Difference between revisions of "Part:BBa K4604025"
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This BioBrick was used to assess the toxicity of MazF with the additional RBS from the <a href="https://parts.igem.org/Part:BBa_K4604031">AdoCbl riboswitch</a> and compare it to <a href="https://parts.igem.org/Part:BBa_K4604024">piG_23</a>.</html>
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This BioBrick was used to assess the toxicity of MazF with the additional RBS from the <a href="https://parts.igem.org/Part:BBa_K4604031">AdoCbl riboswitch</a> and compare it to <a href="https://parts.igem.org/Part:BBa_K4604024">piG_23</a> using growth measurements and colony-forming-units (CFU).</html>
  
 
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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 (Figure 24). 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.
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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.
  
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<td valign="top"> <span style="font-size:smaller;"><b>Figure 3A: <i>E. coli</i> MG1655 colony forming units (CFU) assay.</b> CFU/mL values for each timepoint for piG_23</span> </td>
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<td valign="top"> <span style="font-size:smaller;"><b>Figure 3B: <i>E. coli</i> MG1655 colony forming units (CFU) assay.</b> CFU/mL values for each timepoint for piG_23b</td>
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<td valign="top"> <span style="font-size:smaller;"><b>Figure 3C: <i>E. coli</i> MG1655 colony forming units (CFU) assay.</b> CFU/mL values for each timepoint for control pGGAselect</td>
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Figure 25: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for piG_23 (A), piG_23b (B) and control pGGAselect (C)
 
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After researching we found a publication showing that mazF-mediated cleavage of mazF mRNA leads to increased temporal variability in length of cells in isogenic populations of E. coli [5], while overexpressing mazF. By taking microscopy images of cells containing piG_23 and piG_23b we were able to compare the toxic effect of mazF overexpression regarding length in individual cell lineages after 100 ng/mL DOX induction and without induction. We measured the cell size of about 25 to 40 cells using Fiji software (Figure 26). At time point 0, a similar cell length of about 2.2 µm was measured for all setups, except for non-induced piG_23b cells, which were slightly larger. This could, however, not be explained. After 3 hours, significantly smaller cells were observed for induced piG_23 samples with a size of 1.5 µm compared to non-induced cells (2.3 µm). There was no difference in cell length for cells containing piG_23b (induced and non-induced) at the same time point.
 
  
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Conclusion
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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.  
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With this experiment, we demonstrated that simply by replacing the RBS of the riboswitch with a stronger RBS, induction with DOX leads to growth inhibition and cell death. Regarding the measurement of the cell length, the piG_23 samples that we used were not grown in M9 as the piG_23b samples, but in LB medium (previous experiment). Even if this means that the results are only limitedly comparable, we observed a difference in cell length for non-induced and induced piG_23 cells. We were now very interested to see if we would obtain the same toxic effect if we add the strong RBS to the riboswitch. Unfortunately this was out of the capabilities of our iGEM project.
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===Usage and Biology===
 
  
 
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Revision as of 20:56, 11 October 2023


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]