Before our test the whole system, we measured the strength of promoters which regulate the experssion of genes in our oscillator. As there is a natural ratio of the three proteins-KaiA, KaiB and KaiC in cyanobacteria. And a certain ratio of the three proteins has advantage of modeling.
We can draw a conclution about the strength of the three promoters. So we combine ''TEF1'' promoter with ''KaiA'' and ''TEF1'' terminator, ''PGK1'' promoter with ''KaiB'' and ''PGK1'' terminator and ''TDH3'' promoter with ''AD-KaiC'' and ''ADH1'' terminator. Similarly, we combina ''TEF1'' promoter with ''CikA'' and ''TEF1'' terminator, ''PGK1'' promoter with ''RpaA'' and ''PGK1'' terminator and ''TDH3'' promoter with ''BD-SasA'' and ''ADH1'' terminator. And here is only one combination of our experiment. To get more combinations, please see the part from BBa_K2637027 to BBa_K2637038.You can see more about our project to click here.[http://2018.igem.org/Team:Tianjin]
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==Characterization of Life Tik Tok==
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When we start to measure the datas of experiment group, we set up some control groups at the same time. To prove that our oscillator works surely because of the yeast two-hybrid system, we constructed some bacteria in which there are only one fusion protein of yeast two-hybrid system and a reporter gene or a combination. And we choose 3 or 4 reporter genes as parallel experiment groups.
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[[Image:ganggangfluc1.jpeg|center|frame|300px|<b>Figure 4. </b> The result of oscillator measured by Fluc reporter together with the control groups]]
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[[Image:ganggangfluc2.jpeg|center|frame|300px|<b>Figure 5. </b> The result of oscillator measured by Fluc reporter subtracting the 1st control groups]]
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[[Image:ganggangfluc3.jpeg|center|frame|300px|<b>Figure 6. </b> The result of oscillator measured by Fluc reporter subtracting the 2nd control groups]]
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In the above figures, pABaC is the plasmid with ''AD-KaiC'' fused gene and make up a yeast two-hybrid system with the plasmid
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pbCiRS, which is with ''BD-CikA'' fused gene. p1F is the plasmid with Fluc reporter whose promoter is Gal1. In the 1st figure, the experimental group have black datas and the first control group and the second group have green and red datas, respectively. We substracted the control groups to gat a more objective result.
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As we predicted and looked at the results in other literatures, this oscillatory system is unstable.However, even though the fluctuations are not obvious on the second day, better waveforms could be observed on the first and third days.In addition, on the third day we get very high peak values, and in the wave trough experimental report gene expression is closer to the control group and the blank group, this indicates that in subjective day, yeast two-hybrid system works and results the peak value, in subjective night, it doesn't work and causes the similar results to the control groups and the background.
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Therefore, the overall experimental results show that we successfully reconstructed the oscillator in yeast.
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And the following figures are the results we measured with live Nanoluc. We get the similar conclusion to the Fluc group.
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[[Image:gangganglive1.jpeg|center|frame|300px|<b>Figure 7. </b> The result of oscillator measured by the 1st live Nanoluc reporter together with the control groups]]
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[[Image:gangganglive2.jpeg|center|frame|300px|<b>Figure 8. </b> The result of oscillator measured by the 1st live Nanoluc reporter subtracting the 1st control groups]]
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[[Image:gangganglive3.jpeg|center|frame|300px|<b>Figure 9. </b> The result of oscillator measured by the 1st live Nanoluc reporter subtracting the 2nd control groups]]
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[[Image:gangganglive4.jpeg|center|frame|300px|<b>Figure 10. </b> The result of oscillator measured by the 2nd live Nanoluc reporter together with the control groups]]
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[[Image:gangganglive5.jpeg|center|frame|300px|<b>Figure 11. </b> The result of oscillator measured by the 2nd live Nanoluc reporter subtracting the 1st control groups]]
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[[Image:gangganglive6.jpeg|center|frame|300px|<b>Figure 12. </b> The result of oscillator measured by the 2nd live Nanoluc reporter subtracting the 2nd control groups]]
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You can see more details of our results [http://2018.igem.org/Team:Tianjin/Demonstrate here]
Revision as of 06:46, 16 October 2018
AD-KaiC, Gal4 activation domain fused with KaiC
This fusion protein is made up of Gal4 activation domain and KaiC and fuctions as a part of yeast two-hybrid system. When KaiC binds to another part of the system-SasA, CikA, or KaiB, AD simultaneously binds to upstream genes and initiates the transcription of downstream genes.To see more about the sequence or other things, visit BBa_K2637007 and BBa_K2637003.
Notes: You can select this part and part BBa_K2637022 to make up a yeast two-hybrid system and finally constructe an oscillator. And you can add a specific promoter to this part to regulate the expression of your reporter gene. If you want a integrated yeast two-hybrid system, you can select other combination. You can find that on part page in our wiki.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 670 Illegal BglII site found at 1177
[1]Roger Tseng, Nicolette F. Goularte, Archana Chavan, Jansen Luu, Susan E. Cohen, Yong-Gang Chang, Joel Heisler, Sheng Li, Alicia K. Michael, Sarvind Tripathi, Susan S. Golden, Andy LiWang, Carrie L. Partch, Structural basis of the day-night transition in a bacterial circadian clock. Science, 1174-1180 (2017).
[2]Joost Snijder, Jan M. Schuller, Anika Wiegard, Philip Lössl, Nicolas Schmelling, Ilka M. Axmann, Jürgen M. Plitzko, Friedrich Förster, Albert J. R. Heck, Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state. Science, 1181-1184 (2017).
[3]Joseph S.Markson, Joseph R.Piechura, Anna M.Puszynska, Erin K.O’Shea, Circadian Control of Global Gene Expression by the Cyanobacterial Master Regulator RpaA. Cell, 1396-1408 (2013).
[4]Jun O. Liu,et al.Everything you need to know about the yeast two-hybrid system.[J]. Nature Structural Biology, 1998, 535-536
[5]Hideo Iwasaki,* Stanly B. Hideo, Iwasaki. Sensory Histidine Kinase, SasA, Necessary to Sustain Robust Circadian Oscillation in Cyanobacteria.[J]. cell, 2000, 101(2): 223-233
