Shuimoliuyun (Talk | contribs) |
|||
Line 3: | Line 3: | ||
<head> | <head> | ||
− | + | <!-- Bootstrap Core CSS --> | |
− | + | <link href="https://cdn.bootcss.com/bootstrap/3.3.7/css/bootstrap.min.css" rel="stylesheet"> | |
− | + | <!-- Custom CSS --> | |
− | + | <link href="https://2017.igem.org/Template:NPU-China/css?action=raw&ctype=text/css" rel="stylesheet"> | |
− | + | <!-- Custom Fonts --> | |
− | + | <link href="https://cdn.bootcss.com/font-awesome/4.7.0/css/font-awesome.min.css" rel="stylesheet"> | |
− | + | <!-- jQuery --> | |
− | + | <script src="https://cdn.bootcss.com/jquery/3.2.1/jquery.min.js"></script> | |
− | + | <!-- Bootstrap Core JavaScript --> | |
− | + | <script src="https://cdn.bootcss.com/bootstrap/3.3.7/js/bootstrap.min.js"></script> | |
− | + | <!-- HTML5 Shim and Respond.js IE8 support of HTML5 elements and media queries --> | |
− | + | <!-- WARNING: Respond.js doesn't work if you view the page via file:// --> | |
− | + | <!--[if lt IE 9]> | |
<script src="https://oss.maxcdn.com/libs/html5shiv/3.7.0/html5shiv.js"></script> | <script src="https://oss.maxcdn.com/libs/html5shiv/3.7.0/html5shiv.js"></script> | ||
<script src="https://oss.maxcdn.com/libs/respond.js/1.4.2/respond.min.js"></script> | <script src="https://oss.maxcdn.com/libs/respond.js/1.4.2/respond.min.js"></script> | ||
<![endif]--> | <![endif]--> | ||
− | + | <style> | |
− | + | </style> | |
</head> | </head> | ||
− | <body> | + | <body data-spy="scroll" data-target="#myScrollspy"> |
+ | <!-- Navigation --> | ||
+ | <nav class="navbar navbar-inverse navbar-fixed-top" role="navigation"> | ||
+ | <div class="container"> | ||
+ | <div class="navbar-header"> | ||
+ | <button type="button" class="navbar-toggle" data-toggle="collapse" data-target="#bs-example-navbar-collapse-1"> | ||
+ | <span class="sr-only">Toggle navigation</span> | ||
+ | <span class="icon-bar"></span> | ||
+ | <span class="icon-bar"></span> | ||
+ | <span class="icon-bar"></span> | ||
+ | </button> | ||
+ | <a class="navbar-brand" href="https://2017.igem.org/Team:NPU-China"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/29/NPU-logo.png" style="max-width:50px;margin-top:-10px;"> | ||
+ | </a> | ||
+ | </div> | ||
+ | <div class="collapse navbar-collapse" id="bs-example-navbar-collapse-1"> | ||
+ | <ul class="nav navbar-nav navbar-right"> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China">Home</a> | ||
+ | </li> | ||
+ | <li class="dropdown"> | ||
+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Team | ||
+ | <b class="caret"></b> | ||
+ | </a> | ||
+ | <ul class="dropdown-menu"> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Aboutus">About us</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Attributions">Attributions</a> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="dropdown"> | ||
+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Project | ||
+ | <b class="caret"></b> | ||
+ | </a> | ||
+ | <ul class="dropdown-menu"> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Background">Background</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Description">Description</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Design">Design</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Model">Model</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Proofofconcept">Proof of concept</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Demonstrate">Demonstrate</a> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="dropdown"> | ||
+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Parts | ||
+ | <b class="caret"></b> | ||
+ | </a> | ||
+ | <ul class="dropdown-menu"> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/BasicParts">Basic Parts</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/CompositeParts">Composite Parts</a> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Hardware">Hardware</a> | ||
+ | </li> | ||
+ | <li class="dropdown"> | ||
+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">HP | ||
+ | <b class="caret"></b> | ||
+ | </a> | ||
+ | <ul class="dropdown-menu"> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/HP/Silver">Silver</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/HP/Gold_Integrated">Gold</a> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Collaborations">Collaborations</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Achievements">Achievements</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/InterLab">InterLab</a> | ||
+ | </li> | ||
− | + | <li class="dropdown active"> | |
− | + | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Notebook | |
− | + | <b class="caret"></b> | |
− | + | </a> | |
− | + | <ul class="dropdown-menu"> | |
− | + | <li> | |
− | + | <a href="https://2017.igem.org/Team:NPU-China/Labnotes">Labnotes</a> | |
− | + | </li> | |
− | + | <li> | |
− | + | <a href="https://2017.igem.org/Team:NPU-China/Protocols">Protocols</a> | |
− | + | </li> | |
− | + | </ul> | |
− | + | </li> | |
− | + | </ul> | |
− | + | </div> | |
− | + | </div> | |
− | + | </nav> | |
− | + | <div class="batu" style="background: url('https://static.igem.org/mediawiki/2017/f/fe/Npu-background.png') no-repeat fixed; overflow: hidden;"> | |
− | + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/3/3c/%E9%A2%98%E7%9B%AE%E9%80%9A%E6%A0%8Fdemonstrate.jpg"> | |
− | + | <!-- Page Content --> | |
− | + | <div class="container"> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | <!-- Page Heading/Breadcrumbs --> | |
− | + | ||
− | + | <!-- /.row --> | |
− | + | ||
− | + | <!-- Content Row --> | |
− | + | <div class="row"> | |
− | + | <!-- Sidebar Column --> | |
− | + | <div class="col-md-3" id="myScrollspy" style="font-size:12px;line-height:10px;padding-top: 100px; margin-top: -50px;"> | |
− | + | <ul class="nav nav-pills nav-stacked" data-spy="affix" style="width:250px; position:fixed;"> | |
− | + | <li class="active"> | |
− | + | <a href="#section-1">DNA gel extraction</a> | |
− | + | </li> | |
− | + | <li> | |
− | + | <a href="#section-2">Electrophoresis</a> | |
− | </ | + | </li> |
+ | <li> | ||
+ | <a href="#section-3">Gibson assenbly</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-4">HPLC</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-5">Knock out genes of Ecoli</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-6">Crispr-cas9</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-7">Plasmid preparation</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-8">Plasmid transformation</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-9">Point mutation</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-10">Reagents</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-11">Whole cell catalysis</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-12">the LiAc SS carrier DNA PEG method</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-14">Measure protein concentration</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#section-13">References</a> | ||
+ | </li> | ||
+ | </ul> | ||
</div> | </div> | ||
− | + | <!-- Content Column --> | |
+ | <div class="col-md-9"> | ||
− | + | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">DNA gel extraction</h2> | |
− | + | <h4> | |
− | + | 1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube. | |
− | + | <br /> 2. Add a 3X sample volume of Buffer DE-A. the weight of gel is equivalent to a 100ul volume. | |
− | < | + | <br /> 3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved(no more |
− | + | than 10 min) | |
− | + | <br /> 4. Add 0.5X Buffer DE-A volume of Buffer DE-B, mix. | |
− | + | <br /> 5. Place a miniprep colume into a 2ml microfuge tube. Transfer the solubilized agarose from step 4 into the | |
− | + | column. Centrifuge at 12,000xg for 1 min. | |
− | + | <br /> 6. Discard the filtrate. Add 500ul of Buffer W1. Centrifuge at 12,000xg for 30s. | |
− | + | <br /> 7. Discard the filtrate. Add 700ul of Buffer W2. Centrifuge at 12,000xg for 30s. | |
− | + | <br /> 8. Repeat wash with W2. Centrifuge at 12,000xg for 1min. | |
− | + | <br /> 9. Discard the filtrate. Centrifuge at 12,000xg for 1min. | |
− | + | <br /> 10. Transfer the miniprep column into a clean 1.5ml microfuge tube. Add 25-30ul of Eluent or deionized water | |
− | + | to the center of the membrane. Let it stand for 1min at room temperature. Centrifuge at 12,000xg for 1min. (pre-warming | |
− | + | the Eluent at 65℃) | |
− | + | <br /> | |
− | + | </h4> | |
− | + | <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">Electrophoresis</h2> | |
− | + | <h4>Agarose-Electrophoresis is used in order to see if the PCR product is correct and seperate DNA by the number of | |
− | + | base pairs. | |
− | + | <br /> ●marker was the 2 kb Plus DNA Ladder | |
− | + | <br /> ●fill pockets with 5 µl DNA ladder or 10 µl sample volume with 6x loading buffer | |
+ | <br /> ●running conditions: 130 V for 30-40 minutes | ||
+ | <br /> | ||
+ | </h4> | ||
+ | <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">Gibson assembly</h2> | ||
+ | <h4> | ||
+ | 1. Set up the reaction. | ||
+ | <br /> V(ul)=(0.02*bp)/concentration(ng/ul) | ||
+ | <br /> 2. Add the fragments into Gibson system. | ||
+ | <br /> 3. Incubate samples in a thermocycler at 50 °C for 1h. | ||
+ | <br /> 4. Purify the product using DNA purification kit. | ||
+ | <br /> 5. Transform the product into the competent cells of E.coli BL21 , following the transformation protocol. | ||
+ | <br /> | ||
+ | <br /> </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
+ | <h4> </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
+ | <h4> </h4> | ||
+ | <h4> </h4> | ||
+ | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">HPLC</h2> | ||
+ | <h4> | ||
+ | for acrylic acid | ||
+ | </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
+ | <h4> | ||
+ | <br /> Samples have to be centrifuged at 12,000xg for 5 min in order to remove solids (cells/precipitates). | ||
+ | <br /> | ||
+ | <br /> All instruments we used is ultra-high performance liquid chromatography mass spectrometry instrument ABSciex | ||
+ | 5600 TripleTOF. | ||
+ | <br /> a) HPLC: | ||
+ | <br /> Colum: Waters SunFire C18(4.6×250 mm) | ||
+ | <br /> Detector wavelength: 210 nm | ||
+ | <br /> Flowing phase: 90% 10mM ammonium acetate/ 10% methanol | ||
+ | <br /> Colum temperature: 35℃ | ||
+ | <br /> Flow rate: 1mL/min | ||
+ | <br /> b) MS detecting condition: | ||
+ | <br /> Anion mode | ||
+ | <br /> Ion Source Gas1: 55 psi | ||
+ | <br /> Ion Source Gas2: 55 psi | ||
+ | <br /> Curtain Gas: 35 psi | ||
+ | <br /> IonSpray Voltage Floating: 4500 v | ||
+ | <br /> Interface Heater Temperature: 550℃ | ||
+ | <br /> | ||
+ | <br /> | ||
+ | </h4> | ||
+ | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">Knock out genes of E ▪ coli (MG1655)</h2> | ||
+ | <h4>1. Pre-chill 1.5ml and 2ml microcentrifuge tubes, deionized water, 10% glycerol. Add 1ml LB to 2ml microcentrifuge | ||
+ | tubes. | ||
+ | <br /> 2. When OD600=0.6, incubate competent cells on ice for 20 min. | ||
+ | <br /> 3. Transfer the competent cells to 50 mL pre-chilled centrifuge tube. Centrifuge at 5,500r/min, 4 ℃ for 5 min. | ||
+ | <br /> 4. Discard the filtrate. Add 30ml of pre-chilled deionized water, resuspend the cells gently. | ||
+ | <br /> 5. Centrifuge at 5,500r/min, 4 ℃ for 5 min. Discard the filtrate. Repeat wash with deionized water. | ||
+ | <br /> 6. Discard the filtrate. Add 30ml of pre-chilled 10% glycerol, resuspend the cells gently. | ||
+ | <br /> 7. Centrifuge at 6,500r/min, 4 ℃ for 5 min. Discard the filtrate. Repeat wash with 10% glycerol. | ||
+ | <br /> 8. Discard the filtrate and leave over 1ml 10% glycerol to resuspend the competent cells, pipet 80ul into each | ||
+ | 1.5ml EP tube, add 5ul of DNA and carefully mix with the competent cells. Let it stand for 2min. | ||
+ | <br /> 9. Add electrocompetent to DNA on ice. Move the mixture to the cuvette. Dry and shock the cells(2500V). Add | ||
+ | 1ml of LB medium. Incubate at 30℃ for 4 hours shaking at 220rpm, pipet 100ul from each tube onto the appropriate | ||
+ | plate, and spread the mixture evenly across the plate. Incubate at 30℃ overnight. Position the plates with the | ||
+ | agar side at the top, and the lid at the bottom. | ||
+ | <br /> | ||
+ | </h4> | ||
+ | <h2 id="section-6" style="padding-top: 100px; margin-top: -50px;">Knock out the genes of Saccharomyces cerevisiae with Crispr-Cas9</h2> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c6/Sxt_%284%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
+ | <h4> </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
+ | <h4> | ||
+ | <br /> 2. Purify the PCR product with a DNA purification kit. | ||
+ | <br /> 3. Add the appropriate amount of DMT enzyme, hold for one hour at 37 ° C. | ||
+ | <br /> 4. transform the DNA into competent cells. | ||
+ | <br /> 50ul competent cell + 15ul purified DNA,incubate on ice for 30min,heat shock 45s,incubate on ice for 2min,add | ||
+ | LB medium and incubate for 1h. | ||
+ | <br /> 5. Pipet 100ul from each tube onto the plate with resistance, and spread the mixture evenly across the plate. | ||
+ | Incubate for 12h. Position the plates with the agar side at the top, and the lid at the bottom. | ||
+ | <br /> 6. use a sterile pipet tip to pick Saccharomyces cerevisiae from plates,throw the tip into the tubes of 5 ml | ||
+ | of LB + antibiotics,incubate in a rotary shaker. Prepare plasmid with kit for sequencing. | ||
+ | <br /> 7. Transfer plasmid and fragment into Saccharomyces cerevisiae using the LiAc SS carrier DNA PEG method.</h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e0/Sxt_%286%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
+ | <h4> | ||
+ | <br /> 8. Prepare the template: use a sterile toothpick to pick Saccharomyces cerevisiae from plates,put the toothpick | ||
+ | into 100ul 20mMNaOH and mix,99° C boiling for 30min. </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
+ | <h4> </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width:60%;"> | ||
+ | </a> | ||
− | + | <h4> </h4> | |
− | + | <h2 id="section-7" style="padding-top: 100px; margin-top: -50px;">plasmid preparation</h2> | |
− | + | <h4>Tiangen mini plasmid kit | |
− | + | <br /> | |
− | + | </h4> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | <h2 id="section-8" style="padding-top: 100px; margin-top: -50px;">Plasmid transformation</h2> | |
− | + | <h4>1. Pipette 50µl of competent cells and 2µl of plasmid into 1.5ml tube | |
− | + | <br /> 2. Heat shock tubes at 42°C for 30s | |
− | + | <br /> 3. Incubate on ice for 2min | |
− | + | <br /> 4. Pipette 250µl LB media to each transformation | |
− | + | <br /> 5. Incubate at 37°C for 1h | |
− | + | <br /> 6. Plating | |
− | + | <br /> 7. Pick single colonies | |
− | + | <br /> Reference: http://parts.igem.org/Help:Protocols/Transformation | |
− | + | <br /> | |
+ | </h4> | ||
− | + | <h2 id="section-9" style="padding-top: 100px; margin-top: -50px;">Point mutation</h2> | |
− | |||
− | |||
− | |||
− | + | <img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width:60%;"> | |
+ | </a> | ||
+ | <h4> </h4> | ||
− | + | <img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width:60%;"> | |
− | + | </a> | |
− | + | <h4> | |
− | + | <br /> 2. Purify the PCR product with a DNA purification kit. | |
− | + | <br /> 3. Add the appropriate amount of DMT enzyme, hold for one hour at 37 ° C. | |
− | + | <br /> 4. Transform 5μl digested DNA into competent cells DH5α, incubate on ice for 30min. | |
− | + | <br /> 42° C heat shock, 45s. Incubate on ice for 2min. | |
− | + | <br /> add 200μl of LB. incubate at 37 °C for 1 h, 220rpm/min. | |
− | + | <br /> 5. pipet 200μl from each tube onto the plate with appropriate resistance, and spread the mixture evenly across | |
− | + | the plate. Incubate at 37℃ overnight. Position the plates with the agar side at the top, and the lid at the bottom. | |
− | + | <br /> 6. Select single colonies for sequencing. | |
− | + | <br /> | |
− | + | </h4> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | <h2 id="section-10" style="padding-top: 100px; margin-top: -50px;">Reagents</h2> | |
− | + | <h4>1. LB medium(lysogeny broth) | |
− | + | <br /> The recipe for 1l LB media is as follows: | |
− | + | <br /> Tryptone 10g/L | |
+ | <br /> Yeast extract 5g/L | ||
+ | <br /> NaCl 10g/L | ||
+ | <br /> 2. 0.1mM Kanamycin | ||
+ | <br /> MW of Kanamycin:582.58 | ||
+ | <br /> Store at -20℃ | ||
+ | <br /> 3. LB plate | ||
+ | <br /> The recipe for 1l LB plate is as follows: | ||
+ | <br /> Tryptone 10g/L | ||
+ | <br /> Yeast extract 5g/L | ||
+ | <br /> NaCl 10g/L | ||
+ | <br /> 15g Agar | ||
+ | <br /> Add appropriate amount of resistance. | ||
+ | <br /> 4. 2YT medium | ||
+ | <br /> The recipe for 1l LB plate is as follows: | ||
+ | <br /> Tryptone 16g/L | ||
+ | <br /> Yeast extract 10g/L | ||
+ | <br /> NaCl 5g/L | ||
+ | <br /> 5. 0.5mM IPTG | ||
+ | <br /> MW of IPTG:238.30 | ||
+ | <br /> Store at -20℃ | ||
+ | <br /> 6. 50mM PBS buffer,PH8.0 | ||
+ | <br /> A:0.05mol/L Na2HPO4 | ||
+ | <br /> B:0.05mol/L KH2P04 | ||
+ | <br /> 137mMNaCl,2.7mMKCl,10mMNa2HPO4,2mMKH2PO4 for 1L. | ||
+ | <br /> | ||
+ | </h4> | ||
− | + | <h2 id="section-11" style="padding-top: 100px; margin-top: -50px;">Whole-cell catalysis</h2> | |
− | + | <h4> | |
− | + | Whole cell catalysis means using complete biological organisms (ie, whole cells, tissues or even individuals) as a catalyst. | |
− | + | The essence is using enzymes in cells for catalysis. The method is a kind of biocatalytic technology between | |
− | + | fermentation and extract enzyme for catalysis. The advantage of whole cell catalysis is that the intracellular | |
− | + | complete multi-enzyme system can achieve the cascade reaction of enzyme, so as to make up the deficiency of cascade | |
− | + | reaction in reaction which only use pure enzyme and improve the catalytic efficiency. While eliminating the complex | |
− | + | process in enzyme purification, it is easier to carry out the reaction and lower production costs. <br> | |
− | + | 1. Prepare | |
− | + | sterile tubes of 5 ml of 2YT+antibiotics. Use a sterile pipet tip to pick bacteria from plates. Throw the tip | |
− | + | into the tubes. Incubate in a rotary shaker at37℃ for 3-4h. | |
− | + | <br /> 2. Transfer 120µl of bacteria from a slant culture into an Erlenmeyer flask containing 60 mL LB medium with | |
− | + | appropriate resistance, incubate at 37 °C. | |
− | + | <br /> 3. When the OD600 reaches 0.6-0.8, the induction of IPTG (0.5 mM) should be carried out. Incubate at 30 °C on | |
− | + | a rotary shaker incubator at 220 rpm for 14 h. | |
− | + | <br /> 4. Harvest the bacteria(6000rpm/min 7min). Wash with 30ml PBS. | |
− | + | <br /> 5. The biocatalytic reaction mixture contained 10% glycerol, E▪ coli and 50mM PBS. Reaction time gradient: 8h, | |
− | + | 16h, 32h. | |
− | + | <br /> 6. Use HPLC for further analysis. | |
− | + | <br /> | |
− | + | <br /> Reference: Li N, He Y, Chen Y, et al. Production of cyclic adenosine-3′,5′-monophosphate by whole cell catalysis | |
− | + | using recombinant Escherichia coli, overexpressing adenylate cyclase[J]. Korean Journal of Chemical Engineering, | |
− | + | 2013, 30(4):913-917. | |
− | + | <br /> | |
− | + | </h4> | |
− | + | <h2 id="section-12" style="padding-top: 100px; margin-top: -50px;">the LiAc SS carrier DNA PEG method</h2> | |
− | + | <h4> | |
− | + | 1.use a sterile pipet tip to pick Saccharomyces cerevisiae from plates,throw the tip into the tubes of appropriate medium,incubate | |
− | + | in a rotary shaker for 12h. | |
− | + | <br /> 2.measure OD600, transfer x(x=(50×0.2)/(OD600×dilution ratio)) ml Saccharomyces cerevisiae into 50ml YPAD. | |
− | + | <br /> 3.incubate for 4-5h to make OD600 reaches 0.8-0.9. | |
− | + | <br /> 4.boil ssDNA. | |
− | + | <br /> 5.Centrifuge at 3000g for 5 min. Discard the filtrate. Repeat washes with 25ml deionized water twice. | |
− | + | <br /> 6.Transfer the cells to 1.5 mL centrifuge tube. Add 1ml of deionized water, resuspend the cells gently. | |
− | + | <br /> 7.Centrifuge at 13000rpm for 30s. Discard the filtrate. | |
− | + | <br /> 8.Add 1ml of deionized water, resuspend the cells. Pipet 100ul into each 1.5 mL centrifuge tube. | |
− | + | <br /> 9.Centrifuge using a Mini Centrifuge. Discard the filtrate. | |
− | + | <br /> 10.System for transformation:</h4> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/7/70/Sxt_%2811%29.png" style="max-width:60%;"> | |
− | + | </a> | |
− | + | <h4> | |
− | + | <br /> 11.Incubate at 30℃ for 20min. | |
− | + | <br /> 12.42℃ heat shock for 40min. pipet 100ul from each tube onto the appropriate plate, and spread the mixture evenly | |
− | + | across the plate. Incubate at 30℃ for 2-3 days. Position the plates with the agar side at the top, and the lid | |
− | + | at the bottom. | |
− | + | <br /> 13.Prepare plasmid for sequencing. | |
− | + | <br /> | |
− | + | ||
− | + | </h4> | |
+ | |||
+ | <h2 id="section-14" style="padding-top: 100px; margin-top: -50px;">Measure protein concentration</h2> | ||
+ | <h4> | ||
+ | We used Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to measure protein concentration. | ||
+ | <br> 1. Measure OD at 280 nm to get rough protein concentration, and then diluted the protein to 0.5-1 mg / mL. | ||
+ | <br> 2. Prepare the reaction solution: reagent A and B in the BCA Protein Assay Kit are mixed in a 50: 1 ratio. | ||
+ | <br> 3. Pipette 200 uL of reaction solution in the coated wells | ||
+ | <br> 4. Pipette 25 uL of diluted protein, mixed it with the reaction solution. Hold at 37 ℃ for 30 min. | ||
+ | <br> 5. Measure OD at 562 nm. Protein concentration is measured according to the protein standard curve. | ||
+ | <br> | ||
+ | |||
+ | </h4> | ||
+ | |||
+ | |||
+ | <h2 id="section-13" style="padding-top: 100px; margin-top: -50px;">References</h2> | ||
+ | <h4> | ||
+ | http://parts.igem.org/Help:Protocols/Transformation | ||
+ | <br /> https://2015.igem.org/Team:Aachen/Project/Overview | ||
+ | <br /> http://www.zymoresearch.com/category/all-products | ||
+ | <br /> http://www.corning.com/worldwide/en/products/life-sciences/resources/brands/axygen-brand-products.html | ||
+ | <br /> http://www.tsingke.net/shop/ | ||
+ | <br /> http://www.cwbiotech.com/ | ||
+ | <br /> | ||
+ | </h4> | ||
− | |||
− | |||
</div> | </div> | ||
− | + | </div> | |
− | + | <!-- /.row --> | |
+ | |||
+ | <hr> | ||
+ | |||
+ | <!-- Footer --> | ||
+ | |||
</div> | </div> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/0/0c/Jz.png" class="img-responsive"> | ||
+ | </div> | ||
+ | <!-- /.container --> | ||
+ | |||
+ | |||
+ | |||
</body> | </body> | ||
</html> | </html> |
Revision as of 15:08, 1 November 2017