Composite

Part:BBa_K4604025:Design

Designed by: Hannah Swientek   Group: iGEM23_Freiburg   (2023-10-10)

piG_23b (tetR_riboRBS_mazF)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 701
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 701
    Illegal BglII site found at 710
    Illegal BamHI site found at 1153
    Illegal XhoI site found at 1162
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 1000
    COMPATIBLE WITH RFC[1000]


Cloning strategy

Plasmid piG_23 (BBa_K4604024) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 57°C and an elongation time of 3 minutes. A DpnI digest was done at 37°C overnight, 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 taken for the next steps. Since this was only a change of the 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.