Difference between revisions of "Team:SIAT-SCIE/Safety"

 
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     <p style="text-align: center;margin-top: 80px"><img src="https://static.igem.org/mediawiki/2018/2/2d/T--SIAT-SCIE--SIAT_Safety.png" width="1200px" height="600px"></p>
 
     <p style="text-align: center;margin-top: 80px"><img src="https://static.igem.org/mediawiki/2018/2/2d/T--SIAT-SCIE--SIAT_Safety.png" width="1200px" height="600px"></p>
 
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         <h1>Outer Membrane vesicles</h1>
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         <h1> Risk Group Classification</h1>
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         <p style="font-size: 20px">Outer membrane vesicles (OMVs) are vesicles produced and used as common vehicles in the world of gram-negative-bacteria. Despite their ubiquity, they have been grievously overlooked in the past; budding out as spherical containers of 20 to 500 nm in diameter from the bacterial membrane, they are potentially capable of transporting a wide array of biomolecules that awaits the academia to divulge. As potent transporters, OMVs play an integral role in various biological phenomena, ranging from stress regulations to microbial interactions (1).</p>
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         <p style="font-size: 20px"> According to the Laboratory Bio Safety Manual published by WHO, our laboratory work using infectious microorganisms is classified as Risk Group 1, meaning no or low individual and community risk. Correspondingly, our laboratory is equipped with all the required installations to ensure biosafety in the laboratory. </p>
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        <h1> General Lab Safety</h1>
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        <p style="font-size: 20px"> Every team member must undergo a series of lab safety training for around 1 week’s time in order to attain the permission to work in the laboratory. Information on the location of eye-wash stations, emergency showers, emergency contact person, escape routes and meeting points are all delivered during the training session. The procedures of using lab facilities are all demonstrated by our laboratory instructors, who are all qualified personnels from the laboratory. The proper procedure of handling hazardous or toxic chemical and biological reagents is particularly emphasised. In addition, there will be supervision by personnels from the institute when hazardous substance is to be handled, until the supervisor is convinced of our independence and experience with using these substances. </p>
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         <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/3/36/T--SIAT-SCIE--SIAT_OMV%281%29.png" width="800px" height="400px"></p>
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        <h1> Lab Condition and Environment </h1>
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         <p style="font-size: 20px"> Seeing that the unique properties of OMVs may revolutionise traditional delivery system, our team aims to devise a technique that incorporates the wonders of OMVs and the very frontier technology in genetic engineering — Cas9 proteins — to form a OMV-CRISPR-Cas9 system, which is capable of delivering Cas-9 proteins to target the bacterial genome of bacteria inside mammals’ bodies.  
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        <p style="font-size: 20px"> Our laboratory is newly equipped. All consumables are replenished at a fixed interval. The entire laboratory includes several individual chambers specialised for the preparation of bacteriophage, cultivation of cells, etc., with additional independent entrance guard systems. No entry is allowed to these rooms without permission and prior training. Each team has individual benches. Certain consumables (e.g. gloves, tubes, kits, etc.) are posited in the cabinets in the storage area. There are 4℃ , -20℃ and -80℃ refrigerators available to deposit some of the reagents. </p>
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         </p>
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<br/>
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         <p style="font-size: 20px">As natural kins to bacterial cell membranes, OMVs can be degraded easily while preserving the shape and bioactivity of sensitive Cas9 proteins within, as well as single guide RNAs (sg-RNAs). We expect this technique would open up new possibilities of in vitro genetic engineering, thus providing substantial aid in curing and preventing illnesses such as inflammatory bowel diseases by removing virulence genes from malignant bacteria with this technique.</p>
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<p style="text-align:center; font-size:15px">
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         <h1>Fusobacterium nucleatum</h1>
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    All workbench is required to be cleaned (with disinfectant) and equipments sorted after use.  
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         <p style="font-size: 20px">Our project aims to test the efficiency of our system by applying it to knock out fadA gene of Fusobacterium nucleatum, a strain of bacteria that reside in human alimentary canal. </p>
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</p>
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        <p style="font-size: 20px">F. nucleatum is a gram-negative bacterium prevalently found in mammal oral cavity, and is frequently associated with oral inflammation diseases and various cancers (2). Its virulence stems from its invasion of the human epithelial cells, which induces oncogenic gene expression. The main genetic culprit of its pathogenicity is its fadA gene, which is an adhesion virulence factor that insures the binding of F.nucleatum to the host epithelial cell, thereby enabling the bacterium to invading the host and causing inflammation and cancer. </p>
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         <p style="font-size: 20px">F. nucleatum’s fadA gene is a congenial candidate for our project since, being gram-negative, F.nucleatum possesses abundant OMVs that are central to our designed system. Through knocking out the fadA gene, we are enabled to observe the efficacy of our system’s application in realistic setting.</p>
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        <p style="font-size:20px"> </p>
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         <p style="font-size: 20px"></p>
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         <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/0/07/T--SIAT-SCIE--SIAT_Safety_Lab.png" width="800px" height="400px"></p>
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    </div>
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        <h1> Personal Protection </h1>
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    <article>
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         <p style="font-size: 20px">Here are some basic rules we must follow in the lab[1]:</p>
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        <details>
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        <ul>
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            <summary>Mechanism of FadA protein</summary>
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        <li>Laboratory coveralls, gowns or uniforms must be worn at all times during work in the laboratory.  
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            <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;margin:40px 200px 0px 200px">The FadA protein is activated when its two forms combine and become internalized. The first form is a pre-FadA that is anchored in the cell membrane, whereas the second form is the mature FadA (mFadA) that is secreted out of F. nucleatum. When the two forms combine to form a complex, the protein is capable to help F. nucleatum bind to the host epithelial cell, thus allowing F. nucleatum embark on invading the host cells.</p>
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</li>
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            <h3 style="margin: 20px 200px 20px 200px">Overview (Fig. 1)</h3>
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         <li>Appropriate gloves must be worn for all procedures
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              <p style="text-indent: 20px;font-size: 20px; padding: 10px 10px 10px 10px;margin:0px 200px 0px 200px">We will first express Cas9 and sgRNA in E.coli and then transport them to E.coli’s periplasm(Step 1). By then, they may be packaged by OMVs that bud off from E.coli’s outer membrane(Step 2). Those OMVs will be collected and mixed with our target bacteria, thereby allowing them to fuse with bacteria again, to release the Cas9 proteins and sgRNA(Step 3), and cleave the target gene (Step 4 & 5).  Afterwards, we will test whether the target gene is cleaved by Cas9.
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</li>
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            </p>
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         <li>It is prohibited to wear protective laboratory clothing outside the laboratory
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            <p style="text-align: center"><img src="https://static.igem.org/mediawiki/2018/3/30/T--SIAT-SCIE--SIAT_Description_Figure1.png" height="600px" width="900px"></p>
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</li>
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        </details>
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        <li>Open-toed footwear is prohibited in the laboratory. 

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      </article>
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</li>
 +
        <li>Eating, drinking, smoking, applying cosmetics, and handling contact lenses is 
prohibited in the laboratory working areas. 

 +
</li>
 +
         <li>Storing human foods or drinks anywhere in the laboratory working areas is 
prohibited

 +
</li>
 +
</ul>
 +
         <h1> Utilisation of specialised equipments </h1>
 +
         <p style="font-size: 20px"> Due to the requirement of our project, we need to use some of the specialised equipments in the lab. For example, when separating OMVs from the sample, super-centrifuge is used for its high rotational speed as required by protocol. Because of the tremendous hazard that may result if not used properly, supervision is required. Further more, each piece of equipment in the laboratory are labeled with the name, contact number of the person in charge of it, and precautions. Thus team members would contact the personnel responsible for the machine before a permission is granted to us.
 </p>
 +
         <h1> Disposal of waste in the lab </h1>
 +
         <p style="font-size: 20px">Here are some basic rules we must follow in the lab[1]:</p>
 +
        <ul>
 +
        <li>Proper disposal of experimental waste is vital to maintaining lab safety. In our lab, we generally divide the wastage in to several groups, each group disposed in a certain container to be carried away.[2]
 +
 
 +
</li>
 +
        <li> Contaminated (potentially infectious) materials for disposal such as discarded specimens and cultures are to be placed in leakproof laboratory discard bags posited at each bench in a bright-yellow bin on the ground, with tops secured prior to disposal into waste bins. 

 +
</li>
 +
        <li>Contaminated (infectious) “sharps” —  hypodermic needles, scalpels, knives and broken glass — should be disposed in puncture-proof containers fitted with covers on each bench with a bright yellow and red color and the label of ‘sharp container’ on them. Also, when these containers are 75% full, they are to be emptied and no further wastage should go into these containers.
 +
 
 +
</li>
 +
        <li> Contaminated material for decontamination by autoclaving, washing, reuse, or recycling (e.g. jars for reagents, measuring cylinders, flasks and beads); for materials to be autoclaved, we have specified baskets to place them. All of them will then be collected by certain personnels and decontaminated with autoclaves.
 +
</li>
 +
 
 +
</ul>
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Latest revision as of 16:41, 8 December 2018

Risk Group Classification

According to the Laboratory Bio Safety Manual published by WHO, our laboratory work using infectious microorganisms is classified as Risk Group 1, meaning no or low individual and community risk. Correspondingly, our laboratory is equipped with all the required installations to ensure biosafety in the laboratory.

General Lab Safety

Every team member must undergo a series of lab safety training for around 1 week’s time in order to attain the permission to work in the laboratory. Information on the location of eye-wash stations, emergency showers, emergency contact person, escape routes and meeting points are all delivered during the training session. The procedures of using lab facilities are all demonstrated by our laboratory instructors, who are all qualified personnels from the laboratory. The proper procedure of handling hazardous or toxic chemical and biological reagents is particularly emphasised. In addition, there will be supervision by personnels from the institute when hazardous substance is to be handled, until the supervisor is convinced of our independence and experience with using these substances.

Lab Condition and Environment

Our laboratory is newly equipped. All consumables are replenished at a fixed interval. The entire laboratory includes several individual chambers specialised for the preparation of bacteriophage, cultivation of cells, etc., with additional independent entrance guard systems. No entry is allowed to these rooms without permission and prior training. Each team has individual benches. Certain consumables (e.g. gloves, tubes, kits, etc.) are posited in the cabinets in the storage area. There are 4℃ , -20℃ and -80℃ refrigerators available to deposit some of the reagents.



All workbench is required to be cleaned (with disinfectant) and equipments sorted after use.

Personal Protection

Here are some basic rules we must follow in the lab[1]:

  • Laboratory coveralls, gowns or uniforms must be worn at all times during work in the laboratory.
  • Appropriate gloves must be worn for all procedures
  • It is prohibited to wear protective laboratory clothing outside the laboratory
  • Open-toed footwear is prohibited in the laboratory. 

  • Eating, drinking, smoking, applying cosmetics, and handling contact lenses is 
prohibited in the laboratory working areas. 

  • Storing human foods or drinks anywhere in the laboratory working areas is 
prohibited


Utilisation of specialised equipments

Due to the requirement of our project, we need to use some of the specialised equipments in the lab. For example, when separating OMVs from the sample, super-centrifuge is used for its high rotational speed as required by protocol. Because of the tremendous hazard that may result if not used properly, supervision is required. Further more, each piece of equipment in the laboratory are labeled with the name, contact number of the person in charge of it, and precautions. Thus team members would contact the personnel responsible for the machine before a permission is granted to us.


Disposal of waste in the lab

Here are some basic rules we must follow in the lab[1]:

  • Proper disposal of experimental waste is vital to maintaining lab safety. In our lab, we generally divide the wastage in to several groups, each group disposed in a certain container to be carried away.[2]
  • Contaminated (potentially infectious) materials for disposal such as discarded specimens and cultures are to be placed in leakproof laboratory discard bags posited at each bench in a bright-yellow bin on the ground, with tops secured prior to disposal into waste bins. 

  • Contaminated (infectious) “sharps” — hypodermic needles, scalpels, knives and broken glass — should be disposed in puncture-proof containers fitted with covers on each bench with a bright yellow and red color and the label of ‘sharp container’ on them. Also, when these containers are 75% full, they are to be emptied and no further wastage should go into these containers.
  • Contaminated material for decontamination by autoclaving, washing, reuse, or recycling (e.g. jars for reagents, measuring cylinders, flasks and beads); for materials to be autoclaved, we have specified baskets to place them. All of them will then be collected by certain personnels and decontaminated with autoclaves.