Difference between revisions of "Part:BBa K2627001"
(→‎Characaterization of BBa_K2627001)
 
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<partinfo>BBa_K2627001 short</partinfo>
 
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CcaS belongs to the CcaS-CcaR two-component system, which is a sensor for green light and red light derived from cyanobacterium Synechocystis sp.. CcaS is a sensor protein located on the cell membrane, when it senses the existence of green light or red light, it will autophosphorylate and make the other part of this two-component system--CcaR phosphorylate, thus CcaR can bind to a specific area of DNA, and activate the expression of downstream gene. When green light exists, the gene will be activated while with the existence of red light, the expression will be repressed. Different from the original CcaS derived from Synechocystis sp., we made an improvement of deleting PAS domain on CcaS, making it more sensitive to light, so the expression under green light will be enhanced and the leakage under red light will decrease. In our project, we applied this improved system to increase the sensitivity of the switch as well as the production of PHB.
 
  
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===Usage and Biology===
 
  
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===Usage and Biology===
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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.
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[[File:T--SDU-China--3410.png|600px|center]]
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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.<br>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2627001 parameters</partinfo>
 
<partinfo>BBa_K2627001 parameters</partinfo>
 
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==Results==
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===Characaterization of BBa_K2627001===
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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 at 9h in comparison with BBa_K592001.
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The following figures show the relative fluorescence intensity induced by green light and red light.
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[[File:T--SDU-China---3.png|600px|center|thumb|<p align="justify">'''
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Figure 1. Characterization of part BBa_K2627001 and BBa_K592001 in M9 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).'''</p>]]
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[[File:T--SDU-China--32.png|600px|center|thumb|<p align="justify">'''
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Figure 2. Characterization of part BBa_K2627001 and BBa_K592001 in M9 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).'''</p>]]
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[[File:T--SDU-China--33.png|600px|center|thumb|<p align="justify">'''
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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).'''</p>]]

Latest revision as of 03:59, 18 October 2018


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.

T--SDU-China--3410.png

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 at 9h 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 M9 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 M9 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).