Part:BBa_K1638032

T18 domain of cyaA with cAMP-induced RFP generator

This part contains the T18 domain of the adenylate cyclase cyaA from Bordetella pertussis. This part is intented to be used in the bacterial two-hybrid system. This system is based on the reconstitution of the adenylate cyclase. When protein-coding genes is suffixed to the T25 and T18 domains of cyaA, one can study the interaction of these two proteins. If the conjugated proteins interacts, T25 and T18 will be brought into close proximity. This will enable the two catalytic domains T18 and T25 to synthesize cyclic adenosinemonophosphate (cAMP). The rise in cAMP can trigger the expression of genes by using a cAMP-induced promotor that induce the transcription of red fluorescent protein (RFP). For this reason this part also contains a mRFP generator controlled by PcstA BBa_K861173. The presence of red-fluorescent cells can in turn be used to verify protein-protein interactions [1].

See BBa_K1638033 for the T25 domain.

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 1719
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1260
  Illegal NgoMIV site found at 1670
  Illegal AgeI site found at 712
  Illegal AgeI site found at 824
  Illegal AgeI site found at 1476
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

To validate that our T18 and T25 domain constructs in fact can be used to study protein-protein interactions, we made a control experiment, where the leucine zipper region from the GCN4 yeast protein was fused to the T18 and T25 domains (T18-Zip+T25-Zip). Leucine zippers are known to interact by forming homodimers. If the system indeed works, their interaction will lead to functional complementation between the T18 and T25 domains. This leads to the synthesis of cAMP. By using the cAMP-induced lacZ reporter system, one can observe whether or not there is an interaction. This system is part of the cyaA-deficient Escherichia coli K12-strain BTH101 (MC1061-derived). The lacZ gene encodes a β-Galactosidase which is positively controlled by cAMP.

Four different combinations were sequentially co-transformed into the BTH101-strain¤:

• pSB1C3-T18+pSB1K3-T25
• pSB1C3-T18+pSB1K3-T25-Zip
• pSB1C3-T18-Zip+pSB1K3-T25
• pSB1C3-T18-Zip+pSB1K3-T25-Zip

These transformations were plated out on LB/X-gal plates with appropriate antibiotics (chloramphenicol 25 µg/ml and kanamycin 25 µg/ml) and 40 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-gal produces a blue dye, when cleaved by β-Galactosidase. This will give a characteristic blue phenotype.

Plate streaking of transformed BTH101 on LB/X-gal plates containing pSB1C3-T18+pSB1K3-T25, pSB1C3-T18-Zip+pSB1K3-T25, pSB1C3-T18-Zip+pSB1K3-T25 and pSB1C3-T18-Zip+pSB1K3-T25-Zip. Both the transformations and the streaks of the transformed BTH101 were incubated at 37^oC overnight.

As expected, the results only showed complementation between T18 and T25 when the leucine zipper was fused to both of the domains. These results prove that leucine zippers form homodimers, and that our T18/T25 constructs function as expected. This indicates that the system indeed can be used to study protein-protein interactions.

¤Note: all of the constructs were under control by lac promoter, Plac.

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.