4604017</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, afterwards the DNA was loaded onto a 1% agarose gel. The correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. 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 for correct insertion and no mutation.  
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Plasmid piG_02a (<a href="https://parts.igem.org/Part:BBa_K4604017">BBa_K4604017</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, afterwards the DNA was loaded onto a 1% agarose gel. The correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. 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 for correct insertion and no mutation.  
  
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===Source===
 
===Source===
  
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Modified piG_02a (BBBa_K4604017) using Gibson Assembly.  
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Modified piG_02a (<a href="https://parts.igem.org/Part:BBa_K4604017">BBa_K4604017</a>) using Gibson Assembly.  
  
 
===References===
 
===References===

Revision as of 23:20, 11 October 2023


piG_02b (tetR_riboK12_mazF_mTurq)


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 883
    Illegal AgeI site found at 1376
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2250


Design Notes

Site directed mutagenesis was done between the tet promoter and RBS to remove a EcoRI restriction site. The region downstream of the tetA/R promoter was shortened (in comparison to piG_01a/BBa_K4604016) to counteract the leakiness. ===Cloning of piG_02b=== Plasmid piG_02a (BBa_K4604017) 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, afterwards the DNA was loaded onto a 1% agarose gel. The correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. 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 for correct insertion and no mutation.


Source

Modified piG_02a (<a href="https://parts.igem.org/Part:BBa_K4604017">BBa_K4604017</a>) using Gibson Assembly.

References