Difference between revisions of "Team:OUC-China/Safety"

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<li><a href="#">Lab WORK</a>
 
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China/Experiments">Experiments</a></li>
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China/miniToe">miniToe</a></li>
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China/miniToe Family">miniToe Family</a></li>
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<li><a href="#">HP</a>
 
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Practices</a></li>
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Engagement</a></li>
 
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China/Collaborations">Collaborations</a></li>
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China/Attributions">Attributions</a></li>
 
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<li><a href="https://2018.igem.org/Team:OUC-China/Notebook">Notebook</a></li>
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China/Notebook">Notebook</a></li>
  
 
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<!-- Banner -->
 
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<img src="https://static.igem.org/mediawiki/2018/d/d0/T--OUC-China--designbanner.jpg"alt="banner"width="100%">
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<img src="https://static.igem.org/mediawiki/2018/4/44/T--OUC-China--
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<!-- Features -->
 
<!-- Features -->
 
<section class="box features">
 
<section class="box features">
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<h2 class="major"><span>Design</span></h2>
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<h2  
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class="major"><span>Safety</span></h2>
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<p>
 
<p>
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<h3></h3>
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<br />&emsp;&emsp;Safety is the acutest problem when it comes to genetically modified organism. We
  
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<h3>Background of miniToe Family</h3>
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figured this problem out from three aspects: Project design, Lab work and Shipment.
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<br />The endoribonuclease Csy4 from CRISPR family is the main role of our toolkit. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin. In type I and type III CRISPR systems, the specific Cas6 endoribonuclease splits the pre-crRNAs in a sequence-specific way to generate 60-nucleotide (nt) crRNA products in which segments of the repeat sequence flank the spacer (the target "foreign" nucleic acid sequence). The inactivation of the Cas proteins leads to a total loss of the immune mechanism function.
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<br /><br />
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<br /><br />The Csy4 protein consists of a N-terminal ferredoxin-like domain and a C-terminal domain. This later domain constitutes most of the recognition interactions with the RNA. The RNA adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4. And once the Csy4-RNA complex formed, the structure will stay stable and hard to separate.
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<h3>Project design</h3>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/9/90/T--OUC-China--design1-1.png" height="450">
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<br />&emsp;&emsp;Being responsible for our earth and ourselves is our attitude towards every
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                    <img src="https://static.igem.org/mediawiki/2018/1/16/T--OUC-China--design1-2.png"  height="450">
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<img src="https://static.igem.org/mediawiki/2018/3/3c/T--OUC-China--design1-3.png"  height="450">
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experiment. In our project, all the parts we have utilized are selected from Risk Group 1, none of  
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</div>
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<div align="center"><p>&emsp;&emsp;&emsp;&emsp;Fig.1-1 The structure of Csy4  &emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;
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which are with a Red Flag. What’s more, nonpathogenic bacteria, E.coli DH5α is employed as the main
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Fig.1-2 The structure 22nt hairpin which can be recognized by Csy4.&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;
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Fig.1-3 The Csy4/RNA complex.</p></div>
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chassis for our prior design on the DNA sequence, stem-loop and enzyme. As a matter of fact, when
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&emsp;&emsp;Also,
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（crispr家族蛋白的背景：相较与其他crispr家族其他蛋白质的优点）
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using hairpins and csy4 enzyme to tune the expression of genes, it is relatively safe for
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industrialized applications and scientific research. Based on this, We designed more variants of csy4
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enzymes and hairpins. (See more details in xxx and xxx about the variants of csy4 enzymes and
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hairpins.）So we used these to create a toolkit named miniToe family for synthetic biology which can
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control the expression of the target gene gradient. What’s more, we used the motA gene, one of the  
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genes regulating the motility of E.coli, to verify the feasibility and effectiveness of the toolkit.  
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For the sake of environment, all the chassis organisms will be sterilized before being abandoned to
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prevent the genes from leaking.
 
</p>
 
</p>
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<p>
 
<p>
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<h3>The first system miniToe</h3>
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<h3>Safe lab work</h3>
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<br /><h4 ><font size="3">New method —— miniToe</font></h4>
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<br />&emsp;&emsp;We works at a neat lab named “Lab for Microinnovation and Enterprise”, which is a  
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Based on Csy4's function, we design a new structure named miniToe which can be recognized by Csy4 at the same time. The whole system is a translational activator including three modular parts:
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<br />1. A crRNA to serve as translation suppressor by pairing with RBS as a part of miniToe structure.
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BSL-2 laboratory. Before working in the lab, we are supposed to be trained by the skillful hands, not
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<br />2. A Csy4 site as a linker between crRNA and RBS as a part of miniToe structure,which can be specifically cleaved upon Csy4 expression.
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<br />3. A CRISPR endoribonuclease Csy4.
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only for the general lab safety rules, but also for the standard experimental operation avoiding
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f5/T--OUC-China--design2-1.png" height="400">         
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</div>
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unnecessary damage. No experiment carries no risk to the experimenters, hence we are properly equipped
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with facilities, such as latex gloves, nitrile gloves, goggles, lab coats to protect us from biotic
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and abiotic hazards.
 
<br />
 
<br />
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  <div align="center"><p >Fig.2-1 The structure of miniToe.</p></div>
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<br />
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/0/0d/T--OUC-China--safety001.jpg"
  
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In our project, we use sfGFP as a target gene to test our system. First, we need to make sure the stability of our structure and the formation of hairpin (The Csy4 site) is also crucial. So before the experiment, we focus on the structure of miniToe. We have a prediction of structure of whole circuit as well as the structure of miniToe.
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height="400" >
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<br /><br />
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                    <img src="https://static.igem.org/mediawiki/2018/9/9c/T--OUC-China--Safety01.jpg"
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/5c/T--OUC-China--design2-2.png" width="700" >        
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height="400">
 
</div>  
 
</div>  
 
<br />
 
<br />
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  <div align="center"><p >Fig.2-2 The structure prediction of the whole circuit and miniToe.</p></div>
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  <div align="center"><p style="font-size:80%;color:gray"">Fig.1 Our laboratory and personal safety
  
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To explore the feasibility and function of miniToe, we designed the circuit below as our test system in order to test the function of miniToe structure. We use Ptac as the inducible promoter of Csy4 to control the existence of Csy4 or not. At the same time, we construct the miniToe before the sfGFP which is a symbol of target gene in our circuit. And this circuit is controlled by a constitutive promoter form Anderson family named J23119.
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precautions</p></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/1/16/T--OUC-China--design2-3.png" width="700" >         
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</div>
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&emsp;&emsp;Then for lab safety, a routine inspection is performed twice a day by everyone, not only
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for ensuring normal operation of our lab like supplying experimental consumables and inspecting the  
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running of instruments, but also keeps it away from non-biological hazards, especially the water and
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electricity. For high efficiency, responsibility implements to every team member.
 
<br />
 
<br />
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<div align="center"><p >Fig.2-3 The two plasmids of miniToe test system.</p></div>
 
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  Without Csy4,the crRNA pairs with RBS very well, so the switch just turns off which means that no protein will be produced. Otherwise, with the presence of Csy4, the translation turns on. In this way, the expression of downsteam gene can be regulated.
 
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<br />
 
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/c/ce/T--OUC-China--design2-4.png" width="700" >         
 
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</div>
 
 
<br />
 
<br />
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  <div align="center"><p >Fig.2-4 The working process of miniToe.</p></div>
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/c/c2/T--OUC-China--safety002.jpg"
  
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By experiments we have proved that our system can work well!
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width="400" >
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<br /> <br />
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                    <img src="https://static.igem.org/mediawiki/2018/2/2e/T--OUC-China--safety003.jpg"  
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8b/T--OUC-China--design2-5.png" width="700" >        
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width="400" >
 
</div>  
 
</div>  
 
<br />
 
<br />
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  <div align="center"><p >Fig.2-5 The result of our first system.</p></div>
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  <div align="center"><p style="font-size:80%;color:gray"">Fig.2 Table for routine inspection</p></div>  
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<a href="https://2018.igem.org/Team:OUC-China/Results">Click here for more details!</a><br /> <br />
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<h4><font size="3">The characteristics of miniToe</font></h4>
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<br />1. The Csy4/RNA complexes become really stable once they have formed. It shows that the miniToe can control the state of expression like a switch (ON/OFF). When the switch is turn OFF, the downstream gene expression is completely closed, the reaction grows very slow in the beginning but accelerating rapidly once the complexes have formed.
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<br />2. Compare to the small RNA, the insertion of hairpin provides Csy4 with a recognition and cleavage site so that the Csy4 may enlarge the steric hindrance between crRNA and RBS when we need to release the crRNA for opening the downstream gene expression.
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<br />3. Compare to the toehold switch, miniToe don't need to redesign crRNA over and over again because the crRNA is not paired with CDS.
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</p>
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<p>
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<h3>The second system miniToe family</h3><br />
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<h4><font size="3">miniToe family——The model help us go further </font></h4>
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<br />After testing our first system, miniToe, the dry lab member explore more deeply about our system.
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<br />After building the ODE model, we use it to simulate the dynamics of GFP. Comparing with the experimental data, we find it fits perfectly, which indicates that our model is reliable in our first system. Then we analyze sensitivity of the GFP, it is not too difficult to find that the cleavage rate has an influence in the expression of GFP, which shows that if we change the Csy4 protein we can change the expression of GFP which is a symbol of target gene.
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/9/9f/T--OUC-China--design3-1.png" height="285">
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<br />&emsp;&emsp;Furthermore, several operational zones are divided scientifically for special
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                <img src="https://static.igem.org/mediawiki/2018/c/c4/T--OUC-China--design3-2.png" height="285">
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</div> <br />
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experiments like A zone where is weighing the drugs, preparing the medium and sterilizing, B zone
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<div align="center"><p >&emsp;&emsp;Fig.3-1 The ODE model for the first system&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;
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                  &emsp;&emsp;&emsp;&emsp;  &emsp;&emsp;&emsp;&emsp;  &emsp;&emsp;&emsp;&emsp;  &emsp;&emsp;&emsp;&emsp;  Fig.3-2 The model about sensitivity analysis of the GFP&emsp;&emsp;&emsp;&emsp; &emsp;&emsp;&emsp;&emsp;    &emsp;&emsp;&emsp;  
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where is the recording experimental note and organizing experimental data area, C zone where is a
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</p></div>  
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molecular operation area, D zone where is RNA electrophoresis and a polyacrylamide gel electrophoresis
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<h4><font size="3">The principles of designing mutants </font></h4>
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<br />For Csy4 mutants
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area.
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<br />1. Some key sites in the Csy4 is really crucial for keeping the stable of structure and making sure the functions including recognition and cleavage. The change of those sites may results in big influence on design. By point mutation, we want to get an amount of mutants whose recognition and cleavage rates shows as a "ladder".
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<br />&emsp;&emsp;For us, lab safety is a hot topic and we have come up with a practical lab safety
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<br />For hairpin mutants
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<br />2. Do not to break the recognition site so that we can ensure the function of cleavage. If we break the key site G20 may lead to the damage of cleavage function.
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manual which normalizes our lovely lab.
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<br />3. The stable of secondary structure is vital so we need to focus on each hairpin's Gibbs free energy after design.  
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<br />
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<br />4. Our aim is to obtain different hairpins which have totally different rates be recognized and cleaved. <br /> <br />
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<br />
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<h4><font size="3">Models help us deeply!</font></h4>
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<h3>Practical lab safety manual</h3>
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<br />（此处假装有模型的简述版）<a href="https://2018.igem.org/Team:OUC-China/miniToe">Click here for more details! </a>
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<br />1.Routine inspections must perform between 6:30-8:00pm and 9:30-11:00pm.
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<br />With the help of model, we finally select 4 Csy4 mutants and 5 hairpin mutants. We have tested each mutant and have data support. Then there are 5*6 combinations including wild types. We have tested all of them and have proved that 10 of them work successfully as we expect. So the second system of our project, miniToe family consist of 10 combinations which is designed and selected by us.
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<br />2.Lab cleaning must be done twice a week with disinfectant moping the floor for at least 3times.
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</p>
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<br />3.Well prepared experiment plans should be written before you start.
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<br />4.Lab coats and gloves must be worn when conducting experiments.
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<br />5.Short skirts, shorts, and open shoes must not be worn.
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<p>
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<br />6.If you see a colleague doing something dangerous, point it out to him or her.
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<h3>The third system——miniToe polycistron</h3><br />
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<br />7.Lab coats must not be worn outside laboratories and in public areas.
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<h4><font size="3">Why we do </font></h4>
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<br />8.Know where safety equipment (eyewash, shower and extinguisher) is located.
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<br />Many applications of synthetic biology need the balanced expression of multiple genes. Microorganisms with modified metabolic pathways are employed as a reaction vessel to natural or unnatural products. It involves the introduction of several genes encoding the enzymes of a metabolic pathway. Indeed, pathway optimization requires to adjust the expression of multiple genes at appropriately balanced levels, for example, the synthetic of poly-3-hydroxybutyrate and Mevalonate.  
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<br />9.Food/drink is not allowed in laboratories where chemicals are used stored.
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<br />As is done in the prokaryotes, grouping a cluster of genes into a single polycistron is a convenient mean for regulating genes simultaneously. Thus, for the sake of tuning the expressions of genes within polycistron, we want to develop a tightly regulated by the miniToe structure. We name this system miniToe polycistron which contains several genes in one circuit between different miniToe structures. Our aim for this part is achieving different proportions of output by miniToe in polycistrons compared with normal polycistrons.
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<br />10.Know how to clean up spills of common chemicals and specific chemicals you see, for spilling
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<br /><br /><h4><font size="3">How we do</font> </h4>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f2/T--OUC-China--design4-1.png" height="500">  
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ethidium bromide dealt with sodium hydroxide.
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</div> <br />
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<br />11.Always read the directions before using instruments and read MSDS before handling new
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<div align="center"><p >Fig.4-1 The mechanisms of miniToe polycistron
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</p></div>
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chemicals carefully.
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1. Without the Csy4, the existence of miniToe structure in polycistron will increase the stability when we don't want to open the switch. The crRNA in miniToe is a compliment sequence of RNA to prevent the binding of ribosomes especially for the genes far from 5' terminal.
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<br />12.When post-stain gel electrophoresis, we must wear the gloves that are on the left two and
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<br />2. Also, the miniToe of 3' end is a protection to prevent degradation of nucleotide chain. 
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<br />3. Each miniToe structure works at special recognition and cleavage rates. That will make it possible to regulate the gene behind this miniToe.
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right three.
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<br /><br /> <h4><font size="3">Result </font></h4>
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<br /> <br />
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Finally, we ( 此处要补一下多顺反子的结果后补充 )
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/38/T--OUC-China--Safety004.jpg"  
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</p>
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height="400">
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<p>
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                    <img src="https://static.igem.org/mediawiki/2018/c/cb/T--OUC-China--Safety006.jpg"
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<h3>miniToe Motility detection system</h3><br />
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<h4><font size="3">The application of miniToe——Regulation of motA </font></h4>
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height="400" >
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<br />MiniToe is also a good tool which can be used to study of molecular mechanism. Scientists may puzzle with the functions of certain gene or protein when first discover it. Now one common method to study is knock-out or knock-in. In this way, organisms show some flaws related to the gene which is knocked out. But if we want to know better about the gene functions, we may need the different levels of the gene expressions.
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<br />This year, we used our system to control the motions of E.coli. We have transformed miniToe system into E. coli whose motility is regulated by the motor protein, MotA. MotA provides a channel for the proton gradient required for generation of torque. ΔMotA strains (the motA-deletion strain) can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation. Expression of motA from a plasmid has been shown to restore motility in ΔmotA strains. So we use our miniToe system to control motA protein expression with different levels. We get a strain without motA by knocking out and then transform motA protein under the control of miniToe family. The E.coli will restore motility. It seems that our system has more applications such as regulation of motA.
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<br /><br /><h4><font size="3">How we do</font> </h4>
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<br />E.coli RP6666 (knocked out motA) lacks capacity of motion. By using hairpin detection system to control the translation of the downstream gene, we construct a circuit and put motA downstream of miniToe, then transfer the plasmid into E.coli RP6666. By inducing the production of Csy4, motA could be controlled indirectly, thus making E.coli RP6666 strain regain the capacity of motion.
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<div align="center"><p >Fig.5-1 The process of motility detection system
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<br />In conclusion, we have demonstrated the design of modular translational activators with CRISPR endoribonuclease Csy4 named miniToe. And we have design four systems which is improved step by step.
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    <br />In the future, we still have some ways to perfect our project. First, we would like to enlarge our project by finding more and more mutants. By finding and designing more mutants we may get a larger library which can enlarge the function of our toolkit. Second, we have tested the ratio of regulation in miniToe polycistron. We can use man-made setting to make calibration curve in the future applications which will help us to know our project deeply.
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Revision as of 12:00, 7 October 2018

Team OUC-China: Main
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Safety


  Safety is the acutest problem when it comes to genetically modified organism. We figured this problem out from three aspects: Project design, Lab work and Shipment.

Project design


  Being responsible for our earth and ourselves is our attitude towards every experiment. In our project, all the parts we have utilized are selected from Risk Group 1, none of which are with a Red Flag. What’s more, nonpathogenic bacteria, E.coli DH5α is employed as the main chassis for our prior design on the DNA sequence, stem-loop and enzyme. As a matter of fact, when using hairpins and csy4 enzyme to tune the expression of genes, it is relatively safe for industrialized applications and scientific research. Based on this, We designed more variants of csy4 enzymes and hairpins. (See more details in xxx and xxx about the variants of csy4 enzymes and hairpins.）So we used these to create a toolkit named miniToe family for synthetic biology which can control the expression of the target gene gradient. What’s more, we used the motA gene, one of the genes regulating the motility of E.coli, to verify the feasibility and effectiveness of the toolkit. For the sake of environment, all the chassis organisms will be sterilized before being abandoned to prevent the genes from leaking.

Safe lab work


  We works at a neat lab named “Lab for Microinnovation and Enterprise”, which is a BSL-2 laboratory. Before working in the lab, we are supposed to be trained by the skillful hands, not only for the general lab safety rules, but also for the standard experimental operation avoiding unnecessary damage. No experiment carries no risk to the experimenters, hence we are properly equipped with facilities, such as latex gloves, nitrile gloves, goggles, lab coats to protect us from biotic and abiotic hazards.


Fig.1 Our laboratory and personal safety precautions

  Then for lab safety, a routine inspection is performed twice a day by everyone, not only for ensuring normal operation of our lab like supplying experimental consumables and inspecting the running of instruments, but also keeps it away from non-biological hazards, especially the water and electricity. For high efficiency, responsibility implements to every team member.


Fig.2 Table for routine inspection


  Furthermore, several operational zones are divided scientifically for special experiments like A zone where is weighing the drugs, preparing the medium and sterilizing, B zone where is the recording experimental note and organizing experimental data area, C zone where is a molecular operation area, D zone where is RNA electrophoresis and a polyacrylamide gel electrophoresis area.
  For us, lab safety is a hot topic and we have come up with a practical lab safety manual which normalizes our lovely lab.

Practical lab safety manual


1.Routine inspections must perform between 6:30-8:00pm and 9:30-11:00pm.
2.Lab cleaning must be done twice a week with disinfectant moping the floor for at least 3times.
3.Well prepared experiment plans should be written before you start.
4.Lab coats and gloves must be worn when conducting experiments.
5.Short skirts, shorts, and open shoes must not be worn.
6.If you see a colleague doing something dangerous, point it out to him or her.
7.Lab coats must not be worn outside laboratories and in public areas.
8.Know where safety equipment (eyewash, shower and extinguisher) is located.
9.Food/drink is not allowed in laboratories where chemicals are used stored.
10.Know how to clean up spills of common chemicals and specific chemicals you see, for spilling ethidium bromide dealt with sodium hydroxide.
11.Always read the directions before using instruments and read MSDS before handling new chemicals carefully.
12.When post-stain gel electrophoresis, we must wear the gloves that are on the left two and right three.



  
  
  
  

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