In the cyanobacterium Synechococcus elongatus(PCC 7942), three genes (kaiA, kaiB, and kaiC) are essential components of the circadian clock, whose concentration ratio is also important. According to previous studies, the concentration ratio of these three core proteins, KaiA, KaiB and KaiC, is 1:1:4 (by weight).

Researchers has incubated KaiC with KaiA and KaiB in vitro at a ratio similar to that measured in vivo in the presence of 1 mM ATP. As a result, KaiC phosphorylation has robustly oscillated for at least three cycles without damping. But the amplitude of this in vitro was smaller than that observed in vivo under continuous light conditions.

More specifically, the change in concentration of these three kind of  proteins could exert a complex impact on the KaiABC circadian clock system, or in other words, the circadian oscillation of KaiC phosphorylation. For example, in cyanobacteria, KaiC represses its own (kaiBC) expression, whereas KaiA enhances the expression of kaiBC. Therefore, KaiC and KaiA could be, respectively, regarded as negative and positive factors functioning in circadian feedback regulation of kaiBC expression.

Besides, sufficient and appropriate protein concentrations is necessary for Kai protein interactions and KaiC phosphorylation. To take an example, several researchers have already explored the effect of Kai protein concentration on the rhythm of KaiC phosphorylation. In experiment, the standard mixture (×1 solution) was consisted of 1.2 μM KaiA, 3.5 μM KaiB, and 3.5 μM KaiC. When the proteins were at 2.5× or 5× protein concentrations, the KaiC phosphorylation rhythms were nearly the same as those measured under standard conditions. While lowering the protein concentration to 1/10 of the standard mixture, the amplitude of the KaiC phosphorylation rhythm was reduced and the period of the rhythm was prolonged. What’s more, at 1/20 or 1/40 of the standard concentrations, KaiC would be remained dephosphorylated. In addition, in the case of a decrease of the relative concentrations of KaiA or KaiB to KaiC, the rhythm could change accordingly. While lowering either KaiA or KaiB concentrations to one-fourth or one-third of the standard mixture abolished cycling, the rhythm was still maintained in the presence of a two-thirds concentration of either KaiA or KaiB. However, the period is extended by lowering KaiA concentrations to two-thirds, while lowering the KaiB concentration to two-thirds of the standard couldn’t alter the rhythm.

Owing to a series of reasons like the lack of a complete transcriptional/translational feedback loop(TTFL), the KaiABC system, reconstituted in yeast cells, can not express the correct protein concentrations as in cyanobacteria. Therefore, we intend to get a good command of protein concentrations by selecting promoters of different intensity to construct the corresponding expression cassettes, thus the KaiABC system works better and more stably. Besides, we measure our promoter strength of six core genes convenient for the analysis of the final results.

Based on the research measuring the promoter strength of 14 constitutive promoters by GFP fluorescence intensity[4], we designed our experiment about the measurement of the promoters by constructing mCherry cassette with different promoters to measure the fluorescence intensity of mCherry, representing the promoter strength. Before our experiment, we selected three common promoters which are TDH3 promoter, PGK1 promoter and TEF1 promoter according the data of the paper as our promoters of six core genes. The detailed information of plasmids we constructed were shown on the table 1 below. We assembled our plasmids by yeast homologous recombination. Taking TEF1P-mCherry-TEF1Tcassette for example, we obtained fragments TEF1P, mCherry and TEF1Twith homologous arms by PCR and then we transformed three fragments and linearized vector pRS415 which had been digested by enzymes HindⅢ and NotΙ  into Saccharomyces cerevisiae to do yeast homologous recombination. Through the screening of nutrition label and the verification of PCR, we got the right strains and did further measurement.

Notes for table 1:TEF1P, TEF1T, PGK1P, PGK1T, TDH3P and ADH1T represent TEF1 promoter, TEF1 terminator, PGK1 promoter, PGK1 terminator, TDH3 promoter and ADH1 terminator.

In our experiment, we only measured the promoters we have selected before. Our results were as follows.

What’s more, we constructed  three combinations with two kinds of promoter-gene combinations to character different forms of oscillation. As is shown in the Figure 1, we increased the relative concentration of KaiA protein greatly and decreased the relative concentration of KaiB protein slightly in KaiC-SasA and KaiC-CikA combinations, which shortened the period of the circadian oscillation . In our KaiB-KaiC combination, we increased the relative concentration of KaiA protein greatly.

Our aim is to reconstruct the KaiABC circadian clock system of prokaryotic cyanobacteria in nonciracdian eukaryotic Saccharomyces cerevisiae. First of all, we hope to introduce the core proteins of cyanobacterial circadian clock, KaiA, KaiB and KaiC, into yeast to make them oscillate stably. To prevent KaiC from being trapped in phosphorylation state, we select three auxiliary proteins: SasA, CikA and RpaA. The promoter of prokaryotes can not be directly recognized by yeast, so we abandon the way of using the relevant promoters of the RpaA-mediated downstream reaction which are inherent in cyanobacterial, instead use the yeast two-hybrid system to characterize the KaiABC circadian clock system.

We selected three pairs of periodically binding proteins, KaiC-SasA, KaiC-CikA, and KaiB-KaiC, as the "prey" and "bait" of the yeast two-hybrid system, respectively, to construct fusion proteins with activation domain(AD) or DNA binding domain(BD) of Gal4 protein. Taking for example the KaiC-SasA couple, we assembled three gene expression cassettes of KaiA, KaiB, and AD-KaiC onto the pRS413 plasmid named pABaC, and assembled other three gene expression cassettes of CikA, RpaA and BD-SasA onto the pRS415 plasmid named pCiRbS. Other plasmids involved were pbCiRS (the recombinant pRS415 containing cassettes of BD-CikA, RpaA, SasA), pbCRCi (the recombinant pRS413 containing cassettes of BD-KaiC, RpaA, CikA), paBAS (the recombinant pRS415 containing cassettes of AD-KaiB, KaiA, SasA). When the two plasmids are successfully expressed in yeast, that is to say, when the KaiABC circadian clock system successfully operates, KaiA binds to the CII subunit of KaiC during the subjective daytime, stimulating the autokinase activity of the CII subunit, resulting in the phosphorylation of CII subunit. Residues Ser431 and Thr432 are phosphorylated in turn, during which SasA binds to phosphorylated KaiC, allowing AD and BD to be spatially close enough to activate the promoters Gal1 promoter, Gal2 promoter that regulate downstream genes. When KaiC phosphorylation is complete, the KaiC protein undergoes loop stacking, which causes the binding site of KaiA to be blocked and the binding site of KaiB to be exposed. At the same time, KaiA detaches from KaiC, and KaiB binds to KaiC, stimulating its dephosphorylation. KaiB has a competitive relationship with SasA, which means that SasA falls off from KaiC, causing AD and BD to move away from each other and the related downstream genes not to be started.

In the experiment, we have used the restriction enzyme ligation method, Gibson assembly method and yeast homologous recombination method successively to construct the plasmids mentioned before. Since the experimental results showed that the first two methods were somewhat less efficient than the third one, we finally used the yeast homologous recombination method for plasmids assembly. (Table 1) Taking KaiC-SasA couple as an example, we first used PCR to add corresponding homology arms to the ends of ten gene fragments including TEF1P (the promoter of TEF1, kaiA, TEF1T（the terminator of TEF1）, PGK1P (the promoter of PGK1), kaiB, PGK1T (the terminator of PGK1), TDH3P (the promoter of TDH3), AD, kaiC and ADH1T(the terminator of ADH1, then we introduced the ten gene fragments into the yeast together with the pRS413 plasmid cut by EcoRI and NotI to construct three gene expression cassettes of KaiA(TEF1P-kaiA-TEF1T), KaiB(PGK1P-kaiB-PGK1T), AD-KaiC(TDH3P-AD-kaiC-ADH1T. The right strains containing the recombinant plasmid were screened by nutrition labeling, verified by PCR tag and then the recombinant plasmid was amplified in Escherichia Coli to obtain a large amount. Similarly, a sufficient number of recombinant pRS415 plasmids were obtained using the same way. Finally, we transformed the two kinds of plasmids into the final chassis cell, Saccharomyces cerevisiae BY4741, to complete the construction of the KaiABC system.

 There are many report genes available but not everyone is suitable. We looked up papers and websites, screening massively. With the help of modeling, we primarily chose Fluc, Nanoluc, EYFP, mCherry, mOrange, and  ECFP as our report genes. Then we did verification experiments by linking report genes above with constitutive promoter TDH3 promoter and measured the fluorescence intensity. The detailed information were shown on the table2 below.
Taking TDH3P-mCherry-ADH1T cassette for example, we obtained fragments TDH3P,mCherry and ADH1T with homologous arms by PCR and then we transformed three fragments and linearized vector pRS413 which had been digested by enzymes BamHΙ and Not Ι into Saccharomyces cerevisiae to do yeast homologous recombination.Through the screening of nutrition label and the verification of PCR, we got the right strains for further measurement.

Part of our results is shown on the figure below.

According to our preliminary experiment, we excluded mOrange, ECFP and RFP and finally we chose Nanoluc, Fluc, EYFP and mCherry as our report genes to characterize our system. The reasons were that mOrange showed very low fluorescence intensity and RFP was macroscopic and we didn’t obtain right genes of ECFP.

Except for the selection of report genes, these cassettes play a role in other ways. For example, it was used as a positive control group in our experiment which can help us test our measuring methods and instruments to some degree. Moreover, we measured the degradation curve of mCherry and EYFP, which did a favor for our analysis of the results on the one hand and added new experimental characterization data to part_BBaE2030 and part_BBaE2050 on the other hand.

To characterize the viability of our circadian clock in the Saccharomyces cerevisiae, we constructed reporter plasmids containing the report genes that functions efficiently in Saccharomyces cerevisiae.

Prior to we doing this, our modeling group’s members facilitated us to pick out the fluorescent proteins fitted for our project most. They set up a Evaluation Model, which takes issues like lifetime, quantity yield(QY), bleaching time and strokes into account to select the suitable fluorescent proteins among millions of alternatives. The details about the Evaluation Model can be found in this page. We finally picked out two winners among hundreds of participants: EYFP(BBa_E2030) and mCherry (BBa_E2060).

Besides, thanks to the help of Prof. Li, we decided to simultaneously use the luciferase, a popular choice as a reporter gene. Functional enzyme is created immediately upon translation and the assay is rapid, reliable and easy to perform with ATP, oxygen, and luciferin as substrates. Using luciferase as the genetic reporter in analysis is well suited to laboratory automation and high-throughput applications. As for NanoLuc luciferase, it uses a novel coelenterazine analog to produce high intensity, glow-type luminescence. The luminescent reaction is designed to suppress background luminescence for maximal assay sensitivity. It also possesses a number of physical properties that make it an excellent reporter protein: small, monomeric enzyme, high thermal stability and so on.

Due to the Y2H system, the promoter needs to have less leakage expression and respond sensitively to the combination of AD and BD. After a thorough search, we eventually found a mutant Gal1 promoter, which was designed to functionally reduce false positive conditions. More information can be found in this page. Besides, we selected another Gal2 promoter, which works independently in order to improve the accuracy.

We were successfully cloning four kinds of plasmids carrying the genes of mCherry, EYFP, NanoLuc and Fluc respectively with the Gal1 promoter and the ADH1T terminator by harnessing the principles of yeast homologous recombination. And also other four kinds of plasmids with the Gal2 promoter resembled the above four types, to compare which promoter works better. Moreover, to avoid the appearance of false positive phenomena which are likely to happen in the yeast two-hybrid system, we constructed the plasmids in four genres, and every kind of plasmids contained two cassettes consisting of Gal1 promoter with varying fluorescent proteins and Gal2 promoter with different luciferases. Only when both reporter genes function normally can we ensure that the system succeeds. all circuits are constructed respectively on the plasmid pRS416 and the details can be found in the table.1 below. 

Ultimately, fluorescence spectrophotometer and multilabel reader were performed on our detecting process to analyze the expression of the fluorescent proteins and luciferases of the plasmid we constructed in the Saccharomyces cerevisiae respectively.

We were successfully cloning four kinds of plasmids carrying the genes of mCherry, EYFP, NanoLuc and Fluc respectively with the Gal1 promoter and the ADH1T terminator by harnessing the principles of yeast homologous recombination. And also other four kinds of plasmids with the Gal2 promoter resembled the above four types, to compare which promoter works better. Moreover, to avoid the appearance of false positive phenomena which are likely to happen in the yeast two-hybrid system, we constructed the plasmids in four genres, and every kind of plasmids contained two cassettes consisting of Gal1 promoter with varying fluorescent proteins and Gal2 promoter with different luciferases. Only when both reporter genes function normally can we ensure that the system succeeds. all circuits are constructed respectively on the plasmid pRS416 and the details can be found in the table.1 below. 

Ultimately, fluorescence spectrophotometer and multilabel reader were performed on our detecting process to analyze the expression of the fluorescent proteins and luciferases of the plasmid we constructed in the Saccharomyces cerevisiae respectively.

At last, we get a number of different strains respectively containing a pair of selected recombinant plasmids and one reporter plasmid. (Table 5)