Part:BBa_K2627001

Advanced CcaS with PAS domain knocked out

Usage and Biology

CcaS protein belongs to green/red light sensing two-component system. The two-component system consists of the membrane-associated histidine kinase CcaS and its response regulator CcaR. The activating information stored in light is captured by phytochromes in situ. In phytochromes, a bilin-chromophore (in this case phycocyanobilin) binds at a conserved cysteine within an N-terminal GAF (cyclic GMP phosphodiesterase, adenylyl cyclase, FhlA) domain and imparts reversible photoactivation of signaling activity with maximal responses to 535-nm (green) and 672-nm (red) light. Absorption of green light increases the rate of CcaS autophosphorylation, phosphorylation of CcaR by phosphate transferring, and transcription from the promoter of the phycobilisome linker protein cpcG2, while absorption of red light reverses this process. The lower leakiness is reported to be acquired after removing the second putative promoter in cpcG2, which is thought to be constitutive and contributes to leakiness and low dynamic range.

Part BBa_K2627001 was developed from the original CcaS protein (part BBa_K592001) by removing the two PAS domains, leading to a lower leakage under red light. Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 606
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1400
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Results

Characaterization of BBa_K2627001

Part BBa_K2627001 was characterizaed through measuring relative fluorescence intensity, this measurment was taken place in 24-well plates. Cells were cultured in the seed culture medium for 12 hours before inoculated in 24-well plates in three kinds for culture medium. Then fluorescence intensity of CcaS#4 was measured in comparison with BBa_K592001. The following figures show the relative fluorescence intensity induced by green light and red light.

Figure 1. Characterization of part BBa_K2627001 and BBa_K592001 in LB medium whose carbon source is glucose. Part BBa_K2627001 shows a lower expression index under green light condition and a similar leak index under red light condition. The dynamic ranges of green over red light of different system are: 5.0(Wild type), 3.6(#3).

Figure 2. Characterization of part BBa_K2627001 and BBa_K592001 in LB medium whose carbon source is glycerol. Part BBa_K2627001 shows a similar expression index under green light condition and a similar leak index under red light and dark condition. The dynamic ranges of green over red light of different system are: 2.8(Wild type), 2.7(#3).

Figure 3. Characterization of part BBa_K2627001 and BBa_K592001 in M9 medium whose nitrogen source is yeast extract. Part BBa_K2627001 shows a higher expression index under green light condition and a lower leak index under red light and dark condition. The dynamic ranges of green over red light of different system are: 5.0(Wild type), 3.7(#3).