iGEM 2018 — Team CIEI-BJ

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# Team:CIEI-BJ/Notebook

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Project

[Background](https://2018.igem.org/Team%3ACIEI-BJ/Background)

[Design](https://2018.igem.org/Team%3ACIEI-BJ/Design)

[Results](https://2018.igem.org/Team%3ACIEI-BJ/Results)

[Experiments](https://2018.igem.org/Team%3ACIEI-BJ/Experiments)

[Modeling](https://2018.igem.org/Team%3ACIEI-BJ/Model)

[InterLab](https://2018.igem.org/Team%3ACIEI-BJ/InterLab)

[Human\_Practices](https://2018.igem.org/Team%3ACIEI-BJ/Human_Practices)

Notebook

[Safety](https://2018.igem.org/Team%3ACIEI-BJ/Safety)

[Lab Book](https://2018.igem.org/Team%3ACIEI-BJ/Notebook)

[Protocol](https://2018.igem.org/Team%3ACIEI-BJ/Protocol)

Team

[Gallery](https://2018.igem.org/Team%3ACIEI-BJ/Team)

[Attributions](https://2018.igem.org/Team%3ACIEI-BJ/Attributions)

[Collaborations](https://2018.igem.org/Team%3ACIEI-BJ/Collaborations)

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* [Dry Lab](#a1)* [Wet Lab](#a2)
    + [Beijing Lab](#a3)
    + [Nanjing Lab](#a4)

Dry Lab

January 30th – February 10th ，2018

Our team was basically founded. The team members participated in a 12-day training on fundamental theory and basic experimental skills and started our brainstorming on the project.

May 20th ，2018 Group Meeting

We came out with some ideas about our research direction. After several rounds of discussions, we decided to build up a system to degrade aflatoxin.

May 27th ，2018 Group Meeting

We decided on the exact functions of our system and all of us began to search for background information before finalizing our summary.

July 13th -29th ，2018

Most of team members gathered at the University of Science & Technology Beijing for competition preparation.

July -22nd ，2018 Group Meeting

All the team members and advisors had a meeting to discuss on the focus of the project. A more detailed schedule was made.

August 26th ，2018 Group Meeting

The focus of our project was further clarified after several discussions. Each group of the team reported on their progress. And a specified time plan was agreed on.

September 2nd ，2018 Group Meeting

This time, we mainly focused on improving human practice part and pushed it forward. Our questionnaires and funding proposal were finished online.

October 3rd - 4th ，2018

All team members gathered to work on the poster, wiki and improving our presentation skill. The poster and PowerPoint slides for the final presentation was almost ready.

Wet Lab

Beijing Lab

Soluble expression of ADTZ in Saccharomyces cerevisiae and the degradation of aflatoxin B1 by ADTZ

March 15th, 2018

ADTZ-SC sequence was synthesized and integrated into vector pUC57

March 17th-18th, 2018

The gene was added digested site through PCR

March 24th-25th, 2018

Cut and purify the PCR product (described above) and vector pYES2

April 7th, 2018

The digested product of ADTZ-SC was integrated into vector pYES2

April 8th, 2018

The ligation product was transformed competent cells of *E.coli*, and checked by PCR

April 21st-22nd, 2018

Isolate the plasmid from positive colony

May 5th-6th, 2018

ADTZ-SC-pYES2 plasmid was used to transform yeast strain INVSc1

May 12th -13th, 2018

Check recombinant yeast colony by PCR

June 2nd-3rd, 2018

Yeast cell culture

June 9th-10th, 2018

Extract proteins from the yeast

June 23rd-30th, 2018

Examine if yeast can degrade aflatoxin

July 7th-15th, 2018

pYES2-ADTZ total protein enzyme activity analysis in vitro

**Soluble expression of ADTZ in *E.coli* and the degradation of aflatoxin B1 by ADTZ**

September 23rd , 2018

Design primers for pMAL-c5x- ADTZ construction

September 24th , 2018

Amplify ADTZ fragment and linearized pMAL-c5x fragment by PCR

Purify PCR product.

September 25th , 2018

In-Fusion

Transformation

September 26th , 2018

Colony PCR

September 27th , 2018

Miniprep plasmid pMAL-c5x-ADTZ

Sequence pMAL-c5x-ADTZ

September 29th , 2018

Prepare E.coli BL21 Gold DE(3) competent cells

Transform pMAL-c5x-ADTZ into *E.coli* BL21 Gold DE(3)

September 30th , 2018

Inoculate *E.coli* BL21 Gold DE(3) containing pMAL-c5x-ADTZ into 3mL LB, culture overnight at 37 oC and 200rpm

October 1st, 2018

The overnight culture of the bacteria was inoculated into LB broth containing 2g/L glucose and 100mg/L ampicillin. After 2h culture at 37 oC and 200rpm, recombinant protein production was induced by addition of 0.3 mM IPTG at 20oC and 200rpm for 24h.

October 2nd, 2018

The expressed cells were collected by centrifugation and the pellet was resuspended in H2O about an OD600 of 30. The cell was disrupted by sonication. SDS-PAGE analyzed the expressed MBP-ADTZ fusion protein.

October 3rd, 2018

Perform the degradation reaction of aflatoxin B1 and prepare the samples for HPLC.

October 4th, 2018

HPLC analysis

Nanjing Lab

Verification of the degradation of aflatoxin by the interaction of ScFv1 and ScFv2 which induce the expression of BacC, ADTZ, MNP, MSMEG5998 in yeast

August 10th, 2018

The design of the experiment：

August 20th, 2018

Construction of the vectors:

|  |
| --- |
| pGADT7-pGal1-BacC; |
| pGADT7-pGal1-ADTZ; |
| pGADT7-pGal1-MNP; |
| pGADT7-pGal1-MSMEG5998; |
| pGBKT7-pGal1-Eyfp; |

August 21st, 2018

Screen colonies using carboxybenzyl plates and verify the colonies through PCR with the 2F/2R primer. The gene length is 442bp. Then choos positive colonies to sequencing using primer t7-pro / 3’AD.

August 22nd, 2018

Design the primer ADpGAL1-F/R and BDpGAL1-F/R to amplify ADter and promoter GAL1 which will construct the downstream of ScFv1/2 in order to insert more candidate genes.

August 23rd, 2018

Screen colonies using carboxybenzyl plates and verify the colonies through PCR with the t7-pro / 3’AD primer. The gene length is 1700bp. The sequencing results of positive clones are right.

August 24th-September 3rd, 2018

Transform

|  |
| --- |
| pADT7-ScFv1; |
| pADT7-ScFv1-BacC; |
| pADT7-ScFv1-MNP; |
| pADT7-ScFv1-ADTZ;; |
| pADT7-ScFv1-MSMEG5998 |

to yeast strain AH109 with pBKT7-ScFv2 and pBKT7-ScFv2-eYFP, separately. After transformed three days (8.24 to 8.27), we got the result of pADT7-ScFv1&pBKT7-ScFv2 and pADT7-ScFv1-BacC & pBKT7-ScFv2:

September 3rd, 2018

The yeast cells transformed with two plasmids were cultured in SD-His media with no Aflatoxin B1 (control) or 100 ug/L (100 ppb) AFT B1. According to our design, the presence of AFT B1 should facilitate the association of Gal4 AD and BD, thus driving the expression of His3 protein under the endogenous Gal1 promoter, and enabling yeast growth in SD-His media. The yeast growth (OD 600) was measured every 2 hour for 14 hours on end, and the growth curve clearly showed that, compared to control, the presence of AFT B1 significantly promoted the growth of the yeast. Therefore, the results shows the similar changes with AD-ScFv1 (BBa\_K2247008) and BD-ScFv2 (BBa\_K2247009). Besides this, the candidate genes which may degrade AFB1 shows very hard to growth in SD-His media. Whether these genes are really working, should be further confirmed by more experiments.

September 7th, 2018

The eYFP is working under the AFB1 existing conditions in the yeast cells.

Western bolt to confirm the EYFP protein can be induced in the presence of AFT.

Sponsors

---

[![](https://static.igem.org/mediawiki/2018/e/e7/T--CIEI-BJ--Home--Sponsor--fig1.jpg)](https://croplife.org)
[![](https://static.igem.org/mediawiki/2018/2/28/T--CIEI-BJ--Home--Sponsor--fig2.jpg)](https://hsfilter.b2b.hc360.com)
[![](https://static.igem.org/mediawiki/2018/0/0d/T--CIEI-BJ--Home--Sponsor--fig3.jpg)](http://www.kws.cn)
[![](https://static.igem.org/mediawiki/2018/0/0c/T--CIEI-BJ--Home--Sponsor--fig4.jpg)](http://www.bayer.com.cn)


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

# Team:CIEI-BJ/Experiments

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[Home](https://2018.igem.org/Team%3ACIEI-BJ)

Achievements

[Medals](https://2018.igem.org/Team%3ACIEI-BJ/Medals)

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Project

[Background](https://2018.igem.org/Team%3ACIEI-BJ/Background)

[Design](https://2018.igem.org/Team%3ACIEI-BJ/Design)

[Results](https://2018.igem.org/Team%3ACIEI-BJ/Results)

[Experiments](https://2018.igem.org/Team%3ACIEI-BJ/Experiments)

[Modeling](https://2018.igem.org/Team%3ACIEI-BJ/Model)

[InterLab](https://2018.igem.org/Team%3ACIEI-BJ/InterLab)

[Human\_Practices](https://2018.igem.org/Team%3ACIEI-BJ/Human_Practices)

Notebook

[Safety](https://2018.igem.org/Team%3ACIEI-BJ/Safety)

[Lab Book](https://2018.igem.org/Team%3ACIEI-BJ/Notebook)

[Protocol](https://2018.igem.org/Team%3ACIEI-BJ/Protocol)

Team

[Gallery](https://2018.igem.org/Team%3ACIEI-BJ/Team)

[Attributions](https://2018.igem.org/Team%3ACIEI-BJ/Attributions)

[Collaborations](https://2018.igem.org/Team%3ACIEI-BJ/Collaborations)

![side_top](https://static.igem.org/mediawiki/2018/5/53/T--CIEI-BJ--Team--logo.jpg)

* [Experiments](#a1)
  + [Design of the experiment](#a2)
  + [Construction of the vectors](#a3)
  + [Transform](#a4)
* [OD-Mock OD-AFB1](#a5)* [Soluble expression of ADTZ in *E．coli* and the degradation of aflatoxin B1 by ADTZ](#a6)
    + [construction of pMAL-c5x- ADTZ](#a7)
    + [expression of MBP-ADTZ](#a8)
    + [degradation of aflatoxin B1 by MBP-ADTZ](#a9)
  * [Soluble expression of ADTZ in Saccharomyces cerevisiae and the degradation of aflatoxin B1 by ADTZ](#a10)
    + [ADTZ-SC sequence was synthesized as following:](#a11)
    + [full length of ADTZ-SC with BamHI and XbaI was cloned by PCR](#a12)
    + [BamHI and XbaI digestion of ADTZ-SC and pYES2 empty plasmid](#a13)
    + [transformation of competent cells in *Escherichia coli*](#a14)
    + [plasmid extraction](#a15)
    + [transformation of ADTZ-pYES2 plasmid in yeast](#a16)
    + [yeast colony pcr](#a17)
    + [Yeast cell culture](#a18)
    + [protein extraction from yeast](#a19)
    + [degradation of aflatoxin B1 by ADTZ-pYES2](#a20)
    + [in vitro pYES2-ADTZ total protein enzyme activity analysis](#a21)* [References](#a22)

Verification of the degradation of aflatoxin by the interaction of ScFv1 and ScFv2 which induce the expression of BacC, ADTZ, MNP, MSMEG5998 in yeast

The design of the experiment：

Characterization

![](https://static.igem.org/mediawiki/2018/6/65/T--CIEI-BJ--EX--fig1.png)

Sponsors

---

[![](https://static.igem.org/mediawiki/2018/e/e7/T--CIEI-BJ--Home--Sponsor--fig1.jpg)](https://croplife.org)
[![](https://static.igem.org/mediawiki/2018/2/28/T--CIEI-BJ--Home--Sponsor--fig2.jpg)](https://hsfilter.b2b.hc360.com)
[![](https://static.igem.org/mediawiki/2018/0/0d/T--CIEI-BJ--Home--Sponsor--fig3.jpg)](http://www.kws.cn)
[![](https://static.igem.org/mediawiki/2018/0/0c/T--CIEI-BJ--Home--Sponsor--fig4.jpg)](http://www.bayer.com.cn)

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

# Team:CIEI-BJ/Results

Top

[Home](https://2018.igem.org/Team%3ACIEI-BJ)

Achievements

[Medals](https://2018.igem.org/Team%3ACIEI-BJ/Medals)

[Parts](https://2018.igem.org/Team%3ACIEI-BJ/Parts)

Project

[Background](https://2018.igem.org/Team%3ACIEI-BJ/Background)

[Design](https://2018.igem.org/Team%3ACIEI-BJ/Design)

[Results](https://2018.igem.org/Team%3ACIEI-BJ/Results)

[Experiments](https://2018.igem.org/Team%3ACIEI-BJ/Experiments)

[Modeling](https://2018.igem.org/Team%3ACIEI-BJ/Model)

[InterLab](https://2018.igem.org/Team%3ACIEI-BJ/InterLab)

[Human\_Practices](https://2018.igem.org/Team%3ACIEI-BJ/Human_Practices)

Notebook

[Safety](https://2018.igem.org/Team%3ACIEI-BJ/Safety)

[Lab Book](https://2018.igem.org/Team%3ACIEI-BJ/Notebook)

[Protocol](https://2018.igem.org/Team%3ACIEI-BJ/Protocol)

Team

[Gallery](https://2018.igem.org/Team%3ACIEI-BJ/Team)

[Attributions](https://2018.igem.org/Team%3ACIEI-BJ/Attributions)

[Collaborations](https://2018.igem.org/Team%3ACIEI-BJ/Collaborations)

![side_top](https://static.igem.org/mediawiki/2018/5/53/T--CIEI-BJ--Team--logo.jpg)

* [Results](#a1)
  + [Preparation and parts](#a2)
  + [Operation of the Activation Module](#a3)
  + [Operation of the Detection Module](#a4)
  + [Operation of the Degradation Module](#a5)
  + [Conclusion](#a6)
* [Future Direction](#a7)

Results

From our design, this project includes experiments in 4 sections: the preparation work to construct all the vectors and required parts; the demonstration of the activation by AFT-B1 in yeast, the detection of AFT-B1 in yeast, and the degradation analysis in both bacterium and yeast.

Preparation and parts

1. Enzyme genes

Because the genes for enzymes and antibody fragments used in this projects were not convenient to isolate from their original organisms in the lab, we simply synthesized them. For convenient of further use, we directly cloned the open reading frame (ORF) of each candidate gene in the pMAL-c5x vector (New England BioLabs) downstream the sequence for the maltose binding protein (MBP) fusion tag (Fig. 1).

![](https://static.igem.org/mediawiki/2018/3/3c/T--CIEI-BJ--RE--fig1.PNG)

Sponsors

---

[![](https://static.igem.org/mediawiki/2018/e/e7/T--CIEI-BJ--Home--Sponsor--fig1.jpg)](https://croplife.org)
[![](https://static.igem.org/mediawiki/2018/2/28/T--CIEI-BJ--Home--Sponsor--fig2.jpg)](https://hsfilter.b2b.hc360.com)
[![](https://static.igem.org/mediawiki/2018/0/0d/T--CIEI-BJ--Home--Sponsor--fig3.jpg)](http://www.kws.cn)
[![](https://static.igem.org/mediawiki/2018/0/0c/T--CIEI-BJ--Home--Sponsor--fig4.jpg)](http://www.bayer.com.cn)