Part:BBa_K4604016:Design

piG_01a (leaky_tetR_bluB)

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1647
  

Design Notes

Correct overhangs had to be designed for Golden Gate Cloning. The start codon of the bluB was changed to ATG to act as a start for translation in E. coli.

Cloning of piG_01a

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. The bluB was synthesized by IDT, the sequence for that was taken from the National Library of Medicine (gene ID:61603312) out of sinorhizobium meliloti 2011. 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_01a

fragment	Annealing temp.	Elongation time	Fragment size (in bp)
Tet promoter	61°C	1 min	630
bluB	60°C	1 min	700
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 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 to check for correct insertion and no mutation.

Source

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