iGEM 2019 — Team SCU-China

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SCU-China/Notebook

# LAB

SCU-China Header

[![](https://static.igem.org/mediawiki/2019/5/5d/T--SCU-China--scu-Final-icon.jpg)](https://2019.igem.org/Team%3ASCU-China)

* [HOME](https://2019.igem.org/Team%3ASCU-China)
* PROJECT

  [Description](https://2019.igem.org/Team%3ASCU-China/Description)
  [Design](https://2019.igem.org/Team%3ASCU-China/Design)
  [Results](https://2019.igem.org/Team%3ASCU-China/Results)
  [Parts](https://2019.igem.org/Team%3ASCU-China/Parts)
  [Demostration](https://2019.igem.org/Team%3ASCU-China/Demonstrate)
  [Barricades](https://2019.igem.org/Team%3ASCU-China/Experiments)
* [MODELING](https://2019.igem.org/Team%3ASCU-China/Model)

  [Overview](https://2019.igem.org/Team%3ASCU-China/Model)
  [Comptational Structural Biology](https://2019.igem.org/Team%3ASCU-China/ComstrBio)
  [Enzyme Kinetic Simulation](https://2019.igem.org/Team%3ASCU-China/Enzyme)
  [Delay System Simulation](https://2019.igem.org/Team%3ASCU-China/DelaySystem)
* [HUMAN PRACTICE](https://2019.igem.org/Team%3ASCU-China/Human_Practices)

  [Integrated HP](https://2019.igem.org/Team%3ASCU-China/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3ASCU-China/Collaborations)
  [Meetup](https://2019.igem.org/Team%3ASCU-China/Meetup)
  [Public and engagement](https://2019.igem.org/Team%3ASCU-China/Public)
  [Market](https://2019.igem.org/Team%3ASCU-China/Market)
  [Consultation](https://2019.igem.org/Team%3ASCU-China/Consultation)
* LAB

  [Safety](https://2019.igem.org/Team%3ASCU-China/Safety)
  [Notebook](https://2019.igem.org/Team%3ASCU-China/Notebook)
* TEAM

  [Team members](https://2019.igem.org/Team%3ASCU-China/Team-members)
  [Attributions](https://2019.igem.org/Team%3ASCU-China/Attributions)
  [Team routines](https://2019.igem.org/Team%3ASCU-China/Team-routines)
* [ENTREPRENEURSHIP](https://2019.igem.org/Team%3ASCU-China/Entrepreneurship)
* [MEDALS](https://2019.igem.org/Team%3ASCU-China/Medals)

* ![](https://static.igem.org/mediawiki/2019/7/76/T--SCU-China--scu-wechat.jpg)
* Email:
  SCU-China@163.com
* Address:
  Sichuan University,
   No.24 South Section 1,
   Yihuan Road,
   Chengdu, China,
   610065

# NOTEBOOK

## Protocol

---

## Notebook


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SCU-China/Experiments

# PROJECT

SCU-China Header

[![](https://static.igem.org/mediawiki/2019/5/5d/T--SCU-China--scu-Final-icon.jpg)](https://2019.igem.org/Team%3ASCU-China)

* [HOME](https://2019.igem.org/Team%3ASCU-China)
* PROJECT

  [Description](https://2019.igem.org/Team%3ASCU-China/Description)
  [Design](https://2019.igem.org/Team%3ASCU-China/Design)
  [Results](https://2019.igem.org/Team%3ASCU-China/Results)
  [Parts](https://2019.igem.org/Team%3ASCU-China/Parts)
  [Demostration](https://2019.igem.org/Team%3ASCU-China/Demonstrate)
  [Barricades](https://2019.igem.org/Team%3ASCU-China/Experiments)
* [MODELING](https://2019.igem.org/Team%3ASCU-China/Model)

  [Overview](https://2019.igem.org/Team%3ASCU-China/Model)
  [Comptational Structural Biology](https://2019.igem.org/Team%3ASCU-China/ComstrBio)
  [Enzyme Kinetic Simulation](https://2019.igem.org/Team%3ASCU-China/Enzyme)
  [Delay System Simulation](https://2019.igem.org/Team%3ASCU-China/DelaySystem)
* [HUMAN PRACTICE](https://2019.igem.org/Team%3ASCU-China/Human_Practices)

  [Integrated HP](https://2019.igem.org/Team%3ASCU-China/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3ASCU-China/Collaborations)
  [Meetup](https://2019.igem.org/Team%3ASCU-China/Meetup)
  [Public and engagement](https://2019.igem.org/Team%3ASCU-China/Public)
  [Market](https://2019.igem.org/Team%3ASCU-China/Market)
  [Consultation](https://2019.igem.org/Team%3ASCU-China/Consultation)
* LAB

  [Safety](https://2019.igem.org/Team%3ASCU-China/Safety)
  [Notebook](https://2019.igem.org/Team%3ASCU-China/Notebook)
* TEAM

  [Team members](https://2019.igem.org/Team%3ASCU-China/Team-members)
  [Attributions](https://2019.igem.org/Team%3ASCU-China/Attributions)
  [Team routines](https://2019.igem.org/Team%3ASCU-China/Team-routines)
* [ENTREPRENEURSHIP](https://2019.igem.org/Team%3ASCU-China/Entrepreneurship)
* [MEDALS](https://2019.igem.org/Team%3ASCU-China/Medals)

* ![](https://static.igem.org/mediawiki/2019/7/76/T--SCU-China--scu-wechat.jpg)
* Email:
  SCU-China@163.com
* Address:
  Sichuan University,
   No.24 South Section 1,
   Yihuan Road,
   Chengdu, China,
   610065

* [1. At the Very Beginning](#1)
* [2. Colony PCR](#2)
* [3. AH109](#3)
* [4. The green fluorescence](#4)

# BARRICADES

## The Disaster From Yeast

![](https://static.igem.org/mediawiki/2019/8/8d/T--SCU-China--scu-barricades-1.png)

2019 SCU-China spared no efforts to complete and explore the project: CORegulaTIN. Finally, we synthesized cordycepin and pentostatin successfully in *S. cerevisiae*. Also, we verified our delay expression system step by step.

## At the Very Beginning

![](https://static.igem.org/mediawiki/2019/c/c3/T--SCU-China--scu-barricades-2.png)

Because this is the first year for SCU-China culturing yeast, there was no strain in our lab. Besides, only one or two professors major in the *S. cerevisiae*, so we turned to many people for a desirable yeast strain. At last, we found professor Ke Liu who offered us BY4742, and professor Xiaofeng Zhu applying us with AH109.

At first, we did not notice how big trouble these two strains might cause later.

## Colony PCR

![](https://static.igem.org/mediawiki/2019/f/fe/T--SCU-China--scu-barricades-33.png)

After transforming the plasmids, we constructed after a hard time into *S. cerevisiae*, we expected to confirm the transformants by colony PCR. Unfortunately, we always got negative results in this step. No band appeared on the gel after electrophoresis but DNA ladder. However, [teammates of Tianjing University](https://2019.igem.org/Team%3ASCU-China/Collaborations#Tianjin) had a discussion with us and they asked us to test the moderate time for yeast lysis. 10 min for boiling water bath and 5 min for ice bath as 1 cycle, they advised us to set gradient cycle number for lysing the yeast. We then tried to extend the heating time in the first step of colony PCR, that is, we set 10 min for 98 degrees as the first step. In this way, we successfully confirmed our transformed yeasts by colony PCR.

![](https://static.igem.org/mediawiki/2019/f/f3/T--SCU-China--scu-barricades-4.png)

## 3. AH109

![](https://static.igem.org/mediawiki/2019/4/44/T--SCU-China--scu-barricades-5.png)

We surprisingly found the transforming rate of AH109 was very high! Countless colonies appeared on plates without Ura. There were even biofilms! However, after the later confirmation steps we were no longer excited about it. "There are so many false positive colonies! OMG!" We then spread yeast solution of transformed AH109, which was diluted 20, 50 and 100 times into plates SD-Ura-. There were still close-set colonies appearing on all plates.

Is strain AH109 able to grow on SD plates without Ura？The information of this strain shows that it contain the gene mutation that prevent it grow on SD plates without Ura. We carried out negative control and extra control groups to verify that AH109 cannot grow on SD plates without Ura. But with some reasons still unknow, the control groups appeared no colony.

We consulted professor Liu about this strange phenomenon recently. Shocking reply comes, that strain AH109 does grow in [SD medium even without Ura.](https://2019.igem.org/Team%3ASCU-China/Consultation/Prof#Liu)We then replace strain AH109 with strain YM4271.

![](https://static.igem.org/mediawiki/2019/f/ff/T--SCU-China--scu-barricades-6.png)

## 4. The green fluorescence

![](https://static.igem.org/mediawiki/2019/e/e5/T--SCU-China--scu-barricades-7.png)

It is a common method that using green fluorescence as a reporter. But during the experiments this year, it seemed difficult to make the *S. cerevisiae* BY4741 express green fluorescence. We checked the design once and once again. It was strange why there was no green fluorescence. Later on, on a beautiful sunny day, we found a plate cultured seven days earlier with green fluorescence accidentally. It was a big surprise! But we were not so happy, because it was a simple reason for those transformants that did not shine: the culturing days were not enough!

![](https://static.igem.org/mediawiki/2019/2/27/T--SCU-China--scu-barricades-8.png)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SCU-China/Results

# PROJECT

SCU-China Header

[![](https://static.igem.org/mediawiki/2019/5/5d/T--SCU-China--scu-Final-icon.jpg)](https://2019.igem.org/Team%3ASCU-China)

* [HOME](https://2019.igem.org/Team%3ASCU-China)
* PROJECT

  [Description](https://2019.igem.org/Team%3ASCU-China/Description)
  [Design](https://2019.igem.org/Team%3ASCU-China/Design)
  [Results](https://2019.igem.org/Team%3ASCU-China/Results)
  [Parts](https://2019.igem.org/Team%3ASCU-China/Parts)
  [Demostration](https://2019.igem.org/Team%3ASCU-China/Demonstrate)
  [Barricades](https://2019.igem.org/Team%3ASCU-China/Experiments)
* [MODELING](https://2019.igem.org/Team%3ASCU-China/Model)

  [Overview](https://2019.igem.org/Team%3ASCU-China/Model)
  [Comptational Structural Biology](https://2019.igem.org/Team%3ASCU-China/ComstrBio)
  [Enzyme Kinetic Simulation](https://2019.igem.org/Team%3ASCU-China/Enzyme)
  [Delay System Simulation](https://2019.igem.org/Team%3ASCU-China/DelaySystem)
* [HUMAN PRACTICE](https://2019.igem.org/Team%3ASCU-China/Human_Practices)

  [Integrated HP](https://2019.igem.org/Team%3ASCU-China/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3ASCU-China/Collaborations)
  [Meetup](https://2019.igem.org/Team%3ASCU-China/Meetup)
  [Public and engagement](https://2019.igem.org/Team%3ASCU-China/Public)
  [Market](https://2019.igem.org/Team%3ASCU-China/Market)
  [Consultation](https://2019.igem.org/Team%3ASCU-China/Consultation)
* LAB

  [Safety](https://2019.igem.org/Team%3ASCU-China/Safety)
  [Notebook](https://2019.igem.org/Team%3ASCU-China/Notebook)
* TEAM

  [Team members](https://2019.igem.org/Team%3ASCU-China/Team-members)
  [Attributions](https://2019.igem.org/Team%3ASCU-China/Attributions)
  [Team routines](https://2019.igem.org/Team%3ASCU-China/Team-routines)
* [ENTREPRENEURSHIP](https://2019.igem.org/Team%3ASCU-China/Entrepreneurship)
* [MEDALS](https://2019.igem.org/Team%3ASCU-China/Medals)

* ![](https://static.igem.org/mediawiki/2019/7/76/T--SCU-China--scu-wechat.jpg)
* Email:
  SCU-China@163.com
* Address:
  Sichuan University,
   No.24 South Section 1,
   Yihuan Road,
   Chengdu, China,
   610065

# RESULTS

2019 SCU-China spared no efforts to complete and explore the project: CORegulaTIN. Finally, we synthesized cordycepin and pentostatin successfully in *S. cerevisiae*. Also, we verified our delay expression system step by step.

[Cordycepin](#collapseOne)

## Cordycepin

### Construction of pYES2-cns1 and pYES2-cns2

Since *cns1*  and *cns2* are essential genes for the synthesis of cordycepin (COR), we need to construct plasmids containing *cns1*  and *cns2*. We constructed and amplified the plasmids in *E.coli* and then used *S.cerevisiae* for protein expression. Therefore, we utilized the shuttle vector pYES2-NTA plasmid. *cns1* and *cns2* sequences were inserted into the vector pYES2-NTA, respectively. Then a pYES2-cns1 plasmid (a plasmid containing the *cns1* gene) and a pYES2-cns2 plasmid (a plasmid containing the *cns2* gene) were constructed.

We first performed PCR amplification on the *cns1*  and *cns2* sequences synthesized by Sangon and added 6A sequences to the primer sequences to assist subsequent yeast expression experiments. Due to the time constraint, the 6A sequence was only added upstream *cns1* gene. Since we would construct the fusion protein genes of *cns1* and *cns2*, the addition of the 6A sequence to *cns2* has little effect on the overall experiment. We used the restriction enzymes Hind III and EcoR I to digest the amplified product of *cns1* (Figure. 1a), and cleave the amplified product of cns2 with EcoR I and Xba I (Figure. 1b). Using T4 ligase to ligate the target gene fragment and vector, we constructed pYES2-cns1 plasmid and pYES2-cns2 plasmid. After constrution, we transferred the ligation product into *E.coli* DH5α competent cells, screened them with ampicillin, and verified the construction of pYES2-cns1 plasmid and pYES2-cns2 plasmid by colony PCR (Figure. 3), digestion (Figure. 2) and sequencing.

![](https://static.igem.org/mediawiki/2019/7/7d/T--SCU-China--scu-Result-cns1-2-fig1.png)

**Figure. 1** Digestion of cns1, cns2, pYES2-NTA (a) Digestion of *cns1* and pYES2-NTA. M1 is DL5000 from TAKARA. Lane1-3: Digestion of cns1. Lane4: Digestion of pYES2-NTA. (b) Digestion of *cns2* and pYES2-NTA. M1 is DL10000 from TAKARA.M2 is DL2000 from TAKARA. Lane1: Digestion of pYES2-NTA. Lane2: Digestion of *cns2*

![](https://static.igem.org/mediawiki/2019/0/0d/T--SCU-China--scu-Result-cns1-2-fig2.png)

**Figure. 2** pYES2-cns1 and pYES2-cns2 (a) pYES2-cns1. M1 is DL5000 from TAKARA. M2 is DL10000 from TAKARA. Lane1-6: pYES2-cns1. (b) pYES2-cns2. M1 is DL5000 from TAKARA. Lane1-2: pYES2-NTA. Lane3-4: pYES2-cns2.

![](https://static.igem.org/mediawiki/2019/9/92/T--SCU-China--scu-Result-cns1-2-fig3.png)

**Figure. 3** colony PCR of pYES2-cns1 and pYES2-cns2 (a) colony PCR of pYES2-cns1. M1 is DL5000 from TAKARA. Lane1 and lane3-8 show the positive results. (b) colony PCR of pYES2-cns2. M1 is DL2000 from TAKARA. Lane5 and lane 8 show the positive results.

### Construction of Plasmid pYES2-cns1-cns2

The construction of pYES2-cns1-cns2 plasmid because the co-expression of the *cns1* and *cns2* gene will generate COR in *Saccharomyces cerevisiae*. We constructed pYES2-cns1-cns2 plasmid from pYES2-cns2 plasmid and the PCR products of *cns1*.

The agarose gel shows the semi-digested pYES2-cns1-cns2 plasmid by Hind III and Xho I which will generate 4298bp fragment and 5781bp fragment. The ladder M1 is TaKaRa DL10000 DNA marker (Figure. 4a).

![](https://static.igem.org/mediawiki/2019/7/7a/T--SCU-China--scu-Result-cns1-2-fig4.png)

**Figure. 4** (a) Digestion of pYES2-cns1-cns2. (b) Digestion of pYES2-cns1-linker-cns2

### Construction of pYES2-cns1-linker-cns2 Plasmid

Due to our enzymatic reaction modeling, the fusion protein of *cns1* and *cns2* could enhance the reaction rate and production. It is noted in the literature that Cns1 and Cns2 probably function as heterodimers. The fusion protein linked by the pre-designed linker at the end of cns1 is constructed via deleting a 305bp fragment (containing the CYC1 terminator and the stop codon of cns1) and a 498bp fragment (containing the GAL1 promoter of *cns2*) from the pYES2-cns1-cns2.

![](https://static.igem.org/mediawiki/2019/c/cf/T--SCU-China--scu-Result-cns1-2-consturction-fig5.png)

**Figure. 5** The alignment shows the difference between pYES2-cns1-linker-cns2 (below) and pYES2-cns1-cns2 (above).

The agarose gel shows the comparison between pYES2-cns1-linker-cns2 and its precursor plasmids, and between digested pYES2-cns1-linker-cns2 and pYES2-cns1-linker-cns2 plasmids by BamH I and Xba I which will generate 1113bp fragment and 8263bp fragment, 1611pb fragment and 8362bp fragment respectively. The ladder M1, M2 is the TaKaRa DL5000 and DL10000 DNA marker (Figure. 4b).

### The confirmation of Cns1-linker-Cns2 in transcriptional level

We demonstrated the Cns1-linker-Cns2 expression in transcriptional level by qPCR. As the result, Cns1-linker-Cns2 transcribed successfully in *S. cerevisiae* (Figure. 6).

![](https://static.igem.org/mediawiki/2019/0/03/T--SCU-China--scu-results-COR-fig6.jpg)

**Figure. 6**  The qPCR of Cns1-linker-Cns2, \*\*: P<0.01

### Protein Expression & Function Verification

The transformed yeast cells were grown in YPD liquid medium, and lysed by ultrasonic, releasing internal protein. The lysate was then centrifuged and the supernate was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining. (Figure. 7)

![](https://static.igem.org/mediawiki/2019/a/a8/T--SCU-China--scu-Result-cns1-2-protein-fig1.png)

**Figure. 7**  SDS-PAGE analysis of total protein from empty *S.cerevisiae* BY4741 and modified *S.cerevisiae* BY4741. Lane M: Marker Ladder; Lane 1: *S.cerevisiae* BY4741; Lane 2 and 3: recombinant strain *S.cerevisiae* BY4741. Lane 1, 2, and 3 show bands at the same size (in the red box) consistent with the molecular weight of Cns1 (97kDa).

Then we use western blot to examine protein expression.

We used thin-layer chromatography to detect COR. High Performance Liquid Chromatography is used to quantify the output of COR and PTN comparing with standard samples. (Figure. 8)

![](https://static.igem.org/mediawiki/2019/4/49/T--SCU-China--scu-demonstration-fig2.png)

**Figure. 8** HPLC analysis of COR
Top: Standard sample of 5mM COR.
Middle: Supernatant fraction of Cns1-linker-Cns2 sample.
Bottom: Supernatant fraction of BY4741 sample. The strain BY4741 was culturing in YPDG media for 3 days.

[Pentostatin](#collapseTwo)

## Pentostatin

Cns3 is an important enzyme identified in *Cordyceps. militaris*, which catalyze the production of pentostatin (PTN). To develop the COR/PTN co-production system, 2019 SCU-China has to express it successfully in yeast. And it is a fact that no evidence proves the successful expression of Cns3 in *Saccharomyces cerevisiae*, so we consulted the discoverer of Cns3. According to [the information he offered](https://2019.igem.org/Team%3ASCU-China/Consultation/Prof), we decided to study more about Cns3 and its HisG domain instead of NK domain. ([More details about why we gave up NK domain](https://2019.igem.org/Team%3ASCU-China/Consultation/Prof)). As a result, we determined to transform codon-optimized partial Cns3 containing HisG domain into *S. cerevisiae* to produce PTN.

### Complete Cns3 Protein

In order to validate the expression of Cns3 in *S. cerevisiae*, we need to construct a Cns3 expression plasmid. We cloned *cns3* on the pYES2-NTA vector with a pGAL1 promoter upstream and a CYC1 terminator downstream.

1. We optimized the sequence of Cns3 from NCBI to make it more suitable for *S. cerevisiae* and then synthesized the DNA sequence with the help of Sangon.
2. We inserted the cns3 synthesized DNA sequence into pUC-SP plasmid. Then we transformed the recombinant plasmid into *E.coli* DH5α for amplification and preservation.
3. We constructed pYES2-Cns3 plasmid using HindIII and XbaI double-enzyme digestion. Successful construction of this recombinant plasmid was confirmed by PCR identification, double enzyme digestion, and sequencing of the insert fragment. (Figure. 1)
4. We aimed to transformed pYES2-Cns3 pladmid into the yeast for protein expression. We chose BY4741 as our experimental strain. After 48 hours culture, the colony PCR of yeast containing pYES2-Cns3 was performed, which proved our successful transformation (Figure. 2). Only two samples (3a1 and 3a2) are negative according to the colony PCR.
5. It is of vital importance to verify the expression and function of Cns3. We conducted RT-qPCR and HPLC to detect the existence of Cns3 mRNA and varify the function of Cns3. Before performing the RT-qPCR, we cultivated target yeast cells in fresh 1mL YPG. The results of RT-qPCR and HPLC were as below (Figure. 7 and 8).

![](https://static.igem.org/mediawiki/2019/2/23/T--SCU-China--scu-Result-cns3-protein-fig1.png)
![](https://static.igem.org/mediawiki/2019/8/81/T--SCU-China--scu-Result-cns3-protein-fig1-2.png)

**Figure. 1** The double enzymatic experiment result. 1 and 2 lanes are plasmids extracted from the transformed *E.coli* culture. The Cns3 fragment is 2703 bp as the red arrow shows.

![](https://static.igem.org/mediawiki/2019/d/d7/T--SCU-China--scu-Result-cns3-protein-fig2.png)

**Figure. 2** Colony PCR results of transformed BY4741 & AH109 with plasmid pYES2-cns3. 3-1~3-14 are samples from transformed BY4741. 3a1 and 3a2 are samples from transformed AH109. PC is plasmid pYES2-CNS3. NC1 and NC2 are AH109 and BY4741 without transforming any plasmid.

### The HisG Domain of Cns3

Judging from the reference and the communication with the author, HisG domain of Cns3 is indispensable for PTN production, while the function of NK domain of Cns3 is unclear. Considering that Cns3 is heterogenous protein for yeast, it would be better removing the redundant and functionally uncertain NK domain of Cns3. Additionally, it can reduce metabolic burden. Therefore, in our project, partial Cns3 containing HisG domain was introduced into *S. cerevisiae* to simplify the COR/PTN co-production system.

![](https://static.igem.org/mediawiki/2019/b/b1/T--SCU-China--scu-Result-cns3-protein-fig5-1.png)

**Figure. 3** The plasmid of pYES2-HisG-mf

![](https://static.igem.org/mediawiki/2019/f/f8/T--SCU-China--scu-Result-cns3-protein-fig5-2.png)

**Figure. 4** The plasmid of pYES2-HisG-only.

1. We designed three pairs of PCR primers (called only, mf, and yh) to effectively amplify the fragments of partial Cns3 containing HisG domain from pYES2-Cns3. We used HindIII and XbaI double-enzyme digestion method to construct the plasmid pYES2-Cns3-HisG.
   There are two kinds of partial Cns3 sequences. One is mf-HisG, because it is half of the CNS3 protein containing the HisG domain, which is designed from the reference and has been validated for expression and function in other fungus. The other is further shortened, called only-HisG, which only contains the HisG domain. (Figure. 5)
   The results about *E.coli* colony PCR, PCR identification, double enzyme digestion, and sequencing were as below, proving the successful construction. (Figure. 6)
2. We transfered the plasmids into the yeast BY4741. And we used colony PCR to detect the results. After the colony PCR, we send them to sequencing. And the sequencing was successful.
3. We did RT-qPCR and HPLC to detect the existence of HisG mRNA and the function of HisG. Before we performed the RT-qPCR, we always cultivated target yeasts in new 1mL YPG. The results of RT-qPCR and HPLC were as below (Figure. 7 and Figure. 8).

![](https://static.igem.org/mediawiki/2019/b/be/T--SCU-China--scu-results_figure5-N.png
)

**Figure. 5** The primers of Cns3 for partial amplification. 1 and 3: primers for HisG-MF; 2 and 3: primers for HisG-only.

![](https://static.igem.org/mediawiki/2019/6/6d/T--SCU-China--scu-Result-cns3-protein-fig6-1.png)
![](https://static.igem.org/mediawiki/2019/6/64/T--SCU-China--scu-Result-cns3-protein-fig6.png)

**Figure. 6** The colony PCR of *E.coli* transformed with pYES2-HisG-mf, pYES2-HisG-yh and pYES2-HisG-only. Fragments of HisG-mf and HisG-yh are both 1025 kb in length, and HisG-only is 681. The sequences of HisG-yh and HisG-mf are the same with each other. The target bands are showed by red arrows.

![](https://static.igem.org/mediawiki/2019/0/07/T--SCU-China--Result-pentostatin-Figure7.png)

**Figure. 7** RT-qPCR of Cns3-HisG-mf/Cns3-HisG-only. No-load is the S. cerevisiae without transformed plasmids. The reference gene is PGK1. The RNA of cns3 is detected successfully. Bars are SE.
\*\*: P<0.01, \*\*\*: P<0.001.

![](https://static.igem.org/mediawiki/2019/6/6a/T--SCU-China--scu-demonstration-fig4-1.png)
![](https://static.igem.org/mediawiki/2019/b/b8/T--SCU-China--scu-demonstration-fig4-2.png)
![](https://static.igem.org/mediawiki/2019/2/26/T--SCU-China--scu-demonstration-fig4-3.png)

**Figure. 8** HPLC analysis of PTN
a. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG-MF sample. The strain BY4741 was culturing in YPDG media for 3 days.
b. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG ONLY sample. The strain BY4741 was culturing in YPDG media for 3 days.
c. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of BY4741 sample. The strain BY4741 was culturing in YPDG media for 3 days.

### Constitutive Promoters

In order to accumulate enough pentostatin to inhibit adenosine deaminase before the production of cordycepin, we hope that pentostatin will start to accumulate before the expression of *cns1* and *cns2* being automatically initiated. That is why we introduced a constitutive promoter upstream *cns3* and designed a delay system for *cns1* and *cns2*. We chose 5 yeast endogenous promoters of different strength, one high-strength promoter: pTDH3; two mid-strength promoters: pTEF1 and pTEF2; and two low-strength promoters: pPDA1 and pTPS1.

#### Promoters Amplification

We planned to amplify these 5 promoter fragments from *S. cerevisiae* BY4742 genomic DNA. All the primer sequences with restriction overhangs can be found in the [notebook](https://2019.igem.org/Team%3ASCU-China/Notebook).

![](https://static.igem.org/mediawiki/2019/d/d3/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig1.png)

**Figure. 9** Gradient PCR for pTEF2 and pTDH3. Line 1 Lane 1-6: pTEF2 1-6; Lane 7: NC. Line 2 Lane 1-6: pTDH3 1-6; Lane7: NC. Marker: Takara DL2000.

However, in the gradient PCR reaction, pTEF2 showed faint results, a median annealing temperature of 60 degrees was selected for subsequent reactions; pTDH3 showed negative results at all 8 annealing temperatures, we speculated that differences between genomes of *S.Cerevisiae* BY4742 and the strain used in the reference may attribute to this.

![](https://static.igem.org/mediawiki/2019/7/78/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig2.png)

**Figure. 10** Amplification PCR for constitutive promoters. Lane 1-3: pTEF2 1, 2, NC; Lane 4-6: pPDA1 1, 2, NC; Lane 7-9: pTPS1 1, 2, NC; Lane 10-12: pTEF1 1, 2, NC; Marker: Takara DL2000

We conducted amplification PCR for constitutive promoters except pTDH3, but only pTEF1, pPDA1 and pTPS1 showed favorable results. The bands in the NC group of pPDA1 may be attributed to a fault when adding samples.

#### Constitutive Promoters - yeGFP

In order to perform full characterization of the constitutive promoters, we cloned pTEF1, pPDA1 and pTPS1 into pYES2-NTA vector with a yeGFP coding sequence downstream. We successfully constructed these 3 plasmids and verified them by restriction digestion and sequencing.

![](https://static.igem.org/mediawiki/2019/8/82/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig3.png)

**Figure. 11** Plasmid maps of constitutive promoters-yeGFP

![](https://static.igem.org/mediawiki/2019/2/24/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig4.png)

**Figure. 12** Restriction digestion for pTEF1-yeGFP and pTPS1-yeGFP using SpeI and HindIII. Line 1 Lane1-2: (Plasmid) pTEF1-yeGFP (633bp + 6141bp) pTPS1-yeGFP (613bp + 6141bp); Line 2 Lane1-2: (Digested plasmid) pTEF1-yeGFP pTPS1-yeGFP; Marker:Takara DL2000&DL10000

![](https://static.igem.org/mediawiki/2019/9/94/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig5.png)

**Figure. 13** Enzyme digestion verification for pPDA1-yeGFP (667bp + 6118bp) using EcoRI and HindIII. Lane 1: Plasmid; Lane2: NC; Lane3: EcoRI; Lane4: HindIII; Marker: Takara DL10000

To verify the constitutive promoter-yeGFP plasmids we had constructed, we conducted double-restriction digestion. We digested our pTEF1-yeGFP, pTPS1-yeGFP and pPDA1-yeGFP plasmid samples with SpeI and HindIII. Positive results are shown in Figure 12 and Figure 13. We then sent these samples to sequencing and got positive results.

For pTPS1 and pTEF1, we successfully observed green fluorescence of liquid culture after cultivating engineered *S. cerevisiae* cells for 2 days. (Figure. 17) But green fluorescence was not able to be observed for pPDA1. We think it mybe because of its low transcriptional strength.We successfuly verified the function of pPDA1 in RT-qPCR experiment of cells transformed with pPDA1-cns3. (Figure. 20)

![](https://static.igem.org/mediawiki/2019/6/6a/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig6.png)

**Figure. 14** Yeast colony PCR for pTPS1-yeGFP. Lane 1-6: pTPS1-yeGFP 1-6; Lane7: NC; Marker: Takara DL2000.

![](https://static.igem.org/mediawiki/2019/9/95/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig79.png)

**Figure. 15** Colony PCR for pTEF1-yeGFP. Lane1: NC; Lane 2-7: pTEF1-yeGFP 1-6; Marker: Takara DL2000.

![](https://static.igem.org/mediawiki/2019/0/0f/T--SCU-China--scu-results-colony33.png)

**Figure. 16** Colony PCR for pTEF1-yeGFP; Lane 1: PDA1 NC; Lane2: PDA1 PC; Lane3: Trans2k Plus II Marke; Lane4-6: PDA1 1-3.

![](https://static.igem.org/mediawiki/2019/6/6f/T--SCU-China--scu-results-GFP.png)

**Figure. 17** The green fluorescence of *S. Cerevisiae* BY4741 transformed pYES2-pTEF1-yeGFP and pYES2-pTPS1-yeGFP.

#### Constitutive Promoters-cns3

After characterizing pTEF1, pPDA1 and pTPS1, we planned to apply them to turning on the expression of *cns3*. We cloned these 3 promoters upstream *cns3* on pYES2-NTA vector. We successfully constructed plasmids and verified them by restriction digestion and sequencing.

![](https://static.igem.org/mediawiki/2019/e/e7/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig7.png)

**Figure. 18** Plasmid maps of constitutive promoters-yeGFP

![](https://static.igem.org/mediawiki/2019/a/a8/T--SCU-China--scu-Result-cns3-constitutive-promoters-fig8.png)

**Figure. 19** Restriction digestion for pTPS1-Cns3 (2629bp + 5974bp) and pPDA1-Cns3 (2635bp + 2652bp). Line 1 Lane1-2: (Plasmid) pTPS1-Cns3 and pPDA1-Cns3; Line 2 Lane1-2: (Digested plasmid) pTPS1-Cns3 and pPDA1-Cns3; Marker:Takara DL10000

To verify the constitutive promoters-Cns3 plasmids we construct, we conducted double-restriction digestion. We digested our pPDA1-Cns3 plasmid sample with EcoRI and XbaI, pTPS1-Cns3 plasmid sample with HindIII and XbaI. Positive results are shown in Figure 19. We then sent our samples to sequence and got positive results.

We transformed the constitutive promoters-Cns3 plasmids into S. cerevisiae BY4741 cells. In order to verify there three promoters’ capability of initiating cns3 expression, we performed RT-qPCR to detect cns3 mRNA.(Figure. 20 )Favorable results showed that these sequences could work as promoters as expected because of the successful transcription of Cns3.

![](https://static.igem.org/mediawiki/2019/a/a2/T--SCU-China--scu-results-PTN20.jpg)

**Figure. 20** RT-qPCR of constitutive promoters (pPDA1/pTEF1/pTPS1) - Cns3. NC is the S. cerevisiae without transformed plasmids (constitutive promoters - Cns3). The reference gene is PGK1.
\*: P<0.05, \*\*: P<0.01.

[Delay Expression System](#collapseThree)

## Delay Expression System

In order to ease the burden of *S. cerevisiae*, we want our engineered cells to express Cns1-linker-Cns2 and produce COR after growth plateau. Therefore, we designed a delay expression system applied to automatically initiating the expression of Cns1-linker-Cns2 after *S. cerevisiae* having been cultivated for a while. The verification of this system is divided into three parts, including validating two subsystems, pGAL1 expression system and pMET3 expression system, and the whole delay expression system. Because we use GAL4 as an intermediate of delay expression system, we chose to construct it in a *gal4 Δ S. cerevisiae* strain, AH109 (Changed to YM4271 after consulting with prof. Ke Liu later) to avoid noise-causing by endogenous GAL4.

### The pGAL1 delay expression system

To ensure the growth of yeast and delay the expression of Cns1 and Cns2, we designed GAL4 delay expression system.

GAL1 promoter can be activated by GAL4 protein while GAL 80 is a specific and constantly existed inhibitor of GAL4. Galactose can block this inhibition process through binding to GAL80 consequently activating GAL1 promoter. Meanwhile, glucose is the first intake carbon resource of most *S. cerevisiae* strains. So only when all the glucose has been consumed and galactose is present in the medium, protein will be expressed. In the verification of pGAL1 expression system, we verified that GAL1 promoter could initiate the expression of downstream genes in presence of both GAL4 and galactose. Therefore, we chose a *gal4+ S. cerevisiae* strain, BY4741 to validate pGAL1 expression system.

We constructed yeGFP (offered by iGEM team 2019 Peking) into pYES2-NTA (offered by Prof. Ke Liu’s lab) to test the function of our system (Figure. 1).

![](https://static.igem.org/mediawiki/2019/f/f4/T--SCU-China--scu-Result-delay-system-fig4.png)

**Figure. 1** Map of plasmid pYES2-pGAL1-yeGFP.

We verified our construction by restriction digestion (Figure. 2) and sequcencing. All of the sequencing results aligned perfectly.

![](https://static.igem.org/mediawiki/2019/0/01/T--SCU-China--scu-Result-GAL4-delay-fig2.png)

**Figure. 2** Restriction digestion of plasmid pYES2-pGAL1-yeGFP. Marker: Trans 2K Plus II, Lane 1: pYES2-yeGFP (6651bp), Lane 2: pYES2-yeGFP digestion (digested product size: 717bp).

After that, we introduced pYES2-yeGFP into *S. cerevisiae* BY4741 cells and cultured them on SD-Ura plate for screening. For further verification, we performed colony PCR and all the results were positive. (Figure. 3)

![](https://static.igem.org/mediawiki/2019/c/c1/T--SCU-China--scu-Result-GAL4-delay-fig3.png)

**Figure. 3** Colony PCR of pYES2-yeGFP. Marker: Takara DL2000 DNA marker, Lane 1-5, colony PCR yeGFP (717bp).

Furthermore, to demonstrate the function of our system, we cultured the modified BY4741 cells and empty cells in YPD medium and YPG medium respetively. We verified the function of GAL1 promoter by detecting green fluorescence using stereoscope.

![](https://static.igem.org/mediawiki/parts/e/e6/T--SCU-China--scu-result-delay_expression-Figure_4%28right%29.png)

**Figure. 4** Green fluorescence of S.cerevisiae BY4741 transformed pYES2-yeGFP which was induced by galactose. (a) Fluorescence observation of *S.cerevisiae* BY4741 transformed pYES2-yeGFP. Series 1: BY4741 WT in YPD medium, Series 2: BY4741 WT in YPG medium, Series 3: BY4741 transformed BY4741 in YPD medium, Series 4: Transformed BY4741 in YPG medium. Visible fluorescence in Series 4. (b) The fluorescence measurement of *S.cerevisiae*BY4741 transformed pYES2-yeGFP. YPD: YPD medium, YPG: YPG medium, BY4741 WT: *S.cerevisiae*, BY4741 wildtype, BY4741 transformed: *S.cerevisiae*, BY4741 transformed plasmid pYES2-yeGFP, excitation light is 488nm and emission light is 525nm，\*\*\*: P<0.001.

Overall, we verified the function of GAL1 promoter according to the green fluorescence observed in B3 (Figure. 4) and B4 (Figure. 4) In other words, the GAL1 promoter could initiate the expression of downstream genes (yeGFP in demonstration) with galactose.

### The pMET3 delay expression system

The MET3 promoter is a weak and tightly controlled promoter that has already been used in the construction of conditional lethal strain and heterologous expression in S.Cerevisiae. And the activity of it can be controlled by adding methionine of different concentrations in the medium **(Mao. X et al., 2002)**. To verify the other subsystem, pMET3 expression system, we verified the function of pMET3 by testing its capability of initiating the expression of downstream genes. We planned to perform this in AH109 strain but changed our mind and used YM4271 strain later.

We constructed pMET3 containing plasmid by replacing pGAL1 on pYES2-pGAL1-yeGFP with pMET3 (Figure. 5) . And the successful construction was approved by the result of restriction digestion as followed and sequencing. (Figure. 6).

![](https://static.igem.org/mediawiki/2019/7/77/T--SCU-China--scu-Result-delaysy-stem-fig1.png)

**Figure. 5** The map of constructed plasmid pYES2-pMET3-yeGFP.

![](https://static.igem.org/mediawiki/2019/0/07/T--SCU-China--scu-Result-delay-system-fig2.png)

**Figure. 6** The restriction digestion verification result of pYES2-pMET3-yeGFP. Marker: Trans 2K plus II, Lane 1, 3: the plasmid pYES2-pMET3-yeGFP (6620bp), Lane 2, 4: the plasmid pYES2-pMET3-yeGFP after digesting by EcoRI and XbaI (the backbone is 5869bp, the insertion is 751bp).

Then we introduced the pYES2-pMET3-yeGFP into *S. cerevisiae* AH109. However, we couldn’t screen AH109 with SD-URA medium. After communicating with professor Ke Liu, he advised us to use another strain YM4271 which can be screened by SD-URA medium for our system ([more details](https://2019.igem.org/Team%3ASCU-China/Consultation/Prof#Liu)). Compared with other strains , YM4271, a ura-met+gal80-gal4- strain, is more suitable for our project. Firstly, YM4271 can’t survive without Ura in the medium. Secondly, GAL80 has already been knocked out from YM4271 so that we can simply use glucose as the carbon source, which will lowers the cost of the medium. Thirdly, unlike BY4741, YM4271 is met+ stain, so YM4271 can grow in the met dropout medium.

We introduced pYES2-pMET3-yeGFP into *S. cerevisiae* YM4271 cells and verified the function of pMET3 by detecting green fluorescence (Figure. 7a). And we verified the pMET3 delay expression system with different concentrations of MET (Figure. 7b).

![](https://static.igem.org/mediawiki/2019/b/b3/T--SCU-China--scu-Result-delay-system-fig3.png)
![](https://static.igem.org/mediawiki/2019/4/44/T--SCU-China--Result-delay_expression_system-_Figure_7b.png)

**Figure. 7** (a) The green fluorescence of *S. cerevisiae* YM4271 transformed with pYES2-pMET3-yeGFP.(b) The verification of pMET3 delay expression system with different concentrations of MET.

### The integrated delay expression system

After verifying two subsystem respectively, we planned to integrate these two subsystems together into a completed delay expression system and verify it.

We firstly cloned a GAL4 on pYES2 vector with a pMET3 upstream (pYES2-pMET3-GAL4) (Figure .8).

![](https://static.igem.org/mediawiki/2019/3/39/T--SCU-China--scu-Result-delay-system-fig5.png)

**Figure. 8** The map of constructed plasmid pYES2-pMET3-GAL4

The result of restriction digestion verification shows our successful construction of pYES2-pMET3-GAL4 (Figure. 9).

![](https://static.igem.org/mediawiki/2019/f/f5/T--SCU-China--scu-Result-delay-system-fig6.png)

**Figure. 9** Restriction digestion verification result of pYES2-pMET3-GAL4. Marker: Takara DL10000, Lane 1: the plasmid of pYES2-pMET3-GAL4 (8626bp), Lane 3, 4: the plasmid of pYES2-pMET3-GAL4 after digesting by EcoRI (8626bp).

Subsequently, we constructed the completed delay expression system with a reporter, yeGFP. (pYES2-pMET3-GAL4-pGAL1-yeGFP) (Figure. 10). And the result of restriction digestion verification show our successful construction (Figure. 11). The sequencing result was also right.

![](https://static.igem.org/mediawiki/2019/d/df/T--SCU-China--scu-Result-delay-system-fig7.png)

**Figure. 10** The map of constructed plasmid pYES2-pMET3-GAL4-pGAL1-yeGFP.yeGFP.

![](https://static.igem.org/mediawiki/2019/8/86/T--SCU-China--scu-Result-delay-system-fig8.png)

**Figure. 11** The restriction digestion verification result of pYES2-pMET3-GAL4-pGAL1-yeGFP. Marker: Takara DL10000 and Takara DL15000, Lane 1: the plasmid pYES2-pMET3-GAL4-pGAL1-yeGFP (10384bp), Lane2: the plasmid pYES2-pMET3-GAL4-pGAL1-yeGFP after digesting by BamHI (10384bp).

Because of time constraint, we didn’t actually transform the plasmid we constructed into YM4271 cells.

### Outlook

In the future, we will complete the verification of our delay expression system by transform the plasmid into YM4271 cells and detect green fluorescence using fluorescence microscope. Besides, we will try different initial methionine concentrations to build up a connection between delay time and initial methionine concentration, which we expect to be consistent with our modeling result. [(Detials about moldeling for delay system)](https://2019.igem.org/Team%3ASCU-China/DelaySystem) More importantly, we will apply our delay expression system to the expression of Cns1-linker-Cns2 in our COR producing strain. We expect to realize flexible delayed time of COR production by adjusting the initial methionine added into the medium.

[Reference](#collapseFour)

## Reference

Traven, A., Jelicic, B., & Sopta, M. (2006). Yeast GAL4: a transcriptional paradigm revisited. EM-BO reports, 7(5), 496-499.

Giniger, E., Varnum, S. M., & Ptashne, M. (1985). Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell, 40(4), 767-774.

Mao, X., Hu, Y., Liang, C., & Lu, C. (2002). MET3 Promoter: A Tightly Regulated Promoter and Its Application in Construction of Conditional Lethal Strain. Current Microbiology, 45(1), 37–40. doi:10.1007/s00284-001-0046-0

Reece-Hoyes, J. S., & Walhout, A. J. M. (2018). High-Efficiency Yeast Transformation. Cold Spring Harbor Protocols, 2018(7), pdb.prot094995. doi:10.1101/pdb.prot094995