Difference between revisions of "Part:BBa K4604022:Design"

 
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Since the inherent RBS of the riboswitch might be detremential for the correct folding of said, we decided to add an additional (supposedly stronger) RBS right after the riboswitch, in front of the desired gene.  
 
Since the inherent RBS of the riboswitch might be detremential for the correct folding of said, we decided to add an additional (supposedly stronger) RBS right after the riboswitch, in front of the desired gene.  
  
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===Cloning of piG_10b===
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  Plasmid piG_10a (<a class="link" href="https://parts.igem.org/Part:BBa_K4604021">BBa_K4604021</a>) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 61°C and an elongation time of 5 minutes. A Dpn1 digest was done at 37°C for 3 hours, 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. The incubation time was extended to 25 minutes. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were taken for the next steps. Since this was only the insertion of an additional RBS, screening was not possible. 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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Revision as of 19:26, 10 October 2023


piG_10b (tetR_bluB_T7RBS_riboK12_mazF)


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 1603
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2252


Design Notes

Since the inherent RBS of the riboswitch might be detremential for the correct folding of said, we decided to add an additional (supposedly stronger) RBS right after the riboswitch, in front of the desired gene.

Cloning of piG_10b

Plasmid piG_10a (BBa_K4604021) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 61°C and an elongation time of 5 minutes. A Dpn1 digest was done at 37°C for 3 hours, 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. The incubation time was extended to 25 minutes. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were taken for the next steps. Since this was only the insertion of an additional RBS, screening was not possible. 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 BBa_K4604021 with Gibson assembly. The tet promoter was taken from iGEM Freiburg 2022 (BBa_K4229059). ===References===