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

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<title>2017.igem.org/Team:XMU-China/Safety</title>
 
<title>2017.igem.org/Team:XMU-China/Safety</title>
 
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<link href="https://2017.igem.org/Team:XMU-China/css/SafetyCss?action=raw&ctype=text/css" rel="stylesheet" type="text/css">
 
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<span class="subtitle" id="subtitleone">Introduction</span>
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<span class="subtitle" id="subtitleone">Safe&nbsp;Project&nbsp;Design</span>
 
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<p>The goal of the interlab study is to explore a major question: How close can the numbers be when fluorescence is measured all around the world? So we measure GFP fluorescence in our lab with plate reader (Tecan Infinite M200pro). This year, three devices and one positive control and one negative control were provided by the Registry. The results show increased fluorescence in the stronger promoter expected.</p><br />
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<p>Firstly, E. coli BL21 and E. coli DH5α were the only chassis when we performed the molecular cloning and expression. Our bacteria will be engineered to detect metal ions, e.g., cobalt and mercury. We propose to construct an intermediate to normalize the response level of various ions. An induced expression system will be used to amplify the signal response to those ions. All the plasmids are harmless for humans and none of our parts would raise any safety issue according to the current expertise. Besides, the main part of our product is reusable. </p><br />
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<p>After the experiment, five required devices were created:<br />
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Positive control: I20270 in pSB1C3;<br />
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Negative control: R0040 in pSB1C3;<br />
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Test Device 1: J23101.BCD2.E0040.B0015 in pSB1C3;<br />
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Test Device 2: J23106.BCD2.E0040.B0015 in pSB1C3;<br />
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Test Device 3: J23117.BCD2.E0040.B0015 in pSB1C3;<br />
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Test Device 4: J23101+I13504 in pSB1C3;<br />
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Test Device 5: J23106+I13504 in pSB1C3;<br />
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Test Device 6: J23117+I13504 in pSB1C3.</p>
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<span class="subtitle"id="subtitletwo">Protocol</span>
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<span class="subtitle"id="subtitletwo">Safe&nbsp;Lab&nbsp;Work</span>
 
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<h1>I.&nbsp;Materials</h1>
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<p>The building of laboratory include the location of emergency exits, emergency ventilation system, fire-fighting equipment , first aid kit and so on. Laboratory coat and gloves are necessary when entering the laboratory. All culture fluid and culture medium with bacteria were processed properly to prevent the potential biological pollution. We handled all the biological materials, wastes and equipment strictly following the BSL-1 requirements. Turn off the water and electricity, close the door, window and door when we leave the lab.</p>
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<p>•&nbsp;Competent cells ( Escherichia coli strain DH5α)<br />
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•&nbsp;LB (Luria Bertani) media<br />
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•&nbsp;Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)<br />
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•&nbsp;50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)<br />
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•&nbsp;Incubator at 37°C<br />
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•&nbsp;1.5 ml eppendorf tubes for sample storage<br />
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•&nbsp;Ice bucket with ice<br />
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•&nbsp;Pipettes</p>
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<h1>II.&nbsp;Cell measurement protocol</h1>
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<p>1.Transform 8 plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.<br />
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2.Pick 2 colonies from each of plate and inoculate it on 10mL LB medium and Chloramphenicol. Grow the cells for 16hrs at 37°C and 220rpm.<br />
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3.Cell growth, sampling, and assay.<br />
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4.Obtain initial OD 600 measurement of overnight cultures. Then dilute each sample to an OD of 0.02.<br />
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5.Incubate the cultures at 37°C and 220rpm. Take 100µL (1% of total volume) samples of the cultures at 0, 2, 4 and 6 hours of incubation.<br />
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6.At the end of sampling point measure these samples (OD and Fl measurement).<br />
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7.Measurements of absorbance and fluorescence: <br />
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(1) OD 600<br />
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Device: Plate Reader（Tecan Infinite M200pro） Wavelengths: 600 nm absorption. <br />
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(2) Fluorescence<br />
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Device: Plate Reader（Tecan Infinite M200pro）, 96-well plates. Wavelengths: 485 nm excitation, 530 nm emission.</p>
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<span class="subtitle" id="subtitlethree">Measurements</span>
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<span class="subtitle" id="subtitlethree">Safe&nbsp;Shipment</span>
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<p>We sent our BioBricks through the standard shipping process required by iGEM.</p><br />
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<h1>I.&nbsp;OD 600 Reference point</h1>
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<span class="tableimg"><img class="tableone" src="#"></span><br />
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<span class="table">Table 1. Absorbance at 600nm for LUDOX and H2O</span> 
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<h1>II.&nbsp;Fluorescein standard curve</h1>
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<span class="table">Table 2. Fluorescence measurements for fluorescein stock solution at decreasing concentration</span><br /><br />
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<span class="tableimg"><img class="tablethree" src="https://static.igem.org/mediawiki/2017/d/dc/T--XMU-China--Interlab-Table3.png"></span><br />
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<span class="table">Figure 3. The Fluorescein Standard Curve in plate reader with 485nm excitation, 530nm emission</span>
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Revision as of 15:09, 8 October 2017

2017.igem.org/Team:XMU-China/Safety

Safety
Safe Project Design

Firstly, E. coli BL21 and E. coli DH5α were the only chassis when we performed the molecular cloning and expression. Our bacteria will be engineered to detect metal ions, e.g., cobalt and mercury. We propose to construct an intermediate to normalize the response level of various ions. An induced expression system will be used to amplify the signal response to those ions. All the plasmids are harmless for humans and none of our parts would raise any safety issue according to the current expertise. Besides, the main part of our product is reusable.


Safe Lab Work

The building of laboratory include the location of emergency exits, emergency ventilation system, fire-fighting equipment , first aid kit and so on. Laboratory coat and gloves are necessary when entering the laboratory. All culture fluid and culture medium with bacteria were processed properly to prevent the potential biological pollution. We handled all the biological materials, wastes and equipment strictly following the BSL-1 requirements. Turn off the water and electricity, close the door, window and door when we leave the lab.

Safe Shipment

We sent our BioBricks through the standard shipping process required by iGEM.