Part:BBa_K3191201
pBAD + CheZ + YbaQ
This part places cheZ with a YbaQ degron sequence under a pBAD promoter. A degron is included to prevent overexpression of CheZ and improve motility. The pBAD promoter encourages chemotaxis in the presence of arabinose.
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Figure 1. Average area of diffusion assays under varying arabinose concentrations without the YbaQ degron.
Figure 2. Average area of diffusion assays with the YbaQ degron under varying arabinose concentrations.
After characterization, we concluded our degron was successful, however overall motility failed.
The strain UU2685 E. coli cells without the transformed CheZ system exhibited the expected lack of motility. The cells transformed with our CheZ parts, pBAD+CheZ and pBAD+CheZ+YbaQ displayed significant motility, even without the induction of arabinose.
The pBAD+CheZ showed consistent diffusion motility for both 0% and 0.02% induction. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.092, indicating a lack of significance in the area of diffusion. This contradicts the hypothesis of pBAD+CheZ relying on arabinose induction for motility. The reasoning behind this lack of significance is inconclusive, but may be a result of arabinose contamination from an unaccounted source such as the cell media. An additional possibility is our overestimation of promoter regulation. The pBAD+CheZ induced with 0.2% arabinose displayed motility significantly different (p-value of 0.005) from the combined motility of uninduced pBAD+CheZ and pBAD+CheZ induced with 0.02% arabinose. It was concluded this was likely a result of the greater concentration of arabinose providing the cells with more sugar to consume over time, permitting a greater displacement.
Figure 3. Motility of pBAD+CheZ construct with 0.2% arabinose.
Figure 4. Motility of pBAD+CheZ construct with 0.02% arabinose.
Figure 5. Motility of pBAD+CheZ construct with 0% arabinose.
The pBAD+CheZ+YbaQ induced with 0.02% arabinose produced an area of diffusion comparable to that of pBAD+CheZ+YbaQ when induced with 0% arabinose. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.978, indicating a lack of significant difference. The lack of significance can be attributed to similar conditions discussed in the pBAD+CheZ analysis when the same trend was observed. The pBAD+CheZ+YbaQ induced with 0.2% arabinose was significantly more motile than the motilities of pBAD+CheZ+YbaQ with no induction and 0.02% induction combined. This was maintained by a p-value of 0.0039 generated by a t-test similar to the one mentioned above.
Figure 6.</strong> Motility of pBAD+CheZ+YbaQ construct with 0.2% arabinose.
Figure 7. Motility of pBAD+CheZ+YbaQ construct with 0.02% arabinose.
Figure 8. Motility of pBAD+CheZ+YbaQ construct with 0% arabinose.
The motility observed for the pBAD+CheZ construct with no arabinose induction was registered as a false positive; the lack of arabinose should have yielded no motility. This implies the presence of background CheZ. However, the pBAD+CheZ+YbaQ displayed significantly less motility (two-tail t-test p-value of 0.02) indicating a decrease in the CheZ protein with the presence of the degron. We have concluded this indicates the degron is successful in degrading excess CheZ to allow for the efficient function of chemotaxis. The inhibition of excess CheZ is imperative to the success of the motility mechanism because it ensures that MRSA is precisely targeted as intended. Under optimal conditions (an induction of 0.2% arabinose), pBAD+CheZ showed no significant difference (p-value of 0.766) in motility compared to pBAD+CheZ+YbaQ.
While there was significant diffusion of the CheZ parts as detailed above, there were no indicators of successful directionality. The cells proliferated in a circular region that contested any form of directed motility.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
None |