Composite

Part:BBa_K4604021:Design

Designed by: Hannah Swientek   Group: iGEM23_Freiburg   (2023-10-07)
Revision as of 22:40, 11 October 2023 by HannahSwientek (Talk | contribs)

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piG_10a (tetR_bluB_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 2238


Design Notes

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Cloning of piG_10a

Plasmid piG_03 (BBa_K4604019 was used as a backbone and amplified according to the protocol for the Q5 polymerase with an annealing temperature 56°C and elongation time of 4 minutes. For the PCR of the insert (bluB) we also used the general protocol for the Q5 polymerase with an annealing temperature of 65°C and an elongation time of 1 minute. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto an 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

piG_03 (BBa_K4604019) was used as a backbone, bluB (BBa_K4604005) was inserted via Gibson Assembly.