Part:BBa_K4604018

piG_02b (tetR_riboK12_mazF_mTurq)

BioBrick piG_02b is a plasmid consisting of the tet promoter/repressor, an AdoCbl riboswitch, mazF fused togther with mTurquoise and the rrnB terminator. The backbone we used in the experiments is pGGAselect. The tet promoter is a BioBrick from iGEM Freiburg 2022 (BBa_K4229059). This is an improved version of BioBrick BBa_K4604017.

Characterization

To indirectly check for mazF-mTurquoise expression a fluorescent measurement was done and compared to BBa_K4604019, namely piG_03.

Figure 1: Fluorescence measurement of E. coli MG1655 containing piG_02b/piG_03 over 48 hours in M9 medium. Cultures were induced at OD600 = 0.5 with the indicated concentration of DOX. Fluorescence of cultures (Ex: 434 nm; Em: 474 nm) was measured over a course of 48 hours using Spectramax Plate Reader.

Next, a growth assay was performed comparing this BioBrick to BBa_K4604019, namely piG_03.

Figure 2: Growth assay of E. coli MG1655 containing piG_02b/piG_03 in M9 medium. Cultures were induced at OD600 = 0.5 with the indicated concentration of DOX. Growth of cultures were investigated over a course of 48 hours by OD600- measurement using Spectramax Plate Reader.

We also performed a western blot to detect MazF expresion.

Figure 3: MazF expression for different inducer concentrations. Detection of recombinantly expressed, FLAG-tagged mazF toxin fused to mTurquoise with SDS-PAGE followed by western blot. Detection of MazF was performed with an anti-FLAG antibody. Loading control: RNA polymerase beta-subunit. Time point 12 hours after induction with indicated concentration of DOX.

In conclusion, we did not observe significant cell death or growth inhibition when measuring OD600 (figure 2) even for higher DOX concentrations. Unfortunately, we were not able to evaluate the CFUs from this experiment because there was a bacterial lawn on the plates due to the high growth rate. Those experiments were repeated three times without any growth inhibition or cell death observed. However, when we measured fluorescence we saw an increase in fluorescence with higher DOX concentrations (Figure 1). This would suggest that the lack of cell death is not due to missing expression. This was also confirmed by western blot (Figure 3) where we detected bands for both toxin-mTurquoise fusion as well as the toxin alone. However, the increase in the intensity of the bands with higher DOX concentrations was only mild. We hypothesized that this could be the result of a subculture in our glycerol stock that either does not produce Toxin or has found a way to inhibit Toxin expression.

Sequence and Features

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 883
  Illegal AgeI site found at 1376
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2250