Difference between revisions of "Team:Tsinghua/Experiments"

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<title>Yeasy AFT project page</title>
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<h1>Experiments</h1>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.  
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            <li><a href="#background" class="page-scroll">background</a></li>
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            <li><a href="#how_it_works" class="page-scroll">how it works</a></li>
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            <li><a href="#solution" class="page-scroll">Solution</a></li>
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            <li><a href="#features" class="page-scroll">features</a></li>
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            <li><a href="#result" class="page-scroll">result</a></li>
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            <a href="https://2017.igem.org/Team:Tsinghua" class="btn btn-trial">main page</a>
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<h5>What should this page contain?</h5>
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<section class="video">
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<ul>
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<li> Protocols </li>
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<li> Experiments </li>
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<li> Documentation of the development of your project </li>
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                    <a href="#background" class="btn btn-trial red">Start Intro</a>
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            <h3 class="section-heading">How aflatoxin hazards people</h3>
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                    <p class="percentage">Class<br><span>I</span><br>cancerogen</p>
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                    <p class="tagline">Aflatoxin is known as the most toxic carcinogen. <br>It can covalently bind <br>adenine in DNA, thus causing mutations.</p>
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                    <p class="percentage">In nature <br><span>12</span><br>kinds of Aflatoxin</p>
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                    <p class="tagline">with in which B1 is the most toxic <br> and can be found in maize, peanuts, cotton seeds and many nuts.</p>
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                    <p class="percentage">Up to <br><span>155,000 </span><br> liver cancer-incidents due to Aflatoxin</p>
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                    <p class="tagline">It can also cause stomach <br> cancer and esophageal cancer</p>
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            <h3 class="section-heading">how YeasyAFT can test<br class="hidden-xs">and how they help people?</h3>
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                    <p class="percentage"><span>Quick</span><br>  detection</p>
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                    <p class="tagline">The detection of AFT can be accomplished in hours, <br> which is distinguished from those prevalent biochemical methods.</p>
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                    <p class="percentage"><span>Low</span><br> cost</p>
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                    <p class="tagline">Utilization of genetic-engineered <br>yeast allows quite low cost.</p>
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                    <p class="percentage"><span>High </span><br> sensitivity</p>
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                    <p class="tagline">We use single-chain Fv antibody to detect AFT <br>, and the signal is enlarged by yeast-two-hybrid system and hexose transporter.</p>
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<h5>Inspiration</h5>
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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            <h3 class="section-heading text-center">Background</h3>
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</ul>
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                    <p class="add-review">Aflatoxins (AFT) </p >
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                    <p class="description">Aflatoxins (AFT) are poisonous carcinogens that are produced by certain molds (<I>Aspergillus flavus</I> and <I>Aspergillus parasiticus</I>) which grow in soil, decaying vegetation, hay, and grains. They are regularly found in improperly stored staple commodities such as cassava, chili peppers, corn, cotton seed, millet, peanuts, rice, sesame seeds, sorghum, sunflower seeds, tree nuts, wheat, and a variety of spices. When contaminated food is processed, aflatoxins enter the general food supply where they have been found in both pet and human foods, as well as in feedstock for agricultural animals. Animals fed with contaminated food can pass aflatoxin transformation products into eggs, milk products, and meat[1]
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                    <img src=https://static.igem.org/mediawiki/2017/3/35/T--Tsinghua--project_aft_background.png class="img-responsive img-tab" alt="Figure">
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                    <img src=https://static.igem.org/mediawiki/2017/2/26/T--Tsinghua--project_af_background.gif class="img-responsive" alt="Figure">
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                    <p class="add-review">History of AFT</p>
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                    <p class="description">AFT was coined around 1960 after its discovery as the source of "Turkey X disease"[2], which killed about 100,000 turkeys in England. In animals, after oxidized by cytochrome P450 enzyme in liver cells, AFT can covalently bind adenine in DNA, thus causing mutations. It is listed in Group I carcinogen by the International Agency for Research on Cancer (IARC)[3]. The carcinogenicity of AFT is 900 times more than that of dimethylaminoazobenzene, one of the most famous carcinogen. In China, incidence and mortality of stomach cancer, esophageal cancer and liver cancer is extremely high, second only to lung cancer.
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                    So far, methods for mycotoxins detection mainly include thin layer chromatography, liquid chromatography, and immunological methods, such as enzyme-linked immuno-sorbent assay (ELISA), membrane-based immunoassay, fluorescent polarization, and so on[4]. However, these time-consuming methods cannot meet our demands for daily detection, taking into consideration that AFT can be nearly everywhere. What we need is to detect AFT in everyday foods such as cooking oil. To achieve such detection in our daily life, a simple and straightforward method is in great demand.
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            <h3 class="section-heading text-center">overview</h3>
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                <p class="description">With the rapid development of synthetic biology, we propose to construct a biosensor system for AFT detection. Saccharomyces cerevisiae is chosen as the model organism to design such a biosensor, for its rapid growth speed, minimal pathogenicity,  ensembled selectable markers, well-defined genetic system, and most importantly,  highly versatile DNA transformation system together with a highly efficient homologous recombination system[5]. Furthermore, we can easily make it into dry powder or test paper for daily use.</p>
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                <p class="description ">
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                The system is composed of two components: sensor part and reporter part. In sensor part, we use single-chain Fv antibody (scFv) against AFT, fused with “trigger protein”, as a monitor. Once ScFv detect AFT, it can activate downstream gene expression in reporter part. For convenience of readout, we choose hexose transporter (HXT) as reporter gene.
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            <h3 class="section-heading text-center">reference</h3>
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            <div class="row space50">
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                [1] Fratamico P M, Bhunia A K, Smith J L, et al. Foodborne pathogens: microbiology and molecular biology.[J]. Foodborne Pathogens Microbiology & Molecular Biology, 2005, 6:334.<br>
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                [2] Wannop C C. The histopathology of Turkey 'X' disease in Great Britain.[J]. Avian Diseases, 1961, 5(4):371-381.<br>
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                [3] IARC monographs on the evaluation of carcino-genic risks to human. Some traditional herbal medicine, some mycotoxins, naphthalene and styrene. IARC, France.<br>
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                [4] Placinta CM, D’Mello JPF, Macdonald AMC (1999) A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim Feed Sci Technol 78:21–37<br>
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                [5] Sherman, F. (1991). Getting started with yeast. Methods in enzymology, 194, 3-21.<br>
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            <h3 class="section-heading text-center">How it works</h3>
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                <div class="col-md-6 col-sm-6 arrow">
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                    <img src=https://static.igem.org/mediawiki/2017/c/c8/T--Tsinghua--project_model_biosensor.png class="img-responsive center-block" alt="Figure">
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                    <p class="instructions space50">Yeast two-hybrid systems<br class="hidden-sm"> to capture aflatoxins signals</p>
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                <div class="col-md-6 col-sm-6 arrow">
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                    <img src=https://static.igem.org/mediawiki/2017/e/e5/T--Tsinghua--project_report_construction.png class="img-responsive center-block" alt="Figure">
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                    <p class="instructions space50">HXT converts AFT level<br class="hidden-sm"> to glucose level</p>
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            <h3 class="section-heading text-center">project solution</h3>
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                <div class="col-md-7 col-sm-7">
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                    <p class="add-review">Sensor<br>
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                        part </p>
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                    <p class="description">The sensor part of the AFT detection system was based on the yeast two-hybrid technique. The DNA-binding domain (BD) and the transcription-activation domain (AD) of GAL4, the activator, were incorporated with two different single-chain antibodies of AFT (ScFv1 and ScFv2), correspondingly. Without external AFT, GAL4 the activator was incomplete and unable to activate the expression of the downstream structure gene, hexose transporter (HXT). When AFT was added to the culture, the transcription-activation domain would be recruited to the promoter through the interactions between AFT and both single-chain antibodies, and HXT would be produced.<br class="hidden-xs hidden-sm">
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                    <img src=https://static.igem.org/mediawiki/2017/3/3e/T--Tsinghua--project_sensor_part.png class="img-responsive img-tab" alt="Figure">
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                    <img src=https://static.igem.org/mediawiki/2017/3/3e/T--Tsinghua--project_sensor_part.pngclass="img-responsive img-tab" alt="Figure">
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                </div>
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                    <p class="add-review">Reporter<br>
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                        part </p>
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                    <p class="description">We chose HXT as the reporter gene because the change in the glucose concentration of the culture would be easy to detect by glucometer. To ensure that the autonomous glycometabolism would not interfere with our system, auxotroph that lacked glucose transporters was used, which utilized the maltose instead.<br>
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                    The auxotroph was cultivated in a culture with glucose as the only carbon source. Without the existence of AFT in the culture, the auxotroph would be unable to transport glucose, thus no significant change in the glucose concentration of the culture would be observed. When AFT was added, HXT would be produced in the auxotroph and glucose would be transported and utilized, thus a change in the glucose concentration of the culture would be detected by the glucometer. Furthermore, the concentration of AFT in the culture would be reflected by the consumption rate of the glucose. 
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            <h3 class="section-heading text-center">Features</h3>
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                    <div class="features-list">
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                        <div class="star-container">
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                            <img src=https://static.igem.org/mediawiki/2017/2/20/T--Tsinghua--project_coin_90.png class="img-responsive center-block whitestar">
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                            <img src=https://static.igem.org/mediawiki/2017/0/0f/T--Tsinghua--project_coin_90_green.png class="img-responsive center-block green-star">
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                        <div class="content-middle">
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                            <p class="feature-title ">Lower cost</p>
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                        </div>
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                        <p class="details">Send invites via sms/email<br class="hidden-sm hidden-xs"> to review your business <br class="hidden-sm hidden-xs"> </p>
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                    <div class="features-list">
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                            <img src="https://static.igem.org/mediawiki/2017/6/68/T--Tsinghua--project_puzzle_90.png" class="img-responsive center-block whitestar">
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                            <img src="https://static.igem.org/mediawiki/2017/1/11/T--Tsinghua--project_puzzle_90_green.png" class="img-responsive center-block green-star">
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                            <p class="feature-title ">Easy Procedure</p>
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                    <p class="add-review">Background<br>
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                        effectively <br>
 +
                        inspects aflatoxins</p >
 +
                    <p class="description">Yeast strain：JDY26/27<br>
 +
Genotype：ade2-101  trp1-901 leu2-3.112 his3∆200 ura3-52 gal4∆ gal80∆ SPAL::URA3 LYS2::GAL1-HIS GAL2-ADE2 met2:GAL7-LacZ (or GAL1-LacZ) can1R
 +
<br>96-well, SC-His, adding gradient diluted AFT
 +
<br>Test OD600
 +
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                    <img src=https://static.igem.org/mediawiki/2017/9/99/T--Tsinghua--project_report_1.png class="img-responsive" alt="Figure">
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                    <p class="add-review">Reporter preliminary <br>experiment</p>
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                    <p class="description">Some words are here to explain the figure and experiments<br class="hidden-xs hidden-sm">
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                    <img src=common/project/HXT_reporter_exp.png class="img-responsive" alt="Figure">
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                    <p class="add-review"></p>
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                    <p class="description">SC-LEU, with glucose as the only carbon source <br>
 +
                    EBY.VW4000 <br>
 +
                    +HXT5  <br>
 +
                    +HXT5  <br>
 +
                    +HXT2  <br>
 +
                    +HXT2  <br>
 +
 +
                    HXT can work normally
 +
                       
 +
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 +
                        construction<br>
 +
                        inspects aflatoxins</p>
 +
                    <p class="description">Knock out GAL4, GAL80 by recombination <br>
 +
Test by PCR <br>
 +
Successfully knocked out <br>
 +
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Latest revision as of 15:53, 26 October 2017

Yeasy AFT project page

Start Intro

How aflatoxin hazards people

Class
I
cancerogen

Aflatoxin is known as the most toxic carcinogen.
It can covalently bind
adenine in DNA, thus causing mutations.

In nature
12
kinds of Aflatoxin

with in which B1 is the most toxic
and can be found in maize, peanuts, cotton seeds and many nuts.

Up to
155,000
liver cancer-incidents due to Aflatoxin

It can also cause stomach
cancer and esophageal cancer

how YeasyAFT can testand how they help people?

Quick
detection

The detection of AFT can be accomplished in hours,
which is distinguished from those prevalent biochemical methods.

Low
cost

Utilization of genetic-engineered
yeast allows quite low cost.

High
sensitivity

We use single-chain Fv antibody to detect AFT
, and the signal is enlarged by yeast-two-hybrid system and hexose transporter.

Background

Aflatoxins (AFT)

Aflatoxins (AFT) are poisonous carcinogens that are produced by certain molds (Aspergillus flavus and Aspergillus parasiticus) which grow in soil, decaying vegetation, hay, and grains. They are regularly found in improperly stored staple commodities such as cassava, chili peppers, corn, cotton seed, millet, peanuts, rice, sesame seeds, sorghum, sunflower seeds, tree nuts, wheat, and a variety of spices. When contaminated food is processed, aflatoxins enter the general food supply where they have been found in both pet and human foods, as well as in feedstock for agricultural animals. Animals fed with contaminated food can pass aflatoxin transformation products into eggs, milk products, and meat[1]

Figure
Figure

History of AFT

AFT was coined around 1960 after its discovery as the source of "Turkey X disease"[2], which killed about 100,000 turkeys in England. In animals, after oxidized by cytochrome P450 enzyme in liver cells, AFT can covalently bind adenine in DNA, thus causing mutations. It is listed in Group I carcinogen by the International Agency for Research on Cancer (IARC)[3]. The carcinogenicity of AFT is 900 times more than that of dimethylaminoazobenzene, one of the most famous carcinogen. In China, incidence and mortality of stomach cancer, esophageal cancer and liver cancer is extremely high, second only to lung cancer.

So far, methods for mycotoxins detection mainly include thin layer chromatography, liquid chromatography, and immunological methods, such as enzyme-linked immuno-sorbent assay (ELISA), membrane-based immunoassay, fluorescent polarization, and so on[4]. However, these time-consuming methods cannot meet our demands for daily detection, taking into consideration that AFT can be nearly everywhere. What we need is to detect AFT in everyday foods such as cooking oil. To achieve such detection in our daily life, a simple and straightforward method is in great demand.

overview

With the rapid development of synthetic biology, we propose to construct a biosensor system for AFT detection. Saccharomyces cerevisiae is chosen as the model organism to design such a biosensor, for its rapid growth speed, minimal pathogenicity, ensembled selectable markers, well-defined genetic system, and most importantly, highly versatile DNA transformation system together with a highly efficient homologous recombination system[5]. Furthermore, we can easily make it into dry powder or test paper for daily use.

The system is composed of two components: sensor part and reporter part. In sensor part, we use single-chain Fv antibody (scFv) against AFT, fused with “trigger protein”, as a monitor. Once ScFv detect AFT, it can activate downstream gene expression in reporter part. For convenience of readout, we choose hexose transporter (HXT) as reporter gene.

reference

[1] Fratamico P M, Bhunia A K, Smith J L, et al. Foodborne pathogens: microbiology and molecular biology.[J]. Foodborne Pathogens Microbiology & Molecular Biology, 2005, 6:334.
[2] Wannop C C. The histopathology of Turkey 'X' disease in Great Britain.[J]. Avian Diseases, 1961, 5(4):371-381.
[3] IARC monographs on the evaluation of carcino-genic risks to human. Some traditional herbal medicine, some mycotoxins, naphthalene and styrene. IARC, France.
[4] Placinta CM, D’Mello JPF, Macdonald AMC (1999) A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim Feed Sci Technol 78:21–37
[5] Sherman, F. (1991). Getting started with yeast. Methods in enzymology, 194, 3-21.

How it works

Figure

Yeast two-hybrid systems to capture aflatoxins signals

Figure

HXT converts AFT level to glucose level

project solution

Sensor
part

The sensor part of the AFT detection system was based on the yeast two-hybrid technique. The DNA-binding domain (BD) and the transcription-activation domain (AD) of GAL4, the activator, were incorporated with two different single-chain antibodies of AFT (ScFv1 and ScFv2), correspondingly. Without external AFT, GAL4 the activator was incomplete and unable to activate the expression of the downstream structure gene, hexose transporter (HXT). When AFT was added to the culture, the transcription-activation domain would be recruited to the promoter through the interactions between AFT and both single-chain antibodies, and HXT would be produced.

Figure
Figure

Reporter
part

We chose HXT as the reporter gene because the change in the glucose concentration of the culture would be easy to detect by glucometer. To ensure that the autonomous glycometabolism would not interfere with our system, auxotroph that lacked glucose transporters was used, which utilized the maltose instead.

The auxotroph was cultivated in a culture with glucose as the only carbon source. Without the existence of AFT in the culture, the auxotroph would be unable to transport glucose, thus no significant change in the glucose concentration of the culture would be observed. When AFT was added, HXT would be produced in the auxotroph and glucose would be transported and utilized, thus a change in the glucose concentration of the culture would be detected by the glucometer. Furthermore, the concentration of AFT in the culture would be reflected by the consumption rate of the glucose.

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Send invites via sms/email to review your business

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Our reviews can be easily added to any website created with any site building platform

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Rating will be displayed in search results with a Google approved widget

Details

Background
effectively
inspects aflatoxins

Yeast strain：JDY26/27
Genotype：ade2-101 trp1-901 leu2-3.112 his3∆200 ura3-52 gal4∆ gal80∆ SPAL::URA3 LYS2::GAL1-HIS GAL2-ADE2 met2:GAL7-LacZ (or GAL1-LacZ) can1R
96-well, SC-His, adding gradient diluted AFT
Test OD600

Figure
Figure

Reporter preliminary
experiment

Some words are here to explain the figure and experiments

Some words are here to explain the figure and experiments

Figure
Figure

SC-LEU, with glucose as the only carbon source
EBY.VW4000
+HXT5
+HXT5
+HXT2
+HXT2
HXT can work normally

Reporter
construction
inspects aflatoxins

Knock out GAL4, GAL80 by recombination
Test by PCR
Successfully knocked out

Figure

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