Difference between revisions of "Team:NCTU Formosa/Protocol"
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| − | + | {{NCTU Formosa/Navigation}} {{NCTU Formosa}} | |
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<head> | <head> | ||
<meta charset="UTF-8"> | <meta charset="UTF-8"> | ||
| − | <title> | + | <title>Untitled Document</title> |
| − | <script src=" | + | <script src="//ajax.googleapis.com/ajax/libs/jquery/3.2.1/jquery.min.js"></script> |
| − | < | + | <link href="template.css" rel="stylesheet" type="text/css"> |
| − | < | + | <script src="template.js" type="text/javascript"></script> |
<meta name="viewport" content="width=device-width, initial-scale=1.0, user-scalable=no, minimum-scale=1.0, maximum-scale=1.0" /> | <meta name="viewport" content="width=device-width, initial-scale=1.0, user-scalable=no, minimum-scale=1.0, maximum-scale=1.0" /> | ||
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body { | body { | ||
margin: 0; | margin: 0; | ||
| Line 2,015: | Line 57: | ||
.subtitle>h5{ | .subtitle>h5{ | ||
| − | |||
font-size: 1.95em; | font-size: 1.95em; | ||
position: relative; | position: relative; | ||
| Line 2,121: | Line 162: | ||
/*----------------------------------------------------------------------------*/ | /*----------------------------------------------------------------------------*/ | ||
| − | + | </style> | |
| − | <!-----------------------------------------------------------------------------> | + | <!-----------------------------------------------------------------------------> |
| − | + | <script> | |
$(document).ready(function(){ | $(document).ready(function(){ | ||
$(".show_pic").click(function(){ | $(".show_pic").click(function(){ | ||
| Line 2,278: | Line 319: | ||
}); | }); | ||
| − | + | ||
| − | <!-----------------------------------------------------------------------------> | + | </script> |
| + | <!-----------------------------------------------------------------------------> | ||
</head> | </head> | ||
| + | |||
<body> | <body> | ||
| Line 2,300: | Line 343: | ||
<div class="show"> | <div class="show"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To prepare solid medium to civilize the fungi </p> |
<div><img class="show_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | <div><img class="show_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | ||
</div> | </div> | ||
<div class="hide"> | <div class="hide"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To prepare solid medium to civilize the fungi </p> |
<h1>Drugs and equipment:</h1> | <h1>Drugs and equipment:</h1> | ||
<ul> | <ul> | ||
| Line 2,318: | Line 361: | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li>The ratio of PDA powder and | + | <li>The ratio of PDA powder and ddH2O is 39g : 1000mL. To prevent the liquid spill out from Serum bottles, we usually have only 300mL of the liquid in a 500mL Serum bottle. </li> |
<li>Loosen the bottle cap for a bit, and put it into the autoclave. </li> | <li>Loosen the bottle cap for a bit, and put it into the autoclave. </li> | ||
<li>Screw the bottle cap tight to cool down. If the liquid becomes solid, heat it up with a microwave oven. </li> | <li>Screw the bottle cap tight to cool down. If the liquid becomes solid, heat it up with a microwave oven. </li> | ||
| Line 2,331: | Line 374: | ||
<div class="subtitle"> | <div class="subtitle"> | ||
<h6>Experiment:</h6> | <h6>Experiment:</h6> | ||
| − | <h5>- | + | <h5>- Dual culture technique </h5> |
</div> | </div> | ||
<div class="exp2"> | <div class="exp2"> | ||
<div class="show2"> | <div class="show2"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To test the antifungal activity of peptides comparing to negative control.</p> |
<div><img class="show2_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | <div><img class="show2_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | ||
</div> | </div> | ||
<div class="hide2"> | <div class="hide2"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To test the antifungal activity of peptides comparing to negative control. </p> |
<h1>Drugs and equipment:</h1> | <h1>Drugs and equipment:</h1> | ||
<ul> | <ul> | ||
| − | <li> | + | <li>Drugs (ex: NaN<sub>3</sub>, peptides…)</li> |
| − | <li> | + | <li>Hole puncher (tips are recommended)</li> |
| + | <li>PDA (Potato dextrose agar) plate</li> | ||
| + | <li>Fungi plates</li> | ||
| + | <li>Parafilm</li> | ||
<li>Alcohol burner</li> | <li>Alcohol burner</li> | ||
| − | <li> | + | <li>Tweezers</li> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</ul> | </ul> | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li> | + | <li>Culture some plates of fungi. </li> |
| − | <li> | + | <li>Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.</li> |
| − | <li> | + | <li>Pick up fungi on the outer cycle (peripheral part) of a plate (the latest part of fungi plate) with tip. Dig into the previous PDA plate and pick up a piece of agar with fungi, remove it onto a new PDA plate, put the piece in the center of the plate.</li> |
| − | <li>Disinfect | + | <li>Disinfect tools with an alcohol burner. Seal up the plate with Parafilm, and label on the name of fungi, date and so on. </li> |
| − | <li>Remove the | + | <li>Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.</li> |
| − | <li> | + | <li>Dig holes on the plate with tweezers, the position of the holes should be 0.5cm far away along the radius from the position of latest mycelium as the picture shows.</li> |
| − | + | <li>Introduce the testing drug into the hole (different concentration of peptides, HEPES for our experiments)</li> | |
| − | <li> | + | <li>Disinfect tools with an alcohol burner. Seal up the plate with Parafilm.</li> |
| − | <li> | + | <li>Observe how the mycelium grow</li> |
| − | + | ||
| + | |||
</ol> | </ol> | ||
| + | |||
<div><img class="hide2_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div> | <div><img class="hide2_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div> | ||
</div> | </div> | ||
| Line 2,380: | Line 423: | ||
<div class="show3"> | <div class="show3"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To test the concentration of spore suspension liquid and calculate germination rate.</p> |
<div><img class="show3_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | <div><img class="show3_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | ||
</div> | </div> | ||
<div class="hide3"> | <div class="hide3"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To test the concentration of spore suspension liquid and calculate germination rate.</p> |
<h1>Drugs and equipment:</h1> | <h1>Drugs and equipment:</h1> | ||
<ul> | <ul> | ||
| Line 2,397: | Line 440: | ||
<li>Fungi plates</li> | <li>Fungi plates</li> | ||
<li>Glass Cell Spreaders</li> | <li>Glass Cell Spreaders</li> | ||
| − | <li> | + | <li>10μl pipet</li> |
<li>Gauze</li> | <li>Gauze</li> | ||
<li>Centrifuge</li> | <li>Centrifuge</li> | ||
| Line 2,410: | Line 453: | ||
<ol> | <ol> | ||
<li>Choose fungi plates that have produced spores.</li> | <li>Choose fungi plates that have produced spores.</li> | ||
| − | <li>(It is recommended to experiment in a laminar flow (hood). Add | + | <li>(It is recommended to experiment in a laminar flow (hood)). Add ddH2O to the plate until water covers the surface of the plate.</li> |
<li>Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.</li> | <li>Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.</li> | ||
<li>Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension liquid with unknown concentration.</li> | <li>Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension liquid with unknown concentration.</li> | ||
| − | <li>Clean the Hemocytometer with 75% alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10mL spore suspension liquid from the tiny chamber beside. | + | <li>Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10mL spore suspension liquid from the tiny chamber beside. |
The spore suspension should cover all the square of the Hemocytometer. </li> | The spore suspension should cover all the square of the Hemocytometer. </li> | ||
<li>Put the Hemocytometer under a microscope, and observe the spore.</li> | <li>Put the Hemocytometer under a microscope, and observe the spore.</li> | ||
<li>Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with concentration | <li>Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with concentration | ||
you know. We would like to prepare spore suspension liquid with the concentration of 1.5*10^5 spores/mL for our experiments.</li> | you know. We would like to prepare spore suspension liquid with the concentration of 1.5*10^5 spores/mL for our experiments.</li> | ||
| − | <li>Mixed | + | <li>Mixed 5μl spore suspension liquid with 5μl 2% glucose solution and 5μl peptide together into a PCR tube (It is recommended for pipetting 50 times to ensure that there is a mix of uniform).</li> |
| − | <li>Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some | + | <li>Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate |
easily.</li> | easily.</li> | ||
| − | + | ||
<li>After incubating under 20℃ for 6 hours, we observe and classify the spores into four grades according to the length of the germination tube, calculate the percentage of each germination grade of each sample.</li> | <li>After incubating under 20℃ for 6 hours, we observe and classify the spores into four grades according to the length of the germination tube, calculate the percentage of each germination grade of each sample.</li> | ||
| − | + | ||
</ol> | </ol> | ||
<div><img class="hide3_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div> | <div><img class="hide3_pic" src="https://static.igem.org/mediawiki/2017/c/cb/Ptp_show.png" style="display:block; margin:auto;"></div> | ||
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<div class="show4"> | <div class="show4"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To separate infected plant tissue and cultivate pathogen fungi</p> |
<div><img class="show4_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | <div><img class="show4_pic" src="https://static.igem.org/mediawiki/2017/4/4f/Ptp_hide.png" style="display:block; margin:auto;"></div> | ||
</div> | </div> | ||
<div class="hide4"> | <div class="hide4"> | ||
<h1>Purpose:</h1> | <h1>Purpose:</h1> | ||
| − | <p> & | + | <p> To separate infected plant tissue and cultivate pathogen fungi</p> |
<h1>Drugs and equipment:</h1> | <h1>Drugs and equipment:</h1> | ||
<ul> | <ul> | ||
| Line 2,456: | Line 499: | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li>Soak the plant tissue to sterilization: All parts of | + | <li>Soak the plant tissue to sterilization: All parts of the plant should soak in alcohol for 1 minute. For leaves, skim over NaClO for a few times; for roots, soak in NaClO for 1 minute; for mature stem parts, soak in NaClO for 20~30 |
seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH<sub>2</sub>O.</li> | seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH<sub>2</sub>O.</li> | ||
<li>Remove all the drugs and tools inside the Hood, disinfect knives and tweezers with an alcohol burner. You may take a PDA plate for cool down.</li> | <li>Remove all the drugs and tools inside the Hood, disinfect knives and tweezers with an alcohol burner. You may take a PDA plate for cool down.</li> | ||
| − | <li>Cut the infected plant tissue for an area of 5mm, remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which is put on one single plate depends on the range | + | <li>Cut the infected plant tissue for an area of 5mm<sup>2</sup>, remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which is put on one single plate depends on the range |
of cutting area.</li> | of cutting area.</li> | ||
<li>Seal up the plate with Parafilm, and label on the name of fungi, date and so on.</li> | <li>Seal up the plate with Parafilm, and label on the name of fungi, date and so on.</li> | ||
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<div class="subtitle"> | <div class="subtitle"> | ||
<h6>Experiment:</h6> | <h6>Experiment:</h6> | ||
| − | <h5> | + | <h5> <i>E. coli</i> DH5α growth rate in the arsenic solution</h5> |
</div> | </div> | ||
<div class="exp6"> | <div class="exp6"> | ||
| Line 2,536: | Line 537: | ||
<ol> | <ol> | ||
<br />Rpm: | <br />Rpm: | ||
| − | <li>Culture 3 tubes of 2mL <i>E. coli</i> overnight by 15mL centrifuge tubes. (2mL SOC, | + | <li>Culture 3 tubes of 2mL <i>E. coli</i> overnight by 15mL centrifuge tubes. (2mL SOC, 2uL AMP)</li> |
| − | <li>Put them to the same tube at the next day, then add 6mL of SOC broth (water bath 37°C for 5 min. before use) and | + | <li>Put them to the same tube at the next day, then add 6mL of SOC broth (water bath 37°C for 5 min. before use) and 12uL AMP for a 2-fold dilution.</li> |
<li>Take 5mL SOC broth for to a 15mL centrifuge tube for blank. </li> | <li>Take 5mL SOC broth for to a 15mL centrifuge tube for blank. </li> | ||
<li>Aliquot the bacterial broth into six 15mL centrifuge tubes (2mL for each), then culture for 2 hours.</li> | <li>Aliquot the bacterial broth into six 15mL centrifuge tubes (2mL for each), then culture for 2 hours.</li> | ||
| Line 2,546: | Line 547: | ||
<br />Rpm80: | <br />Rpm80: | ||
<li>100, 50, 30, 10, 1ppm arsenic solutions, use ddH2O in control group</li> | <li>100, 50, 30, 10, 1ppm arsenic solutions, use ddH2O in control group</li> | ||
| − | <li>Dilute the E. coli broth to 50 fold by SOC broth (0.6mL <i>E. coli</i> broth+0.3mL Arsenic solutuin+ | + | <li>Dilute the <i>E. coli</i> broth to 50 fold by SOC broth (0.6mL <i>E. coli</i> broth+0.3mL Arsenic solutuin+30uL AMP 29.1mL SOC broth）</li> |
<li>Measure OD at 0min. measure every 30 min. Blank is needed every time.</li> | <li>Measure OD at 0min. measure every 30 min. Blank is needed every time.</li> | ||
</ol> | </ol> | ||
| Line 2,575: | Line 576: | ||
<li>CP (1000 X)</li> | <li>CP (1000 X)</li> | ||
<li>Arsenic stock (10^5ppm)</li> | <li>Arsenic stock (10^5ppm)</li> | ||
| − | <li> | + | <li>15 mL tube, 50 mL tube</li> |
<li>96 well plate</li> | <li>96 well plate</li> | ||
</ul> | </ul> | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li>Culture 6 tubes of 2 mL <i>E. coli</i>(chelate) and 2 tubes of <i>E. coli</i>(GFP positive control) overnight by 15 mL centrifuge tubes ( | + | <li>Culture 6 tubes of 2 mL <i>E. coli</i>(chelate) and 2 tubes of <i>E. coli</i>(GFP positive control) overnight by 15 mL centrifuge tubes (2 mL SOC, 2uL CP)</li> |
<li>Prepare another 8 tubes and mark them as the tubes cultured last night</li> | <li>Prepare another 8 tubes and mark them as the tubes cultured last night</li> | ||
| − | <li>Perform a two-fold dilution for the tubes cultured last night and take out | + | <li>Perform a two-fold dilution for the tubes cultured last night and take out 2 mL bacteria liquid of each group to the corresponding tube prepared </li> |
| − | <li>Add | + | <li>Add 2uL CP in each tube.</li> |
<li>Culture the tubes for another two hours</li> | <li>Culture the tubes for another two hours</li> | ||
<li>Measure the OD of each tube (wavelenght 595) </li> | <li>Measure the OD of each tube (wavelenght 595) </li> | ||
| − | <li>Dilute all tubes to | + | <li>Dilute all tubes to OD 0.5 by LB. (More than )</li> |
<li>Prepare and mark 50 mL tubes as chart below. Culture the tubes.</li> | <li>Prepare and mark 50 mL tubes as chart below. Culture the tubes.</li> | ||
<li>Take out the tubes from incubator.</li><br /> | <li>Take out the tubes from incubator.</li><br /> | ||
| Line 2,595: | Line 596: | ||
<li>0.6 mL bacteria liquid OD=0.5</li> | <li>0.6 mL bacteria liquid OD=0.5</li> | ||
<li>0.3 mL arsenic solution</li> | <li>0.3 mL arsenic solution</li> | ||
| − | <li> | + | <li> 30uL CP</li> |
</ol> | </ol> | ||
<br />Step 10-18 should be performed after Xhr X=the time marked on the tube | <br />Step 10-18 should be performed after Xhr X=the time marked on the tube | ||
| − | <li>Centrifuge the | + | <li>Centrifuge the 50 mL tubes by 6000 g/10 min</li> |
<li>Take out the supernatant and preserve in -80°C</li> | <li>Take out the supernatant and preserve in -80°C</li> | ||
| − | <li>Add | + | <li>Add 10 mL PBS to each tube</li> |
<li>Centrifuge by 6000 g/10 min</li> | <li>Centrifuge by 6000 g/10 min</li> | ||
| − | <li> Abandon PBS supernatant and add in | + | <li> Abandon PBS supernatant and add in 15 mL ddH<sub>2</sub>O</li> |
<li>Separate the liquid into fifteen full eppendorfs. Centrifuge by 12000 g/5 min</li> | <li>Separate the liquid into fifteen full eppendorfs. Centrifuge by 12000 g/5 min</li> | ||
| − | <li>Abandon ddH<sub>2</sub>O supernatant and add in | + | <li>Abandon ddH<sub>2</sub>O supernatant and add in 10 mL ddH<sub>2</sub>O</li> |
<li> Centrifuge by 12000 g/5 min</li> | <li> Centrifuge by 12000 g/5 min</li> | ||
<li>Combine the bacteria into one tube</li> | <li>Combine the bacteria into one tube</li> | ||
| Line 2,631: | Line 632: | ||
<li>LB broth</li> | <li>LB broth</li> | ||
<li>Cp (1000 X)</li> | <li>Cp (1000 X)</li> | ||
| − | <li> | + | <li>15 mL tube</li> |
<li>96 well plate</li> | <li>96 well plate</li> | ||
<li>Pipette</li> | <li>Pipette</li> | ||
| Line 2,639: | Line 640: | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li>Culture three tubes of 2mL <i>E. coli</i> GFP biosensor with | + | <li>Culture three tubes of 2mL <i>E. coli</i> GFP biosensor with 2uL CP+ , and culture one tube of 2mL <i>E. coli</i> positive control with 2uL CP+ in the other tube.</li> |
<li>Put both tubes into 37°C incubator for 16hr </li> | <li>Put both tubes into 37°C incubator for 16hr </li> | ||
| − | <li>Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in | + | <li>Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in 2uL CP+ in each tube </li> |
<li>Culture the ten tubes for another 2 hours</li> | <li>Culture the ten tubes for another 2 hours</li> | ||
<li>Measure the Obstacle Density of the GFP biosensor and the positive control </li> | <li>Measure the Obstacle Density of the GFP biosensor and the positive control </li> | ||
<li>Prepare a tube that contains 12mL bacteria liquid, which has a 0.500 Obstacle Density </li> | <li>Prepare a tube that contains 12mL bacteria liquid, which has a 0.500 Obstacle Density </li> | ||
<ul> | <ul> | ||
| − | <li>Note: v=original bacteria amount( | + | <li>Note: v=original bacteria amount(uL) , X=LB amount (uL)</li> |
0.5=v*bacteria OD/v+X | 0.5=v*bacteria OD/v+X | ||
<br />v+X=3000 | <br />v+X=3000 | ||
| Line 2,652: | Line 653: | ||
<li>Prepare tubes as the chart below</li> | <li>Prepare tubes as the chart below</li> | ||
<img src="https://static.igem.org/mediawiki/2017/6/60/Protocol_GFP_1-1.png" width="800" height="250"> | <img src="https://static.igem.org/mediawiki/2017/6/60/Protocol_GFP_1-1.png" width="800" height="250"> | ||
| − | <br /><br /><li>Add in 4.85mL 37°C LB, 0.1mL bacteria liquid, 0.05mL Chinese medicine and | + | <br /><br /><li>Add in 4.85mL 37°C LB, 0.1mL bacteria liquid, 0.05mL Chinese medicine and 5uL CP+</li> |
<li>Put these tubes into 37°C incubator </li> | <li>Put these tubes into 37°C incubator </li> | ||
<li>Measure the Obstacle Density and Fluorescence of each tube after 4, 5, 6 hours.</li> | <li>Measure the Obstacle Density and Fluorescence of each tube after 4, 5, 6 hours.</li> | ||
| Line 2,663: | Line 664: | ||
<div class="subtitle"> | <div class="subtitle"> | ||
<h6>Experiment:</h6> | <h6>Experiment:</h6> | ||
| − | <h5>- Growth rate test of LacZ α biosensor on different concentration of | + | <h5>- Growth rate test of LacZ α biosensor on different concentration of As<sup>5+</sup></h5> |
</div> | </div> | ||
<div class="exp9"> | <div class="exp9"> | ||
| Line 2,733: | Line 734: | ||
<ol> | <ol> | ||
<li>Culture two tubes of <i>E. coli</i> LacZ ⍺ biosensor for four hours</li> | <li>Culture two tubes of <i>E. coli</i> LacZ ⍺ biosensor for four hours</li> | ||
| − | <li>Prepare twenty one | + | <li>Prepare twenty one 15mL tubes, mark them as below</li><br /> |
<img src="https://static.igem.org/mediawiki/2017/c/cb/Protocol_GFP_2-1.png" width="800" height="250"> | <img src="https://static.igem.org/mediawiki/2017/c/cb/Protocol_GFP_2-1.png" width="800" height="250"> | ||
| − | <li>Make each tube contain | + | <li>Make each tube contain 1900uL LB, bacteria liquid, 40uL Xgal and 20uL metal concentration </li> |
<li>Culture the twenty one tubes for 16 hours</li> | <li>Culture the twenty one tubes for 16 hours</li> | ||
<li>Take 200µL of the solution to the 96 well plate. </li> | <li>Take 200µL of the solution to the 96 well plate. </li> | ||
| Line 2,749: | Line 750: | ||
<div class="subtitle"> | <div class="subtitle"> | ||
<h6>Experiment:</h6> | <h6>Experiment:</h6> | ||
| − | <h5>- Specificity test of GFP biosensor in | + | <h5>- Specificity test of GFP biosensor in As<sup>5+</sup>, Cu<sup>2+</sup>, and Pb<sup>2+</sup></h5> |
</div> | </div> | ||
<div class="exp11"> | <div class="exp11"> | ||
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<li>SOC broth</li> | <li>SOC broth</li> | ||
<li>Cp (1000x)</li> | <li>Cp (1000x)</li> | ||
| − | <li>15 | + | <li>15 mL tube</li> |
<li>96 well plate</li> | <li>96 well plate</li> | ||
<li>Pipette</li> | <li>Pipette</li> | ||
| Line 2,774: | Line 775: | ||
<h1>Process:</h1> | <h1>Process:</h1> | ||
<ol> | <ol> | ||
| − | <li>Culture three tubes of 2mL <i>E. coli</i> GFP biosensor with | + | <li>Culture three tubes of 2mL <i>E. coli</i> GFP biosensor with 2uL CP<sup>+</sup> , and culture one tube of 2mL <i>E. coli</i> positive control with 2uL CP<sup>+</sup> in the other tube.</li> |
<li>Put both tubes into 37°C incubator for 16hr</li> | <li>Put both tubes into 37°C incubator for 16hr</li> | ||
| − | <li>Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in | + | <li>Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in 2uL CP<sup>+</sup> in each tube </li> |
<li>Culture the ten tubes for another 2 hours</li> | <li>Culture the ten tubes for another 2 hours</li> | ||
<li>Measure the Obstacle Density of the GFP biosensor and the positive control</li> | <li>Measure the Obstacle Density of the GFP biosensor and the positive control</li> | ||
| − | <li>Prepare a tube that contains | + | <li>Prepare a tube that contains 12mL bacteria liquid, which has a 0.500 Obstacle Density</li> |
<ul> | <ul> | ||
| − | <li>Note: v=original bacteria amount( | + | <li>Note: v=original bacteria amount(uL) , X=SOC amount (uL)</li> |
0.5=v*bacteria OD/v+X | 0.5=v*bacteria OD/v+X | ||
<br/>v+X=3000 | <br/>v+X=3000 | ||
| Line 2,787: | Line 788: | ||
<li>Prepare tubes as the chart below</li> | <li>Prepare tubes as the chart below</li> | ||
<img src="https://static.igem.org/mediawiki/2017/1/1f/Protocol_LacZ_2-1.png" width="800" height="250"><br /> | <img src="https://static.igem.org/mediawiki/2017/1/1f/Protocol_LacZ_2-1.png" width="800" height="250"><br /> | ||
| − | <li>Add in 4. | + | <li>Add in 4.8mL 37°C SOC, 0.1mL bacteria liquid, 0.05 mL ion solution and 5uL CP<sup>+</sup>.</li> |
<li>Put these tubes into 37°C incubator </li> | <li>Put these tubes into 37°C incubator </li> | ||
<li>Measure the Obstacle Density and Fluorescence of each tube after 4 hours.</li> | <li>Measure the Obstacle Density and Fluorescence of each tube after 4 hours.</li> | ||
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Latest revision as of 03:58, 2 November 2017
Wet Lab Protocol
Experiment:
- Preparation for Potato dextrose agar (PDA) plates
Purpose:
To prepare solid medium to civilize the fungi
Purpose:
To prepare solid medium to civilize the fungi
Drugs and equipment:
- PDA powder
- ddH2O
- Petri dishes
- Serum bottle
- Microwave oven
- Autoclave
- Alcohol burner
Process:
- The ratio of PDA powder and ddH2O is 39g : 1000mL. To prevent the liquid spill out from Serum bottles, we usually have only 300mL of the liquid in a 500mL Serum bottle.
- Loosen the bottle cap for a bit, and put it into the autoclave.
- Screw the bottle cap tight to cool down. If the liquid becomes solid, heat it up with a microwave oven.
- Bring the bottle into the Hood, disinfect bottleneck with alcohol burner. Pour out the liquid to plates, each plate can hold 15~20mL of the PDA liquid. Shake the plate smoothly and make the liquid surface flat.
- Keep the plates in normal temperature or in 4 °C refrigerator
Experiment:
- Dual culture technique
Purpose:
To test the antifungal activity of peptides comparing to negative control.
Purpose:
To test the antifungal activity of peptides comparing to negative control.
Drugs and equipment:
- Drugs (ex: NaN3, peptides…)
- Hole puncher (tips are recommended)
- PDA (Potato dextrose agar) plate
- Fungi plates
- Parafilm
- Alcohol burner
- Tweezers
Process:
- Culture some plates of fungi.
- Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.
- Pick up fungi on the outer cycle (peripheral part) of a plate (the latest part of fungi plate) with tip. Dig into the previous PDA plate and pick up a piece of agar with fungi, remove it onto a new PDA plate, put the piece in the center of the plate.
- Disinfect tools with an alcohol burner. Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
- Remove all the drugs and tools inside the Hood, disinfect tools with an alcohol burner. You may take a PDA plate for cooling tools down.
- Dig holes on the plate with tweezers, the position of the holes should be 0.5cm far away along the radius from the position of latest mycelium as the picture shows.
- Introduce the testing drug into the hole (different concentration of peptides, HEPES for our experiments)
- Disinfect tools with an alcohol burner. Seal up the plate with Parafilm.
- Observe how the mycelium grow
Experiment:
- Spore germination percentage
Purpose:
To test the concentration of spore suspension liquid and calculate germination rate.
Purpose:
To test the concentration of spore suspension liquid and calculate germination rate.
Drugs and equipment:
- 75% Alcohol
- 2% glucose solution
- ddH2O
- HEPES buffer
- Peptides
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- 10μl pipet
- Gauze
- Centrifuge
- Beaker*2
- Petri dishes
- Lens cleaning paper
- Microscope
- Slide glass with 2 Cavities
- Counter
Process:
- Choose fungi plates that have produced spores.
- (It is recommended to experiment in a laminar flow (hood)). Add ddH2O to the plate until water covers the surface of the plate.
- Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension liquid with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10mL spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with concentration you know. We would like to prepare spore suspension liquid with the concentration of 1.5*10^5 spores/mL for our experiments.
- Mixed 5μl spore suspension liquid with 5μl 2% glucose solution and 5μl peptide together into a PCR tube (It is recommended for pipetting 50 times to ensure that there is a mix of uniform).
- Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate easily.
- After incubating under 20℃ for 6 hours, we observe and classify the spores into four grades according to the length of the germination tube, calculate the percentage of each germination grade of each sample.
Experiment:
- Separation of infected plant tissue
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Drugs and equipment:
- 75% Alcohol
- 0.5%~1% Sodium hypochlorite (NaClO)
- ddH2O
- PDA (Potato dextrose agar) plate
- Parafilm
- Alcohol burner
- Plants
- Knifes
- Tweezers
Process:
- Soak the plant tissue to sterilization: All parts of the plant should soak in alcohol for 1 minute. For leaves, skim over NaClO for a few times; for roots, soak in NaClO for 1 minute; for mature stem parts, soak in NaClO for 20~30 seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH2O.
- Remove all the drugs and tools inside the Hood, disinfect knives and tweezers with an alcohol burner. You may take a PDA plate for cool down.
- Cut the infected plant tissue for an area of 5mm2, remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which is put on one single plate depends on the range of cutting area.
- Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
- Put the plate in the best environment to observe if the fungi or other species of microorganism grow.
Experiment:
E. coli DH5α growth rate in the arsenic solution
Purpose:
Measure the E. coli DH5α growth rate arsenic solution.
Purpose:
Measure the E. coli DH5α growth rate arsenic solution.
Drugs and equipment:
- DH5α pc DNA (ctrl) on LB agar plates
- SOC broth
- Amp (1000 X)
- 15 mL tube, 50 mL tube
- 96 well plate
Process:
- Culture 3 tubes of 2mL E. coli overnight by 15mL centrifuge tubes. (2mL SOC, 2uL AMP)
- Put them to the same tube at the next day, then add 6mL of SOC broth (water bath 37°C for 5 min. before use) and 12uL AMP for a 2-fold dilution.
- Take 5mL SOC broth for to a 15mL centrifuge tube for blank.
- Aliquot the bacterial broth into six 15mL centrifuge tubes (2mL for each), then culture for 2 hours.
- Measure OD 595
- Dilute to OD 0.312. More than 12mL
Rpm:
- 100, 50, 30, 10, 1ppm arsenic solutions, use ddH2O in control group
- Dilute the E. coli broth to 50 fold by SOC broth (0.6mL E. coli broth+0.3mL Arsenic solutuin+30uL AMP 29.1mL SOC broth）
- Measure OD at 0min. measure every 30 min. Blank is needed every time.
Rpm80:
Experiment:
- Growth curve of E. coli with fMT plasmid in arsenic solution of different concentration
Purpose:
Measure the time curve of E. coli chelating arsenic of 20 ppm.
Purpose:
Measure the time curve of E. coli chelating arsenic of 20 ppm.
Drugs and equipment:
- DH5α as plasmid on LB agar plates
- E. coli (chelate) on LB agar plates
- E. coli (GFP positive control) on LB agar plates
- LB broth
- CP (1000 X)
- Arsenic stock (10^5ppm)
- 15 mL tube, 50 mL tube
- 96 well plate
Process:
- Culture 6 tubes of 2 mL E. coli(chelate) and 2 tubes of E. coli(GFP positive control) overnight by 15 mL centrifuge tubes (2 mL SOC, 2uL CP)
- Prepare another 8 tubes and mark them as the tubes cultured last night
- Perform a two-fold dilution for the tubes cultured last night and take out 2 mL bacteria liquid of each group to the corresponding tube prepared
- Add 2uL CP in each tube.
- Culture the tubes for another two hours
- Measure the OD of each tube (wavelenght 595)
- Dilute all tubes to OD 0.5 by LB. (More than )
- Prepare and mark 50 mL tubes as chart below. Culture the tubes.
- Take out the tubes from incubator.
- 29.1 mL LB
- 0.6 mL bacteria liquid OD=0.5
- 0.3 mL arsenic solution
- 30uL CP
- Centrifuge the 50 mL tubes by 6000 g/10 min
- Take out the supernatant and preserve in -80°C
- Add 10 mL PBS to each tube
- Centrifuge by 6000 g/10 min
- Abandon PBS supernatant and add in 15 mL ddH2O
- Separate the liquid into fifteen full eppendorfs. Centrifuge by 12000 g/5 min
- Abandon ddH2O supernatant and add in 10 mL ddH2O
- Centrifuge by 12000 g/5 min
- Combine the bacteria into one tube
-
Each tube contains
Step 10-18 should be performed after Xhr X=the time marked on the tube
Experiment:
- Function test of GFP biosensor in different concentration of Chinese medicine
Purpose:
Test the fluorescence intensity of GFP biosensor in different concentration of Chinese medicine.
Purpose:
Test the fluorescence intensity of GFP biosensor in different concentration of Chinese medicine.
Drugs and equipment:
- DH5α pc DNA (ctrl) on agar plates
- LB broth
- Cp (1000 X)
- 15 mL tube
- 96 well plate
- Pipette
- As solution
- Chinese medicine
Process:
- Culture three tubes of 2mL E. coli GFP biosensor with 2uL CP+ , and culture one tube of 2mL E. coli positive control with 2uL CP+ in the other tube.
- Put both tubes into 37°C incubator for 16hr
- Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in 2uL CP+ in each tube
- Culture the ten tubes for another 2 hours
- Measure the Obstacle Density of the GFP biosensor and the positive control
- Prepare a tube that contains 12mL bacteria liquid, which has a 0.500 Obstacle Density
- Note: v=original bacteria amount(uL) , X=LB amount (uL) 0.5=v*bacteria OD/v+X
- Prepare tubes as the chart below
- Add in 4.85mL 37°C LB, 0.1mL bacteria liquid, 0.05mL Chinese medicine and 5uL CP+
- Put these tubes into 37°C incubator
- Measure the Obstacle Density and Fluorescence of each tube after 4, 5, 6 hours.
v+X=3000
Experiment:
- Growth rate test of LacZ α biosensor on different concentration of As5+
Purpose:
Test the growth rate of LacZ α biosensor on different concentration of As5+.
Purpose:
Test the growth rate of LacZ α biosensor on different concentration of As5+.
Drugs and equipment:
- DH5⍺ pc DNA(crtl) on agar plate
- SOC broth
- Cp (1000x)
- As5+ solution 100,1000,3000,5000,10000 ppm
- ddH2O
- 15mL tube
- 50mL tube
- 96 well plate
- pipet
Process:
- Take a 15mL tube. Culture 2mL E. coli LacZ ⍺ biosensor by SOC with 2µL Cp.
- Put the tube into 37°C incubator for 15 hr.
- Take 1mL of the bacteria liquid with 1mL SOC, and 2µL Cp into a new 15mL tube to perform a 2-fold solution.
- Put the tube into 37°C incubator for 2hr.
- Take 200µL of the solution to the 96 well plate.
- Measure OD595
- Dilute the bacteria liquid to OD=0.312/1.2mL
- Take 18 15mL tubes.(0ppm,1ppm,10ppm,30ppm,50ppm,100ppm. 3 tube for every concentration)
- Add 29.1mL SOC, 30µL Cp, 300µL As5+ solution, 600µL bacteria liquid to each tube.
- Measure OD595 every 30 minutes for 7.5 hours.
Experiment:
- LacZ ⍺ biosensor pretest
Purpose:
Test if the LacZ ⍺ biosensor can produce blue deposit in different As5+ condition.
Purpose:
Test if the LacZ ⍺ biosensor can produce blue deposit in different As5+ condition.
Drugs and equipment:
- DH5⍺ pc DNA(crtl) on agar plate
- LB broth
- Cp (1000x)
- X-gal
- As5+ solution 1000ppm, 10000ppm
- ddH2O
- 15mL tube
- pipet
- PBS
- 96 well plate
Process:
- Culture two tubes of E. coli LacZ ⍺ biosensor for four hours
- Prepare twenty one 15mL tubes, mark them as below
- Make each tube contain 1900uL LB, bacteria liquid, 40uL Xgal and 20uL metal concentration
- Culture the twenty one tubes for 16 hours
- Take 200µL of the solution to the 96 well plate.
- Measure OD595
- After culturing, centrifuge the bacteria liquid by12000G, 5min
- Remove the supernatant
Experiment:
- Specificity test of GFP biosensor in As5+, Cu2+, and Pb2+
Purpose:
Test the specificity of GFP biosensor in different concentration of As5+, Cu2+, and Pb2+ solutions.
Purpose:
Test the specificity of GFP biosensor in different concentration of As5+, Cu2+, and Pb2+ solutions.
Drugs and equipment:
- DH5⍺ pc DNA(crtl) on agar plate
- SOC broth
- Cp (1000x)
- 15 mL tube
- 96 well plate
- Pipette
- As5+ solution
- Cu2+ solution
- Pb2+ solution
Process:
- Culture three tubes of 2mL E. coli GFP biosensor with 2uL CP+ , and culture one tube of 2mL E. coli positive control with 2uL CP+ in the other tube.
- Put both tubes into 37°C incubator for 16hr
- Make a 2-fold dilution, separate the GFP biosensor into eight tubes and the positive control into two tubes. add in 2uL CP+ in each tube
- Culture the ten tubes for another 2 hours
- Measure the Obstacle Density of the GFP biosensor and the positive control
- Prepare a tube that contains 12mL bacteria liquid, which has a 0.500 Obstacle Density
- Note: v=original bacteria amount(uL) , X=SOC amount (uL) 0.5=v*bacteria OD/v+X
- Prepare tubes as the chart below
- Add in 4.8mL 37°C SOC, 0.1mL bacteria liquid, 0.05 mL ion solution and 5uL CP+.
- Put these tubes into 37°C incubator
- Measure the Obstacle Density and Fluorescence of each tube after 4 hours.
v+X=3000
