Part:BBa_K4604025

piG_23b (tetR_riboRBS_mazF)

This BioBrick consists of the tetR repressor, the RBS taken from the K12 riboswitch (BBa_K4604031), the tet promoter, the modified T7 RBS, mazF and the rrnB terminator.

Characterization

This BioBrick was used to assess the toxicity of MazF with the additional RBS from the AdoCbl riboswitch and compare it to piG_23 using growth measurements and colony-forming-units (CFU).

Figure 1: Growth assay of E. coli MG1655, containing pGGAselect pGGAselect, piG_23 and piG_23b over 24 hours in M9 medium with different DOX concentrations. OD600= 0.5 measurement of culture samples using ThermoScientific NanoDrop 2000c Spectrophotometer.

The piG_23b containing cells, as well as control cells containing pGGAselect, showed a steady increase in growth over time in the non-induced and induced state with 100 ng/mL DOX. This result indicates no difference in growth, while significant growth inhibition can be observed for piG_23 cells induced with 100 ng/ml DOX, as can be seen on piG_23 characterization. Constant OD600 values of around 0.8 indicate a stagnation in growth. Unexpectedly, we observed that piG_23 non-induced also showed signs of growth stagnation, so we replicated the experiment.

Figure 2: Growth assay of E. coli MG1655, containing pGGAselect pGGAselect, piG_23 and piG_23b(+Riboswitch RBS) over 24 hours in M9 medium with different DOX concentrations. OD600= 0.5 measurement of culture samples using ThermoScientific NanoDrop 2000c Spectrophotometer.

Replicate 2 presented in Figure 2 showed a similar growth behavior as Replicate 1. We observed that the bacteria carrying piG_23b reached a higher OD although being induced with the same DOX concentration as the bacteria containing piG_23. In contrast to the last replicate, the non-induced piG_23 grew as expected, reaching similar ODs as the non-induced pGGAselect. However, for this repetition, we observed an unexpected growth of induced pGGAselect. This could, however, not be explained and it was the first time we observed this.

Figure 3A: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for piG_23	Figure 3B: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for piG_23b	Figure 3C: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for control pGGAselect

With these experiments we demonstrated that by replacing the stronger RBS with the one from the riboswitch, induction with DOX leads to inhibited growth inhibition and cell death.

Sequence and Features

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]