Part:BBa_K4604017:Design

piG_02a (leaky_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 727
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 898
  Illegal AgeI site found at 1391
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2265
  

Design Notes

A GS-linker had to be cloned between mazF and mTurquoise to ensure correct folding and functionality of both proteins. A FLAG-tag was fused to mazF to make it detectable in western blot.

Cloning of piG_02a

The tet promoter and rrnB terminator were given to us from iGEM Freiburg 2022. We used one of their plasmids as a template for a PCR but did site directed mutagenesis on the tet promoter to fit the iGEM parts standards. MazF (toxin) together with the riboswitch were synthesized by IDT, the sequence for that was taken from the National Library of Medicine out of E. coli K12 MG1655 (gene ID:947252). The gene for the fluorescent protein mTurquoise was taken from a plasmid by iGEM Freiburg 2022 (BBa_K4229064). For the PCR we used the general protocol for the Q5 polymerase with varying parameters (elongation time and annealing temperature):

Fig. 1: table showing parameters varying from PCR protocol for piG_02a

fragment	Annealing temp.	Elongation time	Fragment size (in bp)
Tet promoter	61°C	1 min	630
mazF	61°C	20 s	330
mTurquoise	60°C	1 min	720
rrnB terminator	61°C	30 s	450

The resulting PCR was loaded onto an 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 via colony PCR. With the potential colony containing the insert we did an overnight culture (5mL LB-medium, 34 mg/mL chloramphenicol) and isolated the DNA after incubation. The resulting plasmid was sent for sequencing for correct insertion and no mutation.

Source

See more about exact sources on the pages of the BioBricks it is made up off.