Composite
Kai 1 3

Part:BBa_K2623013

Designed by: Yi Lin   Group: iGEM18_XMU-China   (2018-09-28)


Kai's phosphorylation core oscllator builder and SfYFP reportor for pKaiBC

Summary

As the first part of Kai circuit, it construct the the KaiC phosphorylation oscillation circuit.
The sequence encoding for the gene is preceded by a inducable promoter BBa_R0011, an RBS BBa_B0034, BBa_K2623001, BBa_K2623002, BBa_K2623003 and followed by a double terminator BBa_B0015 in pSB1C3 for periodic oscillation of KaiC phosphorylation.
The mechanisms by which cellular oscillators keep time and transmit temporal information are poorly understood. In cyanobacteria,the timekeeping aspect of the circadian oscillator, composed of the KaiA, KaiB, and KaiC proteins, involves a cyclic progression of phosphorylation states at Ser431 and Thr432 of KaiC.
Besides, with the aim to connect with BBa_K2623011 and fluorescent signals, we add a specific DNA sequence. The sequence encoding for the gene is preceded by the specific promoter BBa_K2623009, an RBS BBa_B0034, sfYFP BBa_K864100 and followed by a double terminator BBa_B0015 in pSB1C3 to be a part of the Kai circuit, which converts periodic oscillations of protein phosphorylation into periodic fluorescent signals by periodically activated RpaA.

Fig.1 Simplified scheme of the central circadian oscillator system.[1]
Fig.2 Simplified model of the central circadian oscillator and output activities.[1]



Indentification


In order to verify the effectiveness of our Kai system, we verified the Escherichia coli that was transferred into the Kai loop under the fluorescence microscope after synchronization.
Since the Kai system is designed for periodic output of signals in Escherichia coli, we evaluateD the changes of fluorescent signals over time. Here are the curves we made. More details can be found in http://2018.igem.org/Team:XMU-China/Measurement

Fig.3 The bioluminescence at different times in GroupA. The negative value is cause by the value of the control group.
Fig.4 The bioluminescence at different times in GroupB. The negative value is cause by the value of the control group.

Fig.5 The fitting curve of the oscillator in GroupA
Fig.6 The fitting curve of the oscillator in GroupB

Reference

[1]Espinosa J, Boyd J S, Cantos R, et al. Cross-talk and regulatory interactions between the essential response regulator RpaB and cyanobacterial circadian clock output[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(7):2198-203.


More details can be seen in http://2018.igem.org/Team:XMU-China/Results



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 627
    Illegal AgeI site found at 1442
    Illegal AgeI site found at 2153
    Illegal AgeI site found at 2162
    Illegal AgeI site found at 2168
    Illegal AgeI site found at 2492
    Illegal AgeI site found at 2519
  • 1000
    COMPATIBLE WITH RFC[1000]


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