Team:Tianjin/Experiments
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Experiments
Reconstruction of the KaiABC circadian clock system
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 select 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 assemble three gene expression cassettes of KaiA, KaiB, and AD-KaiC onto the pRS413 plasmid named pABaC, and assemble other three gene expression cassettes of CikA, RpaA and BD-SasA onto the pRS415 plasmid named pCiRbS. Other plasmids involved are 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 show that the first two methods are somewhat less efficient than the third one, we finally use the yeast homologous recombination method for plasmids assembly.(Table 1) Taking KaiC-SasA couple as an example, we first use 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 introduce 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 are screened by nutrition labeling, verified by PCR tag and then the recombinant plasmid is amplified in Escherichia Coli to obtain a large amount. Similarly, a sufficient number of recombinant pRS415 plasmids are obtained using the same way. Finally, we transform the two kinds of plasmids into the final chassis cell, Saccharomyces cerevisiae BY4741, to complete the construction of the KaiABC system.
| Table 1 Structure of Recombinant Plasmids | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| pABaC (pRS413) |
KaiA cassette | KaiB cassette | AD-KaiC cassette | ||||||||
| TEF1P | kaiA | TEFIT | PGK1P | KaiB | PGK1T | TDH3P | AD | kaic | ADH1T | ||
| pCiRbS (pRS415) |
CikA cassette | RpaA cassette | BD-SasA cassette | ||||||||
| TEF1P | cikA | TEFIT | PGK1P | KaiB | PGK1T | TDH3P | BD | sasA | ADH1T | ||
| pbCiRS (pRS415) |
BD-CikA cassette | RpaA cassette | SasA cassette | ||||||||
| TEF1P | BD | cikA | TEF1T | PGK1P | rpaA | PGK1T | TDH3p | sasA | ADH1T | ||
| pbCRCi (pRS413) |
BD-KaiC cassette | RpaA cassette | CikA cassette | ||||||||
| TDH3P | BD | kaiC | ADH1T | PGK1P | rpaA | PGK1T | TEF1P | cikA | TEF1T | ||
| paBAS (pRS415) |
AD-KaiB cassette | KaiA cassette | SasA cassette | ||||||||
| TDH3P | AD | kaiB | ADH1T | PGK1P | kaiA | PGK1T | TEF1P | sasA | TEF1T | ||
| Table 2 Final Experimental Strains | |
|---|---|
pbCRCi + paBAS + p1m | pABaC +pbCiRS +p1F |
pbCRCi + paBAS + p1E | pABaC +pbCiRS +p2F |
pbCRCi + paBAS + p1F | pABaC +pbCiRS +p1N |
pbCRCi + paBAS + p1N | pABaC +pbCiRS +p2N |
pbCRCi + p1m | pABaC +pbCiRS +p1m |
pbCRCi + p1E | pABaC +pbCiRS +p2m |
pbCRCi + p1F | pABaC +pbCiRS +p1E |
pbCRCi + p1N | pABaC +pbCiRS +p2E |
paBAS + p1m | pABaC + p1F |
paBAS + p1E | pABaC + p2F |
paBAS + p1F | pABaC + p1m |
paBAS + p1N | pABaC + p1E |
pABaC +pCiRbS +p1F | pABaC + p2N |
pABaC +pCiRbS +p2F | pbCiRS +p1F |
pABaC +pCiRbS +p1N | pbCiRS +p2F |
pABaC +pCiRbS +p2N | pbCiRS +p1m |
pABaC +pCiRbS +p1m | pbCiRS +p1E |
pABaC +pCiRbS +p2m | pbCiRS +p2N |
pABaC +pCiRbS +p1E | pCiRbS +p1F |
pABaC +pCiRbS +p2E | pCiRbS +p2F |
| pCiRbS +p1m |
| pCiRbS +p1E |
| pCiRbS +p2N |
Construction of the pathway for characterization
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.
Table1 The structure of reporter plasmids
| name | number | cassette |
|---|---|---|
p1m | BBa_K2637036 | Gal1p-mCherry-ADH1T |
p1E | BBa_K2637037 | Gal1p-EYFP-ADH1T |
p1N | BBa_K2637038 | Gal1p-NanoLuc-ADH1T |
p1F | \ | Gal1p-Fluc-ADH1T |
p2m | BBa_K2637039 | Gal2p-mCherry-ADH1T |
p2E | BBa_K2637040 | Gal2p-EYFP-ADH1T |
p2N | BBa_K2637041 | Gal2p-NanoLuc-ADH1T |
p2F | \ | Gal2p-Fluc-ADH1T |
p1m2N | BBa_K2637042 | Gal1p-mCherry-ADH1T- Gal2p-NanoLuc-CYC1 |
p1E2N | BBa_K2637043 | Gal1p-EYFP-ADH1T- Gal2p-NanoLuc-CYC1 |
p1m2F | \ | Gal1p-mCherry-ADH1T- Gal2p-Fluc-CYC1 |
p1E2F | \ | Gal1p-EYFP-ADH1T- Gal2p-Fluc-CYC1 |
(Chromatin remodeling): To test whether the main circadian clock of cyanobacteria could have effect on the whole regulation of Saccharomyces cerevisiae, we substituted the genes controlling the chromatin remodeling in the yeast for report genes to seek for the answers. Prior to we constructing the plasmids we needed, we deleted the genes of ISWI1 and ISWI2 which both play significant roles in chromatin remodeling utilizing the CRISPR-cas9 technology. Afterwards, We constructed the pRS416 plasmid containing the two cassettes consisting of Gal1 promoter with ISWI1 and ADH1T terminator and Gal2 promoter with ISWI2 and CYC1 terminator. And if the core oscillator worked, the genes of ISWI1 and ISWI2 would finally be expressed with the help of yeast two-hybrid system.
With these processes all done, we eventually measured the growth curve and observed the shapes of cells which have been modified by use of the Flow cytometry to see if these modifications had periodic effect on the yeast.
Outreach
Chromatin remodeling refers to that the molecular state of chromatin packaging, the histones in nucleosomes, and the corresponding DNA molecules will transform in the process of replication and recombination of gene expression.
This remodeling is mainly through two approaches. First, this could be attained by covalent histone modification of specific enzymes, just like histone acetyltransferase (HATs), deacetylase, methyltransferase, and kinase. Specifically, it refers to the addition or removal of various chemical elements on histones under the catalysis of a specific protein complex called a histone modification complex. These enzymatic modifications include acetylation, methylation, phosphorylation, and ubiquitination occurring primarily at the N-terminal tail of histones. Furthermore, these modifications exert effect on the binding affinity between histones and DNA, thereby loosening or tightening the concentrated DNA surrounding the histones. For example, methylation of specific lysine residues in H3 and H4 leads to DNA being further combined with histones which deters transcription factors from binding to genes inhibiting the expression of DNA. In contrast, histone acetylation relaxes chromatin and exposes DNA to bind to transcription factors, giving rise to increasing gene expression.[1][2][3][4]
Second, ATP-dependent chromatin remodeling complex could move, eject or reconstruct nucleosomes to achieve the aim of remodeling. These protein complexes share a common ATPase domain, which can relocate the position of the nucleosome on the DNA by utilizing the energy of ATP hydrolysis to keep the histone away from DNA or promote the exchange of histone variants, thereby producing nucleosome free the DNA region which will activate the expression of DNA. In addition, some remodeling complexes have DNA translocation activity and can perform specific remodeling tasks. Currently we konw that there are at least five chromatin remodeling families in eukaryotes: SWI / SNF, ISWI, NuRD / Mi-2 / CHD, INO80 and SWR1. Although all remodeling complexes share a common ATPase domain, their function is based on several specific biological processes, just like DNA repair, apoptosis, and so on. This is due to the fact that each remodeling complex has a unique protein domain in its catalytic ATPase region and also has different recruitment subunits.[5][6]
Although there still remains some puzzles of the mechanism of the chromatin remodeling, a chunk of researches embodied that these two approaches indeed play a significant role in chromatin remodeling and altering the chromosome topology.
Therefore, in our project, we selected two types of chromatin remodeling families: SWI / SNF and ISWI to substitute the reporter as the downstream proteins, since these two families have been well studied, especially in the yeast model.
Our Saccharomyces cerevisiae incorporated the heterogeneous KaiABC circadian clock from cynaobacterium Synechococcus elongatus. Once KaiC combined with SasA, the downstream genes initiate expressing by utilizing the yeast two- hybrid system. And due to the fact that the tightness of these two proteins’ combination will change over time, the amount of genes’ expression will alter accordingly. Since we substituted the genes of SWI / SNF and ISWI for the downstream genes, we could eventually measure the growth curve and observe the shapes of cells which have been modified by use of the Flow cytometry to find out if these modifications had periodic effect on the Saccharomyces cerevisiae.
The cell factory with KaiABC circadian clock system
Our S. cerevisiae, which incorporates heterogeneous KaiABC circadian clock system from cyanobacterium Synechococcus elongatus, can be used to construct microbial consortium with two yeasts, making it possible to produce different products in subjective days and nights.
In detail, one of the S. cerevisiae combining KaiC with SasA at subjective dawn is able to initiate the expression of downstream target gene, which can translate into a kind of product with a refreshing effect, such as caffeine. In another S. cerevisiae, KaiC is bound to CikA or KaiB at subjective dusk to generate the expression of another downstream gene, whose translational products can function as sleep-enhancing supplements or mosquito repellent, such as melatonin and limonene. In this way, the KaiABC circadian clock system can be used as a cell factory that alternately and periodically produces two different substances between day and night.
Our cell factory will serve as a platform for a variety of biotechnological applications, by varying the downstream target genes to achieve different functions, such as treatment of circadian rhythm disorders, automatic daily drug delivery, etc. To take an example, as we all know, intestinal microbes have a significant impact on human health such as immunity, emotions, and the body's own biological clock. Therefore, perhaps for the foreseeable future, we can introduce the KaiABC oscillation system or its evolution into a specific kind of intestinal microbes to synthesize and secrete certain chemical substances or biologically active molecules to achieve better regulation of the environment in the human body.
Based on this idea, we conducted experimental exploration of caffeine and limonene using the parts provided by iGEM. Caffeine is a xanthine alkaloid compound, a central nervous stimulant that temporarily dispels drowsiness and restores energy, is used clinically to treat neurasthenia and coma. It is also widely used as an additive for a variety of functional refreshing beverages. Limonene is a volatile monoterpenoid compound and produces a lemon-like odor with the effect of repelling mosquitoes. Besides, limonene could inhibits rat mammary gland and other Tumor development, d-limonene has also been used clinically to dissolve cholesterol-containing gallstones. Owing to the benefits mentioned above, inhaling limonene may also have positive influence on our health, with the potentials to prevent cancer and dissolve gallstones. We originally hoped to construct a caffeine-limonenoe cell factory. Unfortunately, the caffeine gene provided in parts showed unclear problems, deterring us from making this idea into reality.
However, we have successfully assembled gene expression cassettes of limonene synthase onto the pRS416 plasmid named pGLA, and transformed pGLA into the S. cerevisiae we have constructed with the KaiABC system to form the limonene cell factory. The newly-built cell factory working with the KaiABC circadian clock systemhas achieved to periodically produce limonene during subjective nights only to help repel mosquitoes. We have constructed a biobrick(BBa_K2637044) encoding limonene sythase expression cassette, which was verifed via SDS-PAGE to tiral its function. You can see the results from here.
Overall, our cell factory proves to be an novel application for Kai ABC circadian clock system and other similar oscillators. We also hope that our work could provide inspirations for both researchers and companies from food, pharmaceutical or other relevant industries. And we would be extremely honored if anyone could transform our design into reality completely in the future.