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

 
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<div class="psg" >
<span class="psg_ttl">Methods and Materials</span>
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<span class="psg_ttl">Project Safety</span>
<span class="psg_subtitle">Transformation</span>
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<span class="psg_ttl psg_subtitle">Biofilm Scaffold</span>
<p>Transform Escherichia coli DH5&alpha; with these following plasmids (all in pSB1C3):</p>
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<p>Our biofilm scaffold consists of curli which is the protein found on the surface of E.Coli. Curli may facilitate bacteria’s invasion into host cells and activate corresponding cytokines and inflammatory mediators in plasma. But the system we wish to produce will be ultimately cell-free and no living organisms will be included. Besides, curli will be immobilized and covered by Nafion on the electrode, so contact with curli will not happen. Therefore, biofilm scaffold is relatively safe.</p>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Negative control: BBa_R0040</p>
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<span class="psg_ttl psg_subtitle">Blood Samples</span>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Positive control: BBa_I20270</p>
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<p>We have gotten approval from our university authority and collected some blood samples from SIR RUN RUN SHAW HOSPITAL, which is classified as a Grade 3 Class A hospital by Chinese government. The procedure of blood sampling was done by medical personnel using ideal blood collection equipment. Besides, our experiment was conducted under professional doctors' supervision and waste was collected by the specialties in the hospital. (It is also an essential part of <a href="https://2018.igem.org/Team:ZJU-China/Human_Practices">Integrated Human Practice.</a>) </br></p>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 1: BBa_J364000</p>
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<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 2: BBa_J364001</p>
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<p></br>Click <a href="https://static.igem.org/mediawiki/2018/5/51/T--ZJU-China--ofile.pdf">here</a> to see the offical collaboration protocol.</p>  
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 3: BBa_J364002</p>
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<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 4: BBa_J364007</p>
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<p></br>Click  <a href="https://static.igem.org/mediawiki/2018/d/dc/T--ZJU-China--file2.pdf">here</a>  to see the approval from National Demonstration Center for Experimental Biology Education(Zhejiang University).<p>
<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 5: BBa_J364008</p>
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<p style="padding-left: 3em; margin-top: .6em;">&bull; Test Device 6: BBa_J364009</p>
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<p></br>Our project meets ethical requirements. Blood samples we got from hospital are all remaining samples after satisfying patients’ pathological diagnosis need. We keep secret of patients’ privacy information. We conduct experiments with clinical material following our country's laws and our university's rules.</br> </p>
<p></br>Resuspend DNA in selected wells in the Distribution Kit with 10 &micro;L ddH20. Thaw competent cells on ice. Pipette 25 &micro;L of competent cells into 1.5 mL tube per transformation and add 2 &micro;L of resuspended DNA into it. Incubate on ice for 30 min. Heat shock tubes at 42&deg;C for 90 sec. Then incubate on ice for 5 min.</p>
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<p></br>The main issue for work with blood samples is the potential for infection. Clinical samples may be contaminated with pathogens. The high risk, well known viral agents such as Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV) and Hepatitis C Virus (HCV) are not the only agents that may be present in blood. Other viruses such as HTLV1 and B19 as well as various bacterial agents may also be present. Regarding the likely incidence of a pathogen in blood samples, several factors should be considered. These include known medical history of a patient or donor, whether the samples are from individuals showing clinical symptoms of infectious disease, the incidence of the various pathogens that are endemic in the local population or donor group and the type of sample. Fortunately, SIR RUN RUN SHAW HOSPITAL offered us the physiological indices of blood samples and other relevant information. So, the samples we used are from formal approach and relatively less dangerous. To minimize the risks,we have made Standard Operating Procedure (SOP) which is strictly followed in our iGEM lab.</p>
<p></br>Add 1000 &micro;L LB media with Chloramphenicol (1000&times;) to each transformation. Incubate at 37&deg;C for 1 hours, shaking at 200-300 rpm.</p>
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<span class="psg_ttl psg_subtitle">Standard Operating Procedure (SOP)</span>
<p></br>Pipette 100 &micro;L of each transformation onto LB plates (Chloramphenicol, 1000&times;). Spread with sterilized spreader. Incubate transformations overnight (14-18 hours) at 37&deg;C.</br></p>
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<p>As insurance, Blood samples from all patients should be considered infective. The following precautions followed in our lab are also recommended for all health-care workers in clinical laboratories.</p>
<span class="psg_subtitle">Colonies Selection</span>
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<p style="padding-left:.5em; margin-top:.5em;">1. All the experiments where blood is involved must be conducted in a separate room within the laboratory.</p>
<p>Pick 2 single colonies from each of plate and inoculate it on 5-10 mL LB medium with Chloramphenicol (1000&times;). Grow the cells overnight (16-18 hours) at 37&deg;C and 220 rpm.</br></p>
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<p style="padding-left:.5em; margin-top:.5em;">2. All specimens of blood should be put in a well- constructed container with a secure lid to prevent leaking. Care should be taken when dealing with each specimen to avoid contaminating the outside of the container and of the laboratory form accompanying the specimen.</p>
<span class="psg_subtitle">Calibration</span>
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<p style="padding-left:.5em; margin-top:.5em;">3. Lab surface including correlated equipment is decontaminated with chemical disinfectant prior and after disposal.</p>
<p>We used the plate reader Synergy Neo2 for all the measurements and we used black 96 well plates with flat, transparent bottom.</p>
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<p style="padding-left:.5em; margin-top:.5em;">4. Every piece of material that has been in direct contact with blood should be disposed of correctly via the clinical waste route when work activities are completed.</p>
<p style="margin-top: .6em; font-weight: bolder;"></br>&bull; OD600 Reference Point</p>
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<p style="padding-left:.5em; margin-top:.5em;">5. All persons processing blood specimens should wear gloves. Gloves should be changed, and hands washed after completion of specimen processing.</p>
<p style="padding-left: 1em;"></br>Add 100 &micro;l LUDOX into wells A1, B1, C1, D1 and 100 &micro;l of H2O into wells A2, B2, C2, D2. Then measure absorbance at 600 nm of all samples in all standard measurement modes in instrument and turn off the pathlength correction at the same time. The temperature setting was 26.6&deg;C. Record the data.</p>
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<p style="padding-left:.5em; margin-top:.5em;">6. Use of sharp objects (e.g. needles, syringes, scissors) should be limited for fear of injuries and cross infection.</p>
<p style="margin-top: .6em; font-weight: bolder;"></br>&bull; Particle Standard Curve</p>
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<p style="padding-left:.5em; margin-top:.5em;">7. All persons should wash their hands after completing blood specimens’ processing.</p>
<p style="padding-left: 1em;"></br>Obtain the tube labeled &ldquo;Silica Beads&ldquo; from the InterLab test kit and vortex vigorously for 30 seconds. Then immediately pipet 96 &micro;L microspheres into a 1.5 mL eppendorf tube. Add 904 &micro;L of ddH2O to the microspheres and vortex well.</p>
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<p style="padding-left: 1em;"></br>Prepare the serial dilution of microspheres as shown below. Set 4 copies.</p>
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<h5>Fig.1 Dilution of microspheres <sup style="font-family: .8em;">[1]</sup></h5>
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<p style="padding-left: 1em;"></br>Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6&deg;C. Record the data.</p>
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<p style="margin-top: .6em; font-weight: bolder;"></br>&bull; Fluorescence standard curve</p>
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<p style="padding-left: 1em;"></br>Spin down fluorescein stock tube. Prepare 10x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM.</p>
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<p style="padding-left: 1em;"></br>Prepare the serial dilutions of fluorescein as shown below. Set 4 copies.</p>
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<h5>Fig.2 Dilution of fluorescein <sup style="font-family: .8em;">[1]</sup></h5>
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<p style="padding-left: 1em;"></br>Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6&deg;C. Record the data.</p>
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<span class="psg_subtitle">Cell measurement</span>
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<p style="padding-left: 1em;">Make a 1:10 dilution of of the overnight cultures prepared after colony selection in LB medium + Chloramphenicol and measure Abs 600 of these 1:10 diluted cultures. Then dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber or covered with foil to block light). Incubate the cultures at 37&deg;C and 220 rpm. Take 500 µL samples of the cultures from each of the 8 devices, two colonies per device, at 0 and 6 hours of incubation and add them into 96 well plates as shown below. Place samples on ice before measurements.</p>
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<h5>Fig.3 Loading samples <sup style="font-family: .8em;">[1]</sup></h5>
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<p style="padding-left: 1em;"></br>Measure the samples (Abs 600 and fluorescence measurement). The cell measurement was under the same condition with the particle standard curve and the fluorescence standard curve, using the same plate.</p>
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<span class="psg_subtitle">Counting colony-forming units (CFUs)</span>
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<p>Measure the OD600 of cell cultures, making sure to dilute to the linear detection range of the plate reader. Then dilute the overnight culture to OD600 = 0.1 in 1 mL of LB + Cam media. Do this in triplicate for each culture and check the OD600 to make sure it is 0.1.</p>
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<p></br>Do the following serial dilutions as blow.</p>
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<h5>Fig.4 Dilutions <sup style="font-family: .8em;">[1]</sup></h5>
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<p></br>Count the colonies on each plate with fewer than 300 colonies. And multiple the colony count by the final dilution factor on each plate to get the colony forming units (CFU) per 1mL of an OD600 = 0.1 culture.</p>
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<span class="psg_ttl">Results</span>
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<span class="psg_subtitle">OD600 Reference point</span>
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<h5>Tab.1 OD600 reference point</h5>
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<span class="psg_subtitle">Particle Standard Curve</span>
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<h5>Fig.5 Particle standard curve 1 | Fig.6 Particle standard curve 2</h5>
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<span class="psg_subtitle">Fluorescence standard curve</span>
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<h5>Fig.7 Fluorescence standard curve 1 | Fig.8 Fluorescence standard curve 2</h5>
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<span class="psg_subtitle">Cell measurement</span>
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<h5>Tab.2 Raw plate reader measurements of fluorescence raw at 0 Hour</h5>
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<h5>Tab.3 Raw plate reader measurements of fluorescence raw at 6 Hour</h5>
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<h5>Tab.4 Raw data of Abs600 measurement at 0 hour</h5>
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<h5>Tab.5 Raw data of Abs600 measurement at 6 hour</h5>
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<p></br>The results of the CFUs and the flow cytometry data have been submitted in time by online forms and a zip file.</p>
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<span class="psg_ttl">Discussion</span>
 
<p></br>Our experiments have got a great result, showing that the protocol is rather detailed and easy to operate. We are pleased to share our data with other teams around the world and we sincerely hope the Interlab study this year goes well.</p>
 
<span class="psg_ttl">References</span>
 
<p>[1] <a style="color: #131124;" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf</a></p>
 
<p>[2] <a style="color: #131124;" href="https://2018.igem.org/Measurement/InterLab">https://2018.igem.org/Measurement/InterLab</a></p>
 
<p>[3] <a style="color: #131124;" href="https://2018.igem.org/Measurement/InterLab/Plate_Reader">https://2018.igem.org/Measurement/InterLab/Plate_Reader</a></p>
 
<p>[4] <a style="color: #131124;" href="https://2018.igem.org/Measurement/InterLab/Flow_Cytometry">https://2018.igem.org/Measurement/InterLab/Flow_Cytometry</a></p>
 
 
 
 
 
 
 
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<span class="psg_ttl">General Safety</span>
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<p>Working with living modified organisms is always a risk. All standard precautions must be taken. Before starting to work in the lab, every member of our team was asked to attend laboratory safety lesson and take an biosafety examination.</p>
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<span class="psg_ttl psg_subtitle">Wearing</span>
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<p>Everyone wears protective lab coats, enclosed leather shoes, gloves and safety glasses before laboratory activities.</br></p>
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<img src="https://static.igem.org/mediawiki/2018/0/01/T--ZJU-China--Safety1.jpg" style="width:500px">
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<h5 style="color: black;">Fig.1 Working with the Clean Bench</h5>
  
  
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<span class="psg_ttl psg_subtitle">Storage</span>
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<p>The chemical and biological reagents are stored in the proper place and suitable temperature. Hazardous or corrosive chemicals are stored separately.</br></p>
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<img src="https://static.igem.org/mediawiki/2018/2/2b/T--ZJU-China--Safety2.jpg" style="width:500px">
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<h5 style="color: black;">Fig.2 Storage for chemical and biological reagents </h5>
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<span class="psg_ttl psg_subtitle">Waste</span>
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<p>Waste should be classified and disposed of separately.</p>
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<img src="https://static.igem.org/mediawiki/2018/a/ae/T--ZJU-China--waste.png" style="width:500px">
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<h5 style="color: black;">Fig.3 Flow chart of waste  disposal </h5>
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<span class="psg_ttl psg_subtitle">Safety Apparatus</span>
 +
<p>Our iGEM lab is equipped with safety apparatus such as fire extinguishers, emergency showers, eyewash stations, and first aid kits in case of dire situations.</br> </p>
 +
<img src="https://static.igem.org/mediawiki/2018/d/da/T--ZJU-China--Safety3.JPG" style="width:500px">
 +
<h5 style="color: black;">Fig.4 The fire extinguisher</h5>
 +
<img src="https://static.igem.org/mediawiki/2018/5/50/T--ZJU-China--Safety4.jpg" style="width:500px">
 +
<h5 style="color: black;">Fig.5 The fire blanket</h5>
 +
<img src="https://static.igem.org/mediawiki/2018/0/07/T--ZJU-China--Safety5.JPG" style="width:500px">
 +
<h5 style="color: black">Fig.6 The emergency shower</h5>
 +
<img src="https://static.igem.org/mediawiki/2018/0/01/T--ZJU-China--Safety6.JPG" style="width:500px">
 +
<h5 style="color: black;">Fig.7 The eyewash equipment</h5>
 +
<img src="https://static.igem.org/mediawiki/2018/0/02/T--ZJU-China--Safety7.jpg" style="width:500px">
 +
<h5 style="color: black;">Fig.8 The first-aid case</h5>
 +
 +
<span class="psg_ttl">References</span>
 +
<p style="margin-top:.5em;">[1] Michelle M. Barnhart Matthew R. Chapman. Curli Biogenesis and Function. Annual Review of Microbiology(2006):131-147</p>
 +
<p style="margin-top:.5em;">[2] CDC.Recommendations for Prevention of HIV Transmission in Health-Care Settings.MMWR,1987;36</p>
 +
<p style="margin-top:.5em;">[3] Z Botyanszki, PKR Tay, PQ Nguyen, MG Nussbaumer,  NS Joshi.Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology & Bioengineering, 2015, 112 (10) :2016-2024</p>
 
 
<!--<img src="https://static.igem.org/mediawiki/2018/5/54/T--ZJU-China--ip01.png" style="width: 80%;"/>-->
 
<table class="table">
 
<thead><tr>
 
<th><span>Part Number</span></th>
 
<th><span>Relative Strength</span></th>
 
</tr></thead>
 
<tbody>
 
<tr>
 
<td><span>BBa_R0085(wild type)</span></td>
 
<td><span>1</span></td>
 
</tr>
 
<tr>
 
<td><span>BBa_K2721000</span></td>
 
<td><span>20.99</span></td>
 
</tr>
 
<tr>
 
<td><span>BBa_K2721001</span></td>
 
<td><span>17.75</span></td>
 
</tr>
 
<tr>
 
<td><span>BBa_K2721002</span></td>
 
<td><span>7.63</span></td>
 
</tr>
 
<tr>
 
<td><span>BBa_K2721003</span></td>
 
<td><span>13.92</span></td>
 
</tr>
 
</tbody>
 
</table>
 
 
</br></br>
 
</br></br>
 
<!---->
 
<!---->

Latest revision as of 02:50, 18 October 2018

Improve Part
SAFETY 

Our project aims to detect the injury based on a cell-free device with three connected enzymes which fixed on a biofilm scaffold. We use blood specimens with biochemical analysis to calibrate our device and help us with our machine learning modeling to set a diagnosis criterion.



Project Safety Biofilm Scaffold

Our biofilm scaffold consists of curli which is the protein found on the surface of E.Coli. Curli may facilitate bacteria’s invasion into host cells and activate corresponding cytokines and inflammatory mediators in plasma. But the system we wish to produce will be ultimately cell-free and no living organisms will be included. Besides, curli will be immobilized and covered by Nafion on the electrode, so contact with curli will not happen. Therefore, biofilm scaffold is relatively safe.

Blood Samples

We have gotten approval from our university authority and collected some blood samples from SIR RUN RUN SHAW HOSPITAL, which is classified as a Grade 3 Class A hospital by Chinese government. The procedure of blood sampling was done by medical personnel using ideal blood collection equipment. Besides, our experiment was conducted under professional doctors' supervision and waste was collected by the specialties in the hospital. (It is also an essential part of Integrated Human Practice.)


Click here to see the offical collaboration protocol.


Click here to see the approval from National Demonstration Center for Experimental Biology Education(Zhejiang University).


Our project meets ethical requirements. Blood samples we got from hospital are all remaining samples after satisfying patients’ pathological diagnosis need. We keep secret of patients’ privacy information. We conduct experiments with clinical material following our country's laws and our university's rules.


The main issue for work with blood samples is the potential for infection. Clinical samples may be contaminated with pathogens. The high risk, well known viral agents such as Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV) and Hepatitis C Virus (HCV) are not the only agents that may be present in blood. Other viruses such as HTLV1 and B19 as well as various bacterial agents may also be present. Regarding the likely incidence of a pathogen in blood samples, several factors should be considered. These include known medical history of a patient or donor, whether the samples are from individuals showing clinical symptoms of infectious disease, the incidence of the various pathogens that are endemic in the local population or donor group and the type of sample. Fortunately, SIR RUN RUN SHAW HOSPITAL offered us the physiological indices of blood samples and other relevant information. So, the samples we used are from formal approach and relatively less dangerous. To minimize the risks,we have made Standard Operating Procedure (SOP) which is strictly followed in our iGEM lab.

Standard Operating Procedure (SOP)

As insurance, Blood samples from all patients should be considered infective. The following precautions followed in our lab are also recommended for all health-care workers in clinical laboratories.

1. All the experiments where blood is involved must be conducted in a separate room within the laboratory.

2. All specimens of blood should be put in a well- constructed container with a secure lid to prevent leaking. Care should be taken when dealing with each specimen to avoid contaminating the outside of the container and of the laboratory form accompanying the specimen.

3. Lab surface including correlated equipment is decontaminated with chemical disinfectant prior and after disposal.

4. Every piece of material that has been in direct contact with blood should be disposed of correctly via the clinical waste route when work activities are completed.

5. All persons processing blood specimens should wear gloves. Gloves should be changed, and hands washed after completion of specimen processing.

6. Use of sharp objects (e.g. needles, syringes, scissors) should be limited for fear of injuries and cross infection.

7. All persons should wash their hands after completing blood specimens’ processing.

General Safety

Working with living modified organisms is always a risk. All standard precautions must be taken. Before starting to work in the lab, every member of our team was asked to attend laboratory safety lesson and take an biosafety examination.

Wearing

Everyone wears protective lab coats, enclosed leather shoes, gloves and safety glasses before laboratory activities.

Fig.1 Working with the Clean Bench
Storage

The chemical and biological reagents are stored in the proper place and suitable temperature. Hazardous or corrosive chemicals are stored separately.

Fig.2 Storage for chemical and biological reagents
Waste

Waste should be classified and disposed of separately.

Fig.3 Flow chart of waste disposal
Safety Apparatus

Our iGEM lab is equipped with safety apparatus such as fire extinguishers, emergency showers, eyewash stations, and first aid kits in case of dire situations.

Fig.4 The fire extinguisher
Fig.5 The fire blanket
Fig.6 The emergency shower
Fig.7 The eyewash equipment
Fig.8 The first-aid case
References

[1] Michelle M. Barnhart Matthew R. Chapman. Curli Biogenesis and Function. Annual Review of Microbiology(2006):131-147

[2] CDC.Recommendations for Prevention of HIV Transmission in Health-Care Settings.MMWR,1987;36

[3] Z Botyanszki, PKR Tay, PQ Nguyen, MG Nussbaumer, NS Joshi.Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology & Bioengineering, 2015, 112 (10) :2016-2024