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<!--Project content  -->
 
<!--Project content  -->
 
<tr>
 
<tr>
<td> <h3> Project Description </h3></td>
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<td> <h4> Background </h4></td>
 
</tr>
 
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<p>We move the frozen power from Disney to E.coli. We construct E.coli to express RFP to draw a dynamic pattern of snowflake. Once we kindle a spot, the pattern starts and grows up gradually. According to our design, two kinds of E.coli with partly different genetic pathway align alternatively. The two kinds of E.coli interact through two independent quorum sensing systems, LuxI/LuxR/AHL in vibrio fischeri and RpaI/RpaR/pC-HSL in Rhodopseudomonas palustris, while neither of them influencing themselves. A plate for E.coli culture is divided into rectangular grids and every “cell” in the grid is occupied with a colon of E.coli. On-state shows as red fluorescence while off-state shows as nothing illuminating. To kindle the system, we only need to add correspondent signal molecular to the central 4 colons of E.coli in a plate. Finally, there will be a snowflake with red fluorescence starts to grow up from a point automatically. </p>
+
<p>China is one of the biggest agricultural countries over the world. But in our country, farmers still have a lot of problems to deal with. Drought, pests, water pollution, weeds and so on. These problems cause damage to the agriculture industry. So we want to do some help.</p>
 +
<p>We focused on the weeds problem. Usually, farmers use herbicides to kill the weeds. However, the abuse of herbicides in our country have caused a series of secondary prblems. One of them is that the weeds have the resistant to the herbicides. As we all know, if we always use one kind of antibiotic,the bacteria will become drug-fast. So do the weeds. If famers use only use one kind of herbicide, as the time goes by, the weeds will become herbicide resistant. And farmers have to raise the dose to kill them. However, the more dose farmers raise, the stronger the weeds will be. It is a vicious circle. What’s more, raising the dose will cause many other environmental problems. It will raise the poisonous residue and do harm to animals and  human. </p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/4/49/CAU_project_16.jpg" width="500px"></p>
 +
<p>Let’s use the antibiotic as the example again. If we find that the bacteria has the resistant to antibiotic A, then we will antibiotic B to kill it because there is very few chance that the bacteria have double resistance at the same time in the wild. Similarly, if we use two or more kinds of herbicides the resistant weeds will be killed easily. Also the mix use of herbicides can help us solve many other probelems. So, obviously, Creating a genetically modified crop which is resistant to more than one kind of herbicide enable us to utilize a combination of several herbicides in agriculture.So, obviously, Creating a genetically modified crop which is resistant to more than one kind of herbicide enable us to utilize a combination of several herbicides in agriculture.</p>
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 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/6/65/CAU_project_17.jpg" width="500px"></p>
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 +
 
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</tr>
  
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<td><h3> Content</h3></td>
 
</tr>
 
  
 
<tr>
 
<tr>
<td width="60%" valign="top">
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<td width="45%" valign="top">
<p> Overall project summary </p>
+
<h4>How did we come up with it?</h4>
 +
<p>Unlike the microrganism, co-expression of diffrent kinds of genes is hard to achieve for plant(without a counterpart of operon ) . In our project we choose 2A to serve as a linker between 2 proteins. 2A is a short peptide containing 20 contiguous amino acids. And it has an interesting feature.When placed between 2 genes using a single ORF, during the translation process, the attachment between 19th and 20th amino acid will break. Then the translation process will continue. That means 2A  provides us a potential approach to realize gene co-expression.</p>
 +
<p>The major advantages of using the 2A system in the construction of multicistronic vectors are(i)its small size(54-174bp)compared to IRESes,that coexpression of proteins linked by 2A is independent  of the cell type(since structurally highly conserved amongst the eukaryotic ribosomes,structurally highly conserved amongst the eukaryota),and(iii)that multiple 2As may be used, the activity of each being completely independent.</p>
 +
<p>Our project create 4 genetically modified  Arabidopsis thaliana, and categorized them into 2 combinations, one being BAG & GAB, the other BGT & TAB(B=BAR G=GAT T=tfdA, they're 3 common herbicide resistant genes. A=2A linker). We hope 2A will help with the puzzle of herbicide resistance in agriculture.</p>
 +
<h4>Pre analysis</h4>
 +
<p>Before we started to do our in-lab experiments, we did some analysis of 2A system to help us build a better device. According to previous reports and our analysis, these factors will influence the efficiency of 2A cleavage: (i) 2A sequence (ii) upstream sequences of 2A (iii) The length of 2A.</p>
 +
<p>As for the sequence of 2A, we analyze the sequence of several natural different 2A linkers. The result shows below.</p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/5/5b/CAU_project_11.jpg" width="500px"></p>
 +
<p>So there are several conserved residues in 2A sequence, site mutant experiment carried by Ryan shows that mutant of these conserved residues may lead to much lower cleavage efficiency.</p>
 +
<p>In order to know why upstream sequence and length of 2A will influence cleavage efficiency, we firstly have to take a deep look at the mechanism of 2A cleavage.</p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/0/0e/CAU_project_12.jpg" width="500px"></p>
 +
<p>The most acceptable model shows 2A mediates a cotranslational “ribosome skipping” event here, it predicts that the nascent 2A interacts with the exit tunnel of the ribosome to affect the conformational space occupied by the ester bond linking the nascent protein and tRNAGly in the ribosome P-site. The model predicts that the residues may influence activity reside within the exit tunnel of the ribosome. Also the structural data shows that the ribosome exit tunnel may accommodate 30-40 amino acids. So the activity of shorter forms of F2A may be affected by the nature of the C-terminal sequence of the protein upstream .Then we use secondary structural prediction program to predict the 2A region structure, the result showed that it will form an a helices capped that their C termini. And this has been more stringently tested by dynamic molecular modeling studies on FMDV 2A by J.Wilkie.</p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/8/80/CAU_project_13.jpg" width="300px"></p>
 +
<p align="center">Prediction result from Protein Homology/analogY Recognition Engine V 2.0.</p>
 +
<br></br>
 +
<p>To get the most suitable length of 2A, we analyze the stability of 2A linkers with different lengths. Here we use ProtParam to analyze the 2A sequence listed on the paper to calculate the unstable index. The instability index provides an estimate of the stability of your protein in a test tube. Statistical analysis of 12 unstable and 32 stable proteins has revealed [7] that there are certain dipeptides, the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. The authors of this method have assigned a weight value of instability to each of the 400 different dipeptides (DIWV). Using these weight values it is possible to compute an instability index (II) which is defined as:</p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/0/02/CAU_project_15.jpg" width="200px"></p>
 +
<p align="center">DIWV(x[i]x[i+1]) is the instability weight value for the dipeptide starting in position i.</p>
  
<p>In the popular fantasy-comedy film Frozen, produced by Disney early in this year, there is an amazing fairy tale that the princess has the magical ability to make all things frozen,  presenting us a fantastic world related to dreamy snow. Do you believe our cute E.coli has such kind of magical power? Yes, it can be true!</p>
+
<p>A protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein may be unstable.  
<p>
+
The statistical result shows here.</p>
In our project, we want to move the frozen power from Disney to E.coli, that is, a dynamic  pattern of snowflake emitting red fluorescence, which is actually composed of colons of E.coli expressing RFP, starts to appear and grow up gradually from a single point once we kindle it, showing ideally in figure 1.
+
<p align="center"><img src="https://static.igem.org/mediawiki/2015/2/2e/CAU_project_14.jpg" width="500px"></p>
</p>
+
<p>Since 2A with 22 amino acids shows lowest unstable index, it is the most stable one, which will unlikely to be affected by upstream sequences. </p>
<img src="https://static.igem.org/mediawiki/2014/d/d7/Project_Description_01.gif" width="500px">
+
<p>So using the results of our pre analysis, we constructed our device and did our experiments.</p>
<p>Figure 1. A dynamic pattern of snowflake with E.coli </p>
+
<p>How do we realize it?</p>
+
<p>Our design comes from a combination of genetic-engineering modification, belonging to synthetic biology, and Cellular Automaton, an amazing algorithm in math and computing science. </p>
+
<p>We first introduce the concept of Cellular Automaton, CA[1]. There are a lot of classifications of CA and our project applies one of simple bidimensional forms. In our model, the cellular automaton consists of a bidimensional rectangular grid of cells, and every cell is defined as the very basic unit with 2 states, on and off. For a single cell, there are four nearest cells, located in the same distance from the cell, up and down, left and right, which are defined as neighbors. For a CA, its evolution principles play a determining role in altering every cell’s state following the proceeding of time points. To form a pattern of snowflake, the principle is, once a celll’s state is on, it will be always on; if the state of a cell is off, it will turn to be on only when a single neighbor of it is on.</p>
+
<p>Then, how can we use E.coli to simulate the CA above?</p>
+
<p>To guarantee the mechanism can work out and reach a satisfying effect of view, we do the following design:</p>
+
<p>1.A plate for E.coli culture is divided into rectangular grids and every “cell” in the grid is occupied with a single colon of E.coli ;
+
2.On-state shows as red fluorescence, coming from the expression of RFP in E.coli, and companies with the production of signal molecular, while off-state shows as nothing illuminating;
+
3.Two kinds of E.coli with partly different genetic pathway align alternatively. The two kinds of E.coli interact through two independent quorum sensing systems, LuxI/LuxR/AHL in vibrio fischeri and RpaI/RpaR/pC-HSL in Rhodopseudomonas palustris[2], while neither of them influence themselves.
+
4.Based on the work of Ron Weiss(2005)[3], which designs a pathway where receiver cells only respond to signal molecular of medium concentration as figure 2, we design a genetic pathway to fulfill the evolution principles in CA as figure 3.
+
</p>
+
<img src="https://static.igem.org/mediawiki/2014/c/c7/Project_Description_02.jpg" width="500px">
+
<p>It is conceivable that for a “cell”, any one of its 4 neighbors in grid turns on would cause the produced signal molecular diffuses out of E.coli cells and around, accumulating to a certain concentration and influencing the “cell”. We can deduce that if there are more than one neighbors on, the cell would be immersed in a higher concentration of signal molecular. So, if we modulate related genetic elements in work of Ron Weiss to make the so-called medium concentration of signal molecular as what only one on neighbor produces and accumulates, we can fulfill the principle that an off cell responds and turns on only when one of its neighbors is on.</p>
+
<p>Thanks to RFP’s long half-life period, the other evolution principle asking for the maintenance of on state achieves automatically.</p>
+
<p>As described in figure 3, the upper kind of cells only respond to blue signal molecular of medium concentration referring to condition of only one on neighbor, expressing RFP and producing green signal molecular. When green signal molecular diffuses to the lower kind of cells and satisfies the same condition, the lower cells respond to green signal molecular, expressing RFP and producing blue signal molecular to influence the upper kind of cells. </p>
+
<p>To kindle the whole system, we only need to add correspondent signal molecular to 4 colons of E.coli located in the central of the plate. Then, we would observe a snowflake with red fluorescence starts to grow up from a point automatically just as figure 1.</p>
+
  
<ol>
+
<br></br>
<li>Project Details</li>
+
<li>Materials and Methods</li>
+
<li>The Experiments</li>
+
<li>Results</li>
+
<li>Data analysis</li>
+
<li>Conclusions</li>
+
</ol>
+
  
 +
<h4>Our experiment</h4>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/3/33/CAU_project_9.png" width="500px"></p>
 +
<h4>We want to do more</h4>
 +
<p>Bio safety</p>
 +
<p>We have plans to make further modifications to the system, adding a toxic gene in between the two target genes in case of super weed caused by genetic drift. And the application of this vector is restrained within the crops that won't be disturbed by the toxin. In our bettered system, only one herbicide gene is at the risk of drifting because if more genes were to drift into another species, it would definitely include the toxic gene and disrupt the normal life of weeds and thus can't live. The genetic drift rate of each gene is at the end of the day very low, and present prevention methods are proven to be efficient. With our modification, this problem is kept at a even smaller scale.</p>
 +
<p>"Super Crop"</p>
 +
<p>We aims to create a kind of crop that is resistant to different kinds of stress and having other good qualities. Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders. Co-expressing different pesticidal protein in crops using 2A can help create insect-resistant crops.</p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/9/95/CAU_project_5.jpg" width="300px"></p>
 +
<br></br>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/4/40/CAU_project_6.jpg" width="200px"></p>
 +
<p>Also transimitting the LEA protein family can improve the cold-resistant ability of plants.
 +
SOS 1,2,3 cooperate to regulate ion homeostasis under salt stress. SOS family is also under consideration to resist salt stress.
 +
We also want to transform the metabolic pathways. C4 photosynthesis is a series of anatomical and biochemical modifications to the typical C3 pathway that increases the productivity of plants in warm, sunny, and dry conditions. Co-expressing key enzyme of C4 plant in C3 plant may help to modify the classical C3 pathway.</p>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2015/c/cf/CAU_project_7.jpg" width="500px"></p>
 
</td>
 
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<td width="45%" valign="top">
 
<td width="45%" valign="top">
 
<h3>References </h3>
 
<h3>References </h3>
<p>
+
<p>[1]Dill GM, Cajacob CA, Padgette SR. Glyphosate-resistant crops :adoption, use and future considerations. Pest Manag Sci, 2008, 64(4):326-331.
1. Cellular automaton: http://en.wikipedia.org/wiki/Cellular_automaton.
+
 
</p>
 
</p>
<p>
+
<p>[2]Claire Halpin,Susan E. Cooke, Abdellah Barakate,Abdel El Amrani and Martin D. Ryan.Self-processing 2A-polyproteins – a system for co-ordinate expression of multiple proteins in transgenic plants.The Plant Journal (1999) 17(4), 453–459.
2. Genetic pathway of Ron Weiss(2005):Basu S, Gerchman Y, Collins C H, et al. A synthetic multicellular system for programmed pattern formation[J]. Nature, 2005, 434(7037): 1130-1134.
+
 
</p>
 
</p>
<p>
+
<p>[3]Ekterina Minskaia and Martin D.Ryan.Protein Coexpression Using FMDV 2A Effect of "Linker" Residues.BioMed Research International 2013;Article ID 291730</p>
3. quorum sensing system in Rhodopseudomonas palustris:Schaefer A L, Greenberg E P, Oliver C M, et al. A new class of homoserine lactone quorum-sensing signals[J]. Nature, 2008, 454(7204): 595-599.
+
<p>[4]Michelle L. L. Donnelly, Garry Luke,Amit Mehrotra,Xuejun Li,Lorraine E. Hughes,David Gani and Martin D. Ryan.Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’mechanism indicates not a proteolytic reaction, but a novel translational effect : a putative ribosomal ‘skip’.Journal of General Virology (2001), 82, 1013–1025</p>
</p>  
+
<p>[5]Ekaterina Minskaia, John Nicholson and Martin D Ryan.Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.BMC Biotechnology 2013, 13:67</p>
 +
<p>[6]Martin D. Ryan, Michelle Donnelly, Arwel Lewis, Amit P. Mehrotra,John Wilkie, and David Gani.A Model for Nonstoichiometric, Cotranslational Protein Scission in Eukaryotic Ribosomes.Bioorganic Chemistry 27, 55–79 (1999)</p>
 +
<p>[7]Pablo de Felipe, Garry A. Luke, Jeremy D. Brown and Martin D. Ryan.Inhibition of 2A-mediated ‘cleavage’ of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol. J. 2010, 5, 213–223
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</p>
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Latest revision as of 16:02, 18 September 2015

Team:CAU China/Project - 2014.igem.org

 

Team:CAU China/Project

From 2014.igem.org

Background

China is one of the biggest agricultural countries over the world. But in our country, farmers still have a lot of problems to deal with. Drought, pests, water pollution, weeds and so on. These problems cause damage to the agriculture industry. So we want to do some help.

We focused on the weeds problem. Usually, farmers use herbicides to kill the weeds. However, the abuse of herbicides in our country have caused a series of secondary prblems. One of them is that the weeds have the resistant to the herbicides. As we all know, if we always use one kind of antibiotic,the bacteria will become drug-fast. So do the weeds. If famers use only use one kind of herbicide, as the time goes by, the weeds will become herbicide resistant. And farmers have to raise the dose to kill them. However, the more dose farmers raise, the stronger the weeds will be. It is a vicious circle. What’s more, raising the dose will cause many other environmental problems. It will raise the poisonous residue and do harm to animals and human.

Let’s use the antibiotic as the example again. If we find that the bacteria has the resistant to antibiotic A, then we will antibiotic B to kill it because there is very few chance that the bacteria have double resistance at the same time in the wild. Similarly, if we use two or more kinds of herbicides the resistant weeds will be killed easily. Also the mix use of herbicides can help us solve many other probelems. So, obviously, Creating a genetically modified crop which is resistant to more than one kind of herbicide enable us to utilize a combination of several herbicides in agriculture.So, obviously, Creating a genetically modified crop which is resistant to more than one kind of herbicide enable us to utilize a combination of several herbicides in agriculture.

How did we come up with it?

Unlike the microrganism, co-expression of diffrent kinds of genes is hard to achieve for plant(without a counterpart of operon ) . In our project we choose 2A to serve as a linker between 2 proteins. 2A is a short peptide containing 20 contiguous amino acids. And it has an interesting feature.When placed between 2 genes using a single ORF, during the translation process, the attachment between 19th and 20th amino acid will break. Then the translation process will continue. That means 2A provides us a potential approach to realize gene co-expression.

The major advantages of using the 2A system in the construction of multicistronic vectors are(i)its small size(54-174bp)compared to IRESes,that coexpression of proteins linked by 2A is independent of the cell type(since structurally highly conserved amongst the eukaryotic ribosomes,structurally highly conserved amongst the eukaryota),and(iii)that multiple 2As may be used, the activity of each being completely independent.

Our project create 4 genetically modified Arabidopsis thaliana, and categorized them into 2 combinations, one being BAG & GAB, the other BGT & TAB(B=BAR G=GAT T=tfdA, they're 3 common herbicide resistant genes. A=2A linker). We hope 2A will help with the puzzle of herbicide resistance in agriculture.

Pre analysis

Before we started to do our in-lab experiments, we did some analysis of 2A system to help us build a better device. According to previous reports and our analysis, these factors will influence the efficiency of 2A cleavage: (i) 2A sequence (ii) upstream sequences of 2A (iii) The length of 2A.

As for the sequence of 2A, we analyze the sequence of several natural different 2A linkers. The result shows below.

So there are several conserved residues in 2A sequence, site mutant experiment carried by Ryan shows that mutant of these conserved residues may lead to much lower cleavage efficiency.

In order to know why upstream sequence and length of 2A will influence cleavage efficiency, we firstly have to take a deep look at the mechanism of 2A cleavage.

The most acceptable model shows 2A mediates a cotranslational “ribosome skipping” event here, it predicts that the nascent 2A interacts with the exit tunnel of the ribosome to affect the conformational space occupied by the ester bond linking the nascent protein and tRNAGly in the ribosome P-site. The model predicts that the residues may influence activity reside within the exit tunnel of the ribosome. Also the structural data shows that the ribosome exit tunnel may accommodate 30-40 amino acids. So the activity of shorter forms of F2A may be affected by the nature of the C-terminal sequence of the protein upstream .Then we use secondary structural prediction program to predict the 2A region structure, the result showed that it will form an a helices capped that their C termini. And this has been more stringently tested by dynamic molecular modeling studies on FMDV 2A by J.Wilkie.

Prediction result from Protein Homology/analogY Recognition Engine V 2.0.



To get the most suitable length of 2A, we analyze the stability of 2A linkers with different lengths. Here we use ProtParam to analyze the 2A sequence listed on the paper to calculate the unstable index. The instability index provides an estimate of the stability of your protein in a test tube. Statistical analysis of 12 unstable and 32 stable proteins has revealed [7] that there are certain dipeptides, the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. The authors of this method have assigned a weight value of instability to each of the 400 different dipeptides (DIWV). Using these weight values it is possible to compute an instability index (II) which is defined as:

DIWV(x[i]x[i+1]) is the instability weight value for the dipeptide starting in position i.

A protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein may be unstable. The statistical result shows here.

Since 2A with 22 amino acids shows lowest unstable index, it is the most stable one, which will unlikely to be affected by upstream sequences.

So using the results of our pre analysis, we constructed our device and did our experiments.



Our experiment

We want to do more

Bio safety

We have plans to make further modifications to the system, adding a toxic gene in between the two target genes in case of super weed caused by genetic drift. And the application of this vector is restrained within the crops that won't be disturbed by the toxin. In our bettered system, only one herbicide gene is at the risk of drifting because if more genes were to drift into another species, it would definitely include the toxic gene and disrupt the normal life of weeds and thus can't live. The genetic drift rate of each gene is at the end of the day very low, and present prevention methods are proven to be efficient. With our modification, this problem is kept at a even smaller scale.

"Super Crop"

We aims to create a kind of crop that is resistant to different kinds of stress and having other good qualities. Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders. Co-expressing different pesticidal protein in crops using 2A can help create insect-resistant crops.



Also transimitting the LEA protein family can improve the cold-resistant ability of plants. SOS 1,2,3 cooperate to regulate ion homeostasis under salt stress. SOS family is also under consideration to resist salt stress. We also want to transform the metabolic pathways. C4 photosynthesis is a series of anatomical and biochemical modifications to the typical C3 pathway that increases the productivity of plants in warm, sunny, and dry conditions. Co-expressing key enzyme of C4 plant in C3 plant may help to modify the classical C3 pathway.

References

[1]Dill GM, Cajacob CA, Padgette SR. Glyphosate-resistant crops :adoption, use and future considerations. Pest Manag Sci, 2008, 64(4):326-331.

[2]Claire Halpin,Susan E. Cooke, Abdellah Barakate,Abdel El Amrani and Martin D. Ryan.Self-processing 2A-polyproteins – a system for co-ordinate expression of multiple proteins in transgenic plants.The Plant Journal (1999) 17(4), 453–459.

[3]Ekterina Minskaia and Martin D.Ryan.Protein Coexpression Using FMDV 2A Effect of "Linker" Residues.BioMed Research International 2013;Article ID 291730

[4]Michelle L. L. Donnelly, Garry Luke,Amit Mehrotra,Xuejun Li,Lorraine E. Hughes,David Gani and Martin D. Ryan.Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’mechanism indicates not a proteolytic reaction, but a novel translational effect : a putative ribosomal ‘skip’.Journal of General Virology (2001), 82, 1013–1025

[5]Ekaterina Minskaia, John Nicholson and Martin D Ryan.Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.BMC Biotechnology 2013, 13:67

[6]Martin D. Ryan, Michelle Donnelly, Arwel Lewis, Amit P. Mehrotra,John Wilkie, and David Gani.A Model for Nonstoichiometric, Cotranslational Protein Scission in Eukaryotic Ribosomes.Bioorganic Chemistry 27, 55–79 (1999)

[7]Pablo de Felipe, Garry A. Luke, Jeremy D. Brown and Martin D. Ryan.Inhibition of 2A-mediated ‘cleavage’ of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol. J. 2010, 5, 213–223