Part:BBa_K1638038

Yhbj fused to T25 domain of CyaA with cAMP-induced RFP reporter

The RNase adapter protein YhbJ fused to the T25-domain of the adenylate cyclase, CyaA, from Bordetella pertussis. This construct can be used in the two-hybrid screening of protein-protein interactions between YhbJ and another x protein fused to the T18 domain. If a library of peptide aptamers is fused to the T18-domain, one can screen for specific peptide aptamers against YhbJ [1].

Click in on the following parts to dig deeper into the two-hybrid screening and learn how to construct your own experiment for detection of protein-protein interactions:
BBa_K1638033
BBa_K1638005

If you want to do a screening for peptide aptamers we recommend the following parts:
BBa_K1638018
BBa_K1638035

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1851
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 712
  Illegal AgeI site found at 824
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Characterization of cstA promoter, PcstA

Transcriptional activity of PcstA during growth by measurering RNA. Negative control of MG1655ΔcyaA in LB, WT in LB+0.2% glucose, and WT in LB. Samples collected at different OD₆₀₀measurements. Graph shows intensities of mRNA-gfp normalized according to intensity of 5S.

We set up to measure promotor activity of PcstA by measurering levels of gfp-mRNA with bacteria transformed with PcstA-gfp (BBa_K1135002). MG1655ΔcyaA, LB was used as a negative control. Samples were collected at different OD₆₀₀-measurements. A single sample from the negative control was collected at OD₆₀₀ = 0.3. The RNA from the samples was purified and a Northen Blot with gfp and 5S probes was performed.

Generally during the exponential phase of the bacteria, they have a high level of transcriptional activity. However, levels of 5S rRNA are relatively constant at all times. The transcription of gfp increase as the cells enter exponential phase between the two OD₆₀₀ measurements 0.1 and 0.3. As expected, very low levels of gfp can be detected in the negative control. This strain lacks the ability to generate cAMP, and thus very little transcription is induced. The small amounts of gfp could be explained by leakiness of PcstA or that CAP alone initiates some transcription.

In the setup with WT, LB it is quite clear that the amount of gfp rises, compared to WT, LB+0.2% glucose. Transcription is clearly affected by the presence of glucose. One measurement WT, LB OD₆₀₀ = 0.8 stands out. The result is not readily explained, but is probably due to some error. But the tendency of the results correlates with the knowledge of the invert relationship between glucose and cAMP. Glucose signaling will repress adenylate cyclase-activity, thus intracellular levels of cAMP will be low in high-energy states, and little transcription of gfp will be initiated.

References

[1]: Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.