iGEM 2018 — Team Tianjin

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# Team:Tianjin/Notebook

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Team:Tianjin - 2018.igem.org

* PROJECT
  + [BACKGROUND](https://2018.igem.org/Team%3ATianjin/Description)
  + [DESIGN](https://2018.igem.org/Team%3ATianjin/Design)
  + [EXPERIMENTS](https://2018.igem.org/Team%3ATianjin/Experiments)
  + [NOTEBOOK](https://2018.igem.org/Team%3ATianjin/Notebook)
  + [MODEL](https://2018.igem.org/Team%3ATianjin/Model)
  + [MEASUREMENT](https://2018.igem.org/Team%3ATianjin/Measurement)
  + [DEMONSTRATE](https://2018.igem.org/Team%3ATianjin/Demonstrate)
* PARTS
  + [PARTS OVERVIEW](https://2018.igem.org/Team%3ATianjin/Parts)
  + [BASIC PARTS](https://2018.igem.org/Team%3ATianjin/Basic_Part)
  + [COMPOSITE PARTS](https://2018.igem.org/Team%3ATianjin/Composite_Part)
  + [PART COLLECTION](https://2018.igem.org/Team%3ATianjin/Part_Collection)
  + [IMPROVED PART](https://2018.igem.org/Team%3ATianjin/Improve)
* [SAFETY](https://2018.igem.org/Team%3ATianjin/Safety)
* HUMAN PRACTICES
  + [INTEGRATED](https://2018.igem.org/Team%3ATianjin/Human_Practices)
  + [COLLABORATION](https://2018.igem.org/Team%3ATianjin/Collaborations)
  + [PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3ATianjin/Public_Engagement)
* TEAM
  + [MEMBERS](https://2018.igem.org/Team%3ATianjin/Team)
  + [ATTRIBUTIONS](https://2018.igem.org/Team%3ATianjin/Attributions)
* [FOR JUDGES](https://2018.igem.org/Team%3ATianjin/Judging)

NOTEBOOK

[1.Construction of Yeast Two-hybrid System Plasmids](#collapseOne)

[Download](https://static.igem.org/mediawiki/2018/5/51/T--Tianjin--123.pdf)

[2.Construction of Reporter Gene Plasmid](#collapseTwo)

[Download](https://static.igem.org/mediawiki/2018/f/f6/T--Tianjin--rp_gene11.pdf)

[3.Construction of Limonene Plasmid](#collapseThree)

[Download](https://static.igem.org/mediawiki/2018/6/6b/T--Tianjin--ningmengxi.pdf)

[4.Deleting Genes and Assembling Plasmids](#collapseFour)

[Download](https://static.igem.org/mediawiki/2018/f/f1/T--Tianjin--XJN.pdf)

[5.Measurement](#collapseFive)

[Download](https://static.igem.org/mediawiki/2018/c/c4/T--Tianjin--measurement_notebook3.pdf)

IGEMTIANJIN

IGEMTIANJIN2018

HEBO@TJU.EDU.CN

BEIYANG CAMPUS,TIANJIN UNIVERSITY,TIANJIN


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tianjin/Experiments

<!DOCTYPE html>

Team:Tianjin - 2018.igem.org

* PROJECT
  + [BACKGROUND](https://2018.igem.org/Team%3ATianjin/Description)
  + [DESIGN](https://2018.igem.org/Team%3ATianjin/Design)
  + [EXPERIMENTS](https://2018.igem.org/Team%3ATianjin/Experiments)
  + [NOTEBOOK](https://2018.igem.org/Team%3ATianjin/Notebook)
  + [MODEL](https://2018.igem.org/Team%3ATianjin/Model)
  + [MEASUREMENT](https://2018.igem.org/Team%3ATianjin/Measurement)
  + [DEMONSTRATE](https://2018.igem.org/Team%3ATianjin/Demonstrate)
* PARTS
  + [PARTS OVERVIEW](https://2018.igem.org/Team%3ATianjin/Parts)
  + [BASIC PARTS](https://2018.igem.org/Team%3ATianjin/Basic_Part)
  + [COMPOSITE PARTS](https://2018.igem.org/Team%3ATianjin/Composite_Part)
  + [PART COLLECTION](https://2018.igem.org/Team%3ATianjin/Part_Collection)
  + [IMPROVED PART](https://2018.igem.org/Team%3ATianjin/Improve)
* [SAFETY](https://2018.igem.org/Team%3ATianjin/Safety)
* HUMAN PRACTICES
  + [INTEGRATED](https://2018.igem.org/Team%3ATianjin/Human_Practices)
  + [COLLABORATION](https://2018.igem.org/Team%3ATianjin/Collaborations)
  + [PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3ATianjin/Public_Engagement)
* TEAM
  + [MEMBERS](https://2018.igem.org/Team%3ATianjin/Team)
  + [ATTRIBUTIONS](https://2018.igem.org/Team%3ATianjin/Attributions)
* [FOR JUDGES](https://2018.igem.org/Team%3ATianjin/Judging)

EXPERIMENTS

Reconstruction of the KaiABC system

Promoter selection

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* promoteraccording 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*-*TEF1T*cassette for example, we obtained fragments *TEF1P, mCherry* and*TEF1T*with homologous arms by PCR and then we transformed three fragments and linearized vector pRS415 which had been digested by enzymes *Hind*Ⅲ 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 and did further measurement.

| Table 1 Structure of Recombinant Plasmids | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Table 1 Plasmids constructed for the measurement of the promoter strength | |
| plasmids | cassette |
| *pTEmCTE* | *TEF1P*-*mCherry*-*TEF1T* |
| *pPGmCPG* | *PGK1P*-*mCherry*-*PGK1T* |
| *pTDmCAD* | *TDH3P*-*mCherry*-*ADH1T* |

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.

![](https://static.igem.org/mediawiki/2018/8/85/T--Tianjin--experiment1.png)

Figure 1 the promoter strength characterized by mCherry

What’s more, we constructed  [three combinations](#table2) 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 .](https://2018.igem.org/Team%3ATianjin/Demonstrate) In our KaiB-KaiC combination, we increased the relative concentration of KaiA protein greatly.

Plasmids assembly

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 *Not*I 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.

| Table 2 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* |

![](https://static.igem.org/mediawiki/2018/6/6c/T--Tianjin--experiment2.png)

![](https://static.igem.org/mediawiki/2018/1/12/T--Tianjin--experiment3.png)

![](https://static.igem.org/mediawiki/2018/1/19/T--Tianjin--experiment4.png)

![](https://static.igem.org/mediawiki/2018/f/fe/T--Tianjin--experiment5.png)

![](https://static.igem.org/mediawiki/2018/a/ab/T--Tianjin--experiment6.png)

Figure 2 Map of the five plasmids

Construction of the reporter circuits

Reporter genes selection

To characterize the viability of our circadian clock, we required special report genes that functions sensitively in Saccharomyces cerevisiae. There are many report genes available but not everyone is suitable. We looked up papers and websites, screening massively. Our [modeling](https://2018.igem.org/Team%3ATianjin/Model) group facilitated us to rate the fluorescent proteins fitted for our project most. They set up an Evaluation Model, which takes issues like lifetime, quantity yield(QY) and strokes into account to select the suitable fluorescent proteins among millions of alternatives.

Besides, Prof. Li recommended *luciferase* for us. The functional enzyme is created immediately upon translation and the assay is rapid, reliable and easy to perform with ATP, oxygen, and luciferin as substrates. It possesses a number of physical properties that make it an excellent reporter protein: small, monomeric enzyme, high thermal stability and so on.

we primarily chose Fluc, NanoLuc(BBa\_K1680009), EYFP(BBa\_E2030), mCherry(BBa\_E2060), mOrange, and ECFP(BBa\_I13602) as alternatives. Then verification experiments were operated by linking report genes above with constitutive promoter *TDH3 promoter* and measured the fluorescence intensity.

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.

 The detailed information were shown on the table2 below.

| Table 3. Plasmids constructed for the measurement of various fluorescent proteins and luciferases | |
| --- | --- |
| plasmids | cassette |
| *pTDmCAD* | *TDH3P-mCherry-ADH1T* |
| *pTDmOAD* | *TDH3P-mOrange-ADH1T* |
| *pTDEYAD* | *TDH3P-EYFP-ADH1T* |
| *pTDECAD* | *TDH3P-ECFP-ADH1T* |
| *pTDRAD* | *TDH3P-RFP-ADH1T* |
| *pTDFAD* | *TDH3P-Fluc-ADH1T* |
| *pTDNAD* | *TDH3P-Nanoluc-ADH1T* |

Part of our results is shown on the figure below.

![](https://static.igem.org/mediawiki/2018/9/9f/T--Tianjin--experiment7.png)

Figure 3 fluorescence intensity of various fluorescence proteins

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 it takes too long for RFP to degrade and we didn’t obtain right genes of ECFP. EYFP and mCherry were finally picked out, which showed higher fluorescence intensity.

Except for the selection of reporter genes, these cassettes play a role [in other ways](https://2018.igem.org/Team%3ATianjin/Measurement#M1). For example, it was used as a positive control group in measuring. And 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.

Y2H report circuits construction

Due to the Y2H system, the promoter needs to have less leakage expression and respond sensitively to the proteins combination. After thorough search, we eventually found a mutant *Gal1 promoter*([BBa\_K2637059](http://parts.igem.org/Part%3ABBa_K2637059)), which was designed to functionally reduce false positive conditions. Besides, we selected *Gal2 promoter*([BBa\_K2637009](http://parts.igem.org/Part%3ABBa_K2637009)), 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 table4 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* *cerevisia**e* respectively.

![](https://static.igem.org/mediawiki/2018/7/75/T--Tianjin--reporter_p1m.jpg)

Figure 4 Example map of the reporter plasmids

| Table4 The structure of reporter plasmids | | |
| --- | --- | --- |
| name | number | cassette |
| p1m | BBa\_K2637036 | *G**al1p-mCherry-ADH1T* |
| p1E | BBa\_K2637037 | *G**al1p-EYFP-ADH1T* |
| p1N | BBa\_K2637038 | *G**al1p-NanoLuc-ADH1T* |
| p1F | \ | *G**al1p-Fluc-ADH1T* |
| p2m | BBa\_K2637039 | *G**al2p-mCherry-ADH1T* |
| p2E | BBa\_K2637040 | *G**al2p-EYFP-ADH1T* |
| p2N | BBa\_K2637041 | *G**al2p-NanoLuc-ADH1T* |
| p2F | \ | *G**al2p-Fluc-ADH1T* |
| p1m2N | BBa\_K2637042 | *G**al1p-mCherry-ADH1T-**G**al2p-NanoLuc-CYC1* |
| p1E2N | BBa\_K2637043 | *G**al1p-EYFP-ADH1T-**G**al2p-NanoLuc-CYC1* |
| p1m2F | \ | *G**al1p-mCherry-ADH1T-**G**al2p-Fluc-CYC1* |
| p1E2F | \ | *G**al1p-EYFP-ADH1T-**G**al2p-Fluc-CYC1* |

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

| Table 5 Final Experimental Strains  (the strains are named after the plasmids that they contain) | |
| --- | --- |
| Experimental group | Control group |
| pABaC +pbCiRS +p1F | pABaC + p1F |
| pbCiRS +p1F |
| pABaC +pbCiRS +p2F | pABaC + p2F |
| pbCiRS +p2F |
| pABaC +pbCiRS +p1N | pABaC + p1N |
| pbCiRS +p1N |
| pABaC +pbCiRS +p2N | pABaC + p2N |
| pbCiRS +p2N |
| pABaC +pbCiRS +p1m | pABaC + p1m |
| pbCiRS +p1m |
| pABaC +pbCiRS +p2m | pABaC + p2m |
| pbCiRS +p2m |
| pABaC +pbCiRS +p1E | pABaC + p1E |
| pbCiRS +p1E |
| pABaC +pbCiRS +p2E | pABaC + p2E |
| pbCiRS +p2E |
| pABaC +pCiRbS +p1F | pABaC + p1F |
| pCiRbS +p1F |
| pABaC +pCiRbS +p2F | pABaC + p2F |
| pCiRbS +p2F |
| pABaC +pCiRbS +p1N | pABaC + p1N |
| pCiRbS +p1N |
| pABaC +pCiRbS +p2N | pABaC + p2N |
| pCiRbS +p2N |
| pABaC +pCiRbS +p1m | pABaC + p1m |
| pCiRbS +p1m |
| pABaC +pCiRbS +p2m | pABaC + p2m |
| pCiRbS +p2m |
| pABaC +pCiRbS +p1E | pABaC + p1E |
| pCiRbS +p1E |
| pABaC +pCiRbS +p2E | pABaC + p2E |
| pCiRbS +p2E |
| pbCRCi + paBAS + p1N | pbCRCi +p1N |
| paBAS + p1N |
| pbCRCi + paBAS + p1F | pbCRCi +p1F |
| paBAS + p1F |
| pbCRCi + paBAS + p1E | pbCRCi+ p1E |
| paBAS + p1E |
| pbCRCi + paBAS + p1m | pbCRCi +p1m |
| paBAS + p1m |
| pbCRCi + paBAS + p2N | pbCRCi +p2N |
| paBAS + p2N |
| pbCRCi + paBAS + p2F | pbCRCi +p2F |
| paBAS + p2F |

Novel application

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 our [human practice](https://2018.igem.org/Team%3ATianjin/Human_Practices), the survey inspired us the idea. We collected the data, sought help from experts, made market analysis to select the proper products. 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 (we eliminated it later for unsatisfactory effect) 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.

![](https://static.igem.org/mediawiki/2018/7/77/T--Tianjin--Experiment5.1.png)

Figure 5 The schematic diagram of our cell factory

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.

We got the three genes controlling caffeine production (BBa\_K801070, BBa\_K801071 and BBa\_K801072) from the work of [Technical University of Munich in 2012](https://2012.igem.org/Team%3ATU_Munich). Unfortunately, the caffeine genes provided in kits showed unclear problems, deterring us from making this idea into reality. By PCR we found it seems not to contain the right sequence. As we can see in the gel photo below, three 1200bp-long sequences are shorter than supposed.

![](https://static.igem.org/mediawiki/2018/f/fb/T--Tianjin--caffeine.jpg)

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](http://parts.igem.org/Part%3ABBa_K2637044).

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.

# References

[1] Nakajima M, Imai K, et al. Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in Vitro. [J]Science, 2005,308(5720):414-415

[2] Kageyama H, Nishiwaki T ,et al. Cyanobacterial Circadian Pacemaker: Kai Protein Complex Dynamics in the KaiC Phosphorylation Cycle In Vitro. [J]Molecular Cell, 2006, 23(2):161-171

[3]Zwicker D, Lubensky DK, ten Wolde. PR Robust circadian clocks from coupled protein-modification and transcription–translation cycles. [J]Proceedings of the national Academy of Sciences of the United States of America , 2010, 107(52):22540-22545

[4] Jie Sun, Zengyi Shao, Hua Zhao, Nikhil Nair, et al. Cloning and Characterization of a Panel of Constitutive Promoters for Applications in Pathway Engineering in Saccharomyces cerevisiae. [J]Biotechnology and Bioengineering, 2012, 109: 2082–2092.

IGEMTIANJIN

IGEMTIANJIN2018

HEBO@TJU.EDU.CN

BEIYANG CAMPUS,TIANJIN UNIVERSITY,TIANJIN

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tianjin/Results

<!DOCTYPE html>

Team:Tianjin - 2018.igem.org

* PROJECT
  + [DESCRIPTION](https://2018.igem.org/Team%3ATianjin/Description)
  + [DESIGN](https://2018.igem.org/Team%3ATianjin/Design)
  + [EXPERIMENTS](https://2018.igem.org/Team%3ATianjin/Experiments)
  + [NOTEBOOK](https://2018.igem.org/Team%3ATianjin/Notebook)
  + [MODEL](https://2018.igem.org/Team%3ATianjin/Model)
  + [DEMONSTRATE](https://2018.igem.org/Team%3ATianjin/Demonstrate)
* PARTS
  + [PARTS OVERVIEW](https://2018.igem.org/Team%3ATianjin/Parts)
  + [BASIC PARTS](https://2018.igem.org/Team%3ATianjin/Basic_Part)
  + [COMPOSITE PARTS](https://2018.igem.org/Team%3ATianjin/Composite_Part)
  + [PART COLLECTION](https://2018.igem.org/Team%3ATianjin/Part_Collection)
  + [IMPROVED PART](https://2018.igem.org/Team%3ATianjin/Improve)
* [SAFETY](https://2018.igem.org/Team%3ATianjin/Safety)
* HUMAN PRACTICES
  + [INTEGRATED HP](https://2018.igem.org/Team%3ATianjin/Human_Practices)
  + [COLLABORATION](https://2018.igem.org/Team%3ATianjin/Human_Practices)
  + [EDU&PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3ATianjin/Public_Engagement)
* TEAM
  + [MEMBERS](https://2018.igem.org/Team%3ATianjin/Team)
  + [ATTRIBUTIONS](https://2018.igem.org/Team%3ATianjin/Attributions)
* [JUDGING FORM](https://igem.org/2018_Judging_Form?team=Tianjin)

Measurment

[How did we choose our reporter gene?](#collapseOne)

To achieve the best measurement of our oscillating system, we chose two different kinds of reporter gene as our downstream reporter, fluorescent protein gene and luciferase gene, which are both widely used in the biotechnological labs.

Fluorescent protein

In search for the part kit provided by the iGEM headquarter, we found several available fluorescent proteins. Under the instruction of our [fluorescent protein judging model](https://2018.igem.org/Team%3ATianjin/Model), we finally chose mCherry and EYFP as our fluorescent protein reporter.

| Table 1. Chosen fluorescent proteins | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Protein | λem(nm) | λem(nm) | part serial number | site |
| mCherry | 587 | 610 | Part:BBa\_E2060 | 3-7L |
| EYFP | 513 | 527 | Part:BBa\_E2030 | 3-7J |

![](https://static.igem.org/mediawiki/2018/4/40/T--Tianjin--measurement1.jpg)

Figure 1. Mechanism of fluorescent proteins

Luciferase

Luciferase is a popular choice as a reporter for gene expression because functional enzyme is created immediately upon translation and the assay is rapid, reliable and easy to perform.[[1,2]](#re1) The fluorescent output of luciferase gene is due to the chemical reaction between luciferase and corresponding substrate, which can be easily detected by the luminoskan microplate reader.
We used firefly luciferase(Fluc) gene purchased from Promega and Nanoluc luciferase(Nluc) gene submitted by Team Tuebingen 2015 as another two reporters for our KaiABC system.

| Table 2. Chosen luciferase | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| luciferase | λem(nm) | resource | Detail |
| Fluc | 560 | Bought from Promega | pGL4 Luciferase Reporter Vectors |
| Nluc | 460 | Part:BBa\_K1680009 | Kit site:7-3N |

![](https://static.igem.org/mediawiki/2018/2/27/T--Tianjin--measurement2.jpg)

Figure 2. Mechanism of luciferase

![](https://static.igem.org/mediawiki/2018/c/cd/T--Tianjin--measurement3.jpg)

Figure 3.The bioluminescent reaction catalyzed by Fluc and Nluc

[How did we design our measurement plan?](#collapseTwo)

When reconstructed in *Escherichia coli*, KaiABC oscillator showed a periodicity of 24 hours[[3]](#re3). To best verify our system can function in *Saccharomyces cerevisiae* , we planned to measure our system using different reporter at **3-hour intervals for 1 day, 2 days, 3 days respectively****.**
As for the measurement of fluorescent proteins, we not only used the common detecting instrument, fourescence spectrophotometer, but also make an attempt to record the fluorescent variation via the cutting-edge Leica DMi8 inverted microscope.
Another thing worth mentioning is that, we bought all of the luciferase assay system from company Promega, among them there was a new kind of Nanoluc luciferase system----Nano-Glo® Live Cell Assay System, which allows experimenters to detect live cell luminescent signals without lytic process, suitable for the 3-day assay. Despite there is no reference indicating that this product, a commonly used assay system in mammalian cells, could be used in*Saccharomyces cerevisiae*, we decided to take a try and have successfully proved its validity. Consequently, we chose Nluc solely to complete our long time luminescent detection .

| Table3.1 day’s measurement ----- Fluorescent protein detection | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
||  |  |
| --- | --- |
| **Experimental**    **Group** | pABaC +pCiRbS +gal1+mCherry |
| pABaC +pbCiRS +gal1+mCherry |
| pABaC +pCiRbS+gal1+EYFP |
| pABaC +pbCiRS +gal1+EYFP |
| **Negative**    **Control Group** | pABaC +gal1+mCherry |
| pCiRbS +gal1+mCherry |
| pbCiRS +gal1+mCherry |
| pABaC +gal1+EYFP |
| pbCiRS +gal1+EYFP |
| pCiRbS +gal1+EYFP |

![](https://static.igem.org/mediawiki/2018/4/4a/T--Tianjin--measurement4.jpg)

Figure 4. Procedures for measuring mCherry/EYFP via fluorescent spectrophotometer

![](https://static.igem.org/mediawiki/2018/8/8a/T--Tianjin--measurement5.jpg)

Figure 5. Procedures for measuring luciferase via luminaskan microplate reader

Detailed methods are available in our [Experiment](https://2018.igem.org/Team%3ATianjin/Experiments).

| Table 4.2 days’ measurement ----- Fluc&Nluc detection in lytic cells | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Experimental**  **Group** | pABaC +pCiRbS +gal1+Fluc |
|  | pABaC +pbCiRS +gal1+Fluc |
|  | pABaC +pCiRbS +gal2+Nanoluc |
|  | pABaC +pbCiRS +gal2+Nanoluc |
| **Negative**  **Control Group** | pABaC +gal1+Fluc |
|  | pbCiRS +gal1+Fluc |
|  | pCiRbS +gal1+Fluc |
|  | pABaC +gal2+Nanoluc |
|  | pbCiRS +gal2+Nanoluc |
|  | pCiRbS +gal2+Nanoluc |

| Table 5.3 days’ measurement ----- Nluc detection in live cells | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Experimental**  **Group** | pABaC +pCiRbS +gal1+Nanoluc |
| pABaC +pbCiRS +gal1+Nanoluc |
| pABaC +pCiRbS +gal2+Nanoluc |
| pABaC +pbCiRS 1+gal2+Nanoluc |
| **Control Group** | pABaC +gal2+Nanoluc |
| pbCiRS +gal2+Nanoluc |
| pCiRbS +gal2+Nanoluc |
| TDH3P+NanoLuc |

EYFP measurement using Leica DMi8

Leica DMi8 is an excellent inverted microscope for life science research, which is utilized extensively in materials inspection and measurement. For live cell research, the DMi8 platform is a complete solution whether precisely follow the development of a single cell in a dish, screen through multiple assays, obtain single molecule resolution, or tease out behaviors of complex processes.
Combined with easy to use software, high resolution cameras, and brilliant LED illumination, these inverted research systems are flexible to see the expression of the fluorescent protein EYFP in *Saccharomyces cerevisiae*. Through high speed imaging, using the external fluorescent filter wheels, we obtained fast and accurate fluorescent images as following show. It tells that our bacteria that we constructed express EYFP successfully and continuously in oscillating system. Meanwhile, the fluorescence intensity detected was very bright, indicating that the expression of EYFP was at a high level.

![](https://static.igem.org/mediawiki/2018/3/3e/T--Tianjin--measurement6.jpg)

Figure 6. pABaC +pCiRbS+gal1+EYFP photoed by Leica DMi8

[Measuement result](#collapseThree)

![](https://static.igem.org/mediawiki/2018/1/12/T--Tianjin--measurement7.jpg)

Figure 7. Experimental result reported by pABaC+pbCiRS+gal1+Fluc

![](https://static.igem.org/mediawiki/2018/4/45/T--Tianjin--measurement8.jpg)

Figure 8. Experimental result reported by pABaC+pCiRbS+gal1+Fluc

![](https://2018.igem.org/File:T--Tianjin--measurement9.jpg)

Figure 9. Experimental result reported by pABaC+pbCiRS+gal1+Nluc
(obtained by live cell assays)

![](https://static.igem.org/mediawiki/2018/3/36/T--Tianjin--measurement10.jpg)

Figure 10.Experimental result reported by pABaC+pCiRbS+gal1+Nluc
(obtained by live cell assays)

![](https://static.igem.org/mediawiki/2018/d/d6/T--Tianjin--measurement11.jpg)

Figure 11.Experimental result reported by pABaC+pbCiRS+gal2+Nluc
(obtained by live cell assays)

![](https://static.igem.org/mediawiki/2018/7/7a/T--Tianjin--measurement12.jpg)

Figure 12.Experimental result reported by pABaC+pCiRbS+gal2+Nluc
(obtained by live cell assays)

Some basic information can be gained from the above figures:

1. Compared with the corresponding control groups, all the experimental groups have showed apparent oscillatory signals.
2. In the measured 3500 min, the oscillations in 700-2000 min are extremely unstable and nearly absent, while the oscillations for the rest of the time are relatively stable.
3. The cycle is about 500min (about 8.3h).

****Note: final measurement results and analysis should be accessed in [Result](https://2018.igem.org/Team%3ATianjin/Demonstrate) page.****

[Measuement instrument](#collapseFour)

Fluorescent protein:
Hitachi F-2500 Flourescence Spectrophotometer;
Leica DMi8 inverted microscope
Luciferase : Thermoscientific Luminoskan Microplate Reader

# References

[1]Ow, D.W. et al. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and trans-genic plants. Science 234, 856–9.

[2]De Wet, J.R. et al. (1987) Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725–37.

[3]Chen, AH (Chen, Anna H.); Lubkowicz,D (Lubkowicz, David); Yeong, V (Yeong, Vivian); Chang, RL (Chang, Roger L.); Silver, PA(Silver, Pamela A.) Transplantability of a circadian clock to a noncircadian organism. SCIENCE ADVANCES(2015) DOI: 10.1126/sciadv.1500358

IGEMTIANJIN

IGEMTIANJIN2018

HEBO@TJU.EDU.CN

BEIYANG CAMPUS,TIANJIN UNIVERSITY,TIANJIN