Difference between revisions of "Team:Shanghai City United/Experiments"

(Prototype team page)
 
Line 1: Line 1:
−
{{IGEM_TopBar}}
+
<html lang="en">
−
{{Shanghai_City_United}}
+
−
<html>
+
−
 
+
−
<div class="column full_size">
+
−
 
+
−
<h1>Experiments</h1>
+
−
<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
+
−
 
+
−
<p>
+
−
If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a>.
+
−
</p>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
<div class="column two_thirds_size">
+
−
<h3>What should this page contain?</h3>
+
−
<ul>
+
−
<li> Protocols </li>
+
−
<li> Experiments </li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
 
+
−
<div class="column third_size">
+
−
<div class="highlight decoration_A_full">
+
−
<h3>Inspiration</h3>
+
−
<ul>
+
−
<li><a href="https://2019.igem.org/Team:Nantes/Experiments">2019 Nantes </a></li>
+
−
<li><a href="https://2019.igem.org/Team:TU_Eindhoven/Experiments">2019 TU Eindhoven </a></li>
+
−
<li><a href="https://2019.igem.org/Team:Mingdao/Demonstrate">2019 Mingdao </a></li>
+
−
<li><a href="https://2020.igem.org/Team:Amsterdam/Experiments">2020 Amsterdam </a></li>
+
−
<li><a href="https://2020.igem.org/Team:NCTU_Formosa/Experiments">2020 NCTU Formosa </a></li>
+
−
<li><a href="https://2020.igem.org/Team:USAFA/Experiments">2020 USAFA </a></li>
+
−
</ul>
+
−
</div>
+
−
</div>
+
−
 
+
  
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta http-equiv="X-UA-Compatible" content="IE=edge">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>Shanghai_City_United</title>
 +
    <link rel="stylesheet"
 +
        href="https://2021.igem.org/wiki/index.php?title=Template:Shanghai_City_United/Main_CSS&action=raw&ctype=text/css" />
 +
</head>
  
 +
<body>
 +
    <nav class="head-nav clearfix">
 +
        <div class="top-block"></div>
 +
        <div class="top-nav-bar">
 +
            <ul class="clearfix">
 +
                <span class="small-logo"></span>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_City_United">Home</a>
 +
                </li>
 +
                <li class="active">
 +
                    <a href="">Project</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Description"
 +
                                    class="sub-nav-74">Description</a></li>
 +
                            <li class="current-sub-nav"><a href="#" class="sub-nav-74">Experiments</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Results"
 +
                                    class="sub-nav-74">Results</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Proof_Of_Concept"
 +
                                    class="sub-nav-52">Proof Of Concept</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Notebook"
 +
                                    class="sub-nav-52">Notebook</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Safety">Safety</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
                <li>
 +
                    <a href="">Parts</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Collection"
 +
                                    class="sub-nav-74">Parts
 +
                                    Collection</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Engineering"
 +
                                    class="sub-nav-74">Engineering</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
                <li>
 +
                    <a href="">Human Practices</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Human_Practices"
 +
                                    class="sub-nav-74">Integrated Human Practice</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Communication"
 +
                                    class="sub-nav-74">Communication</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Fundraising"
 +
                                    class="sub-nav-74">Fundraising</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_City_United/Implementation">Implementation</a>
 +
                </li>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_City_United/Entrepreneurship">Entrepreneurship</a>
 +
                </li>
 +
                <li>
 +
                    <a href="https://2021.igem.org/Team:Shanghai_City_United/Model">Model</a>
 +
                </li>
 +
                <li>
 +
                    <a href="">Team</a>
 +
                    <div class="sub-nav">
 +
                        <ul>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Members"
 +
                                    class="sub-nav-74">Team
 +
                                    Members</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Attributions"
 +
                                    class="sub-nav-74">Attributions</a></li>
 +
                            <li><a href="https://2021.igem.org/Team:Shanghai_City_United/Collaborations"
 +
                                    class="sub-nav-74">Collaborations</a></li>
 +
                        </ul>
 +
                    </div>
 +
                </li>
 +
            </ul>
 +
        </div>
 +
    </nav>
 +
    <div class="sub-banner">
 +
        <img src="https://static.igem.org/mediawiki/2021/2/23/T--Shanghai_City_United--Description01.png" alt="" />
 +
    </div>
 +
    <div class="sub-content">
 +
        <div class="sub-title">EXPERIMENTS</div>
 +
        <div class="article-title">1. Preparation of LB medium</div>
 +
        <div class="article-content">Materials: </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Yeast extract 5g/L</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Tryptone 10g/L</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;NaCl 10g/L</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Agar(only for solid medium)1.5%</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;H2O (as solvent) 1000ml</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Totally 1000ml (600ml for liquid medium, 400ml for solid
 +
            medium)</div>
 +
        <div class="article-content">Steps:</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. Weighing 5 gram yeast extract</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. Preparing 1000ml, and pouring into a huge measuring
 +
            cylinder</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Adding the yeast extract into water</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Weighing 10 gram Tryptone</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. Adding the tryptone into water</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;6. Weighing 10 gram sodium chlorine</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;7. Adding sodium chlorine into water</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;8. Stirring the solution</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;9. Preparing 5 flasks 3 of them are 250ml, and 2 are 500ml
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;10. Adding 150ml solution into the 250ml flasks, and adding
 +
            300ml solution into 500ml solution</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;11. For solid medium, adding the agar into the bottom of
 +
            the flask first, and than pouring the solution into it.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;12. Use the membrane to seal the flask( double filter, 4
 +
            pores for air recycle)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;13. Put the flask into the High-Pressure Steam
 +
            Sterilization Pot for sterilization(0 is standard pressure 1atm)(condition: 0-0.4, 120 centigrade 30 minute)
 +
        </div>
 +
        <div class="article-title">2. PCR amplification</div>
 +
        <div class="article-content">Materials: </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Primer-F 1μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Primer-R 1μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2X Prime Star Max(DNA polymerase ,Buffer, dNTP) 50μL
 +
            dillute to 1/2 25μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Template 1μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;ddH2O 22μL</div>
 +
        <div class="article-content">In the PCR machine:</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;98℃ 5m pre-denaturation (break the hydrogen bond)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;98℃ 15s break the hydrogen bond</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;55℃ 15s primer connect to the brands</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;72℃ 5s/1k bp extend the DNA strands(the temperature which
 +
            the enzyme most active)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Repeat these three steps 30 times</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;72℃ 3m let the DNA strands completely extend sufficiently
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4℃ preserve the DNA</div>
 +
        <div class="article-content">Process:</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. Primer 5.00 n mol diluter in water</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. 500μL water add into Primer-F</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Split 200μLPrimer-F</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Adding template into 900μLwater(dilute)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. Adding 900μLwater into template</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;6. Adding 22μL ddH2O into the PCR tube</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;7. Adding 25μL Primer Star Max into the PCR tube</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;8. Adding 2 primers</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;9. Adding template</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;10. Marked the number (determine)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;11. Put in the PCR machine(shut the top cover avoid the
 +
            liquid evaporate)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;12. Setting the program has written above.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;13. Waiting for the process finish</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;14. Preserve the DNA.</div>
 +
        <div class="article-title">3. Running nucleic acid electrophoresis </div>
 +
        <div class="article-content"> Mechanism: DNA is negative charge, so it will be attract to the positive
 +
            direction. Also, different DNA contains different number of base pair and shape. The speed of DNA are
 +
            different either. We can compare the model DNA(500bp) with ours product after PCR to determine whether our
 +
            products are that one we need.</div>
 +
        <div class="article-content">A. Nucleic Acid Electrophoresis Gel</div>
 +
        <div class="article-content"> Materials:</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. Agarose 1%</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. TAE buffer 100ml</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Nucleic acid dye 10μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Marker</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. DNA from PCR</div>
 +
        <div class="article-content"> Process:</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. Mix the agarose and 100ml TAE buffer</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. Heated in microwave about 3 min until all the agarose
 +
            melt.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Add Nucleic acid dye into the solution.(double glove
 +
            avoid the dye contact skin)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Pour the heated solution into the mold tank and plate
 +
            and wait it to solid</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. Take the gel(use knife around the gel and pick it up)
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;6. Put the gel in the bottom of the tank</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;7. Pour TAE buffer into the tank until it cover the gel
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;8. Add the marker into the first ole of the gel(do not
 +
            penetrate the gel)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;9. Add Loading buffer into PCR tube(let the DNA get color)
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;10. Add other DNA into the gel.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;11. Clean the surrounding of the tank and cover it</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;12. Energized it with 120V(there are bubbles going out from
 +
            the liquid)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;13. Waiting about 15 min</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;14. Observe the traits of the DNA under the violet light.
 +
        </div>
 +
        <div class="article-title">4. Measuring OD600</div>
 +
        <div class="article-content">Mechanism: The light absorption of bacteria is used to measure the concentration of
 +
            bacterial culture medium, so as to estimate the growth of bacteria. It is usually used to refer to the
 +
            density of bacterial cells.<br />
 +
            Experimental Procedure
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. To measure the concentration, we use Nanodrop by
 +
            measuring light absorption value.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. Use 2ul Micro pipette with ddH2O to clean the water on
 +
            the sampling table, wipe the sweat with dust-free paper, repeat three times.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Drop 2ul DNA sample on the detection platform and the
 +
            internal light penetrate the DNA sample.( We have 100% internal light, if the metering wall shows 20% light,
 +
            then we will know that the DNA sample absorb 80% light.) (A260/A230→calculation of concentration,
 +
            A260/A280→calculation of purity—1.8 is close to pure)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. After the experiment, the ddH2O cleaning process should
 +
            be repeated three times again.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. Building Plasmid (Double Digest&T4 connection)</div>
 +
        <div class="article-content">Lactobacillus’s route:</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. Carry on processing the previous E. coli that carries
 +
            XynA, select white strains that express lac ab enzyme.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. Injecting the selected strains into LB nutrient solution
 +
            and Amp antibiotic solution.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Extracting the plasmids from PMD19-T—XynA. (Which has
 +
            been mentioned in detail later)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Double digestion for different biological parts that
 +
            eventually constructed the final plasmid:</div>
 +
        <div class="article-content">First time: </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Using restriction enzymes to cut
 +
            different parts that serves for the connections. Using NheI and HindIII to cut the part of XynA; Using BamHI
 +
            and NheI to cut the part of PUS; Using BamHI and HindIII to cut the part of PSIP403. Three cutted parts need
 +
            to run the nucleic acid electrophoresis, getting the target length around 500bp for XynA; 300bp for PUS and
 +
            6kbp of PSIP403.
 +
            (Notice worthily, by using the formal strategies on double digestion, we failed on the step of transplanting
 +
            the building plasmids into the Lactobacillus. Excluding the effect of erythromycin, we’ve concluded that two
 +
            restriction sites on the parts may cut off the segment of the genetic expression for ori---a codon initiator
 +
            for the functioning of this plasmid inside the Lactobacillus.)
 +
        </div>
 +
        <div class="article-content">Second time: </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;By first using PCR techniques for
 +
            amplifying the restriction site fragment, we’ve changed one of our restriction enzymes from BamHI to EcoRI.
 +
            (Details for the solutions are provided bellow)</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/9/94/T--Shanghai_City_United--Experiment01.jpg" alt="" />
 +
        </div>
 +
        <div class="article-title">5. T4 connecting system</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp; (PSIP403：PUS : XynA=1:1:1)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;10X T4 Ligase buffer 1μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;psiP403 0.5μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;Pus 5.5μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;XynA 2μL</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;T4Ligase 1μL</div>
 +
        <div class="article-content">Solutions above need to be mixed in a testing tube to react for at least 30 minutes
 +
            under the surrounding temperature of 6 degrees.</div>
 +
        <div class="article-title">6. Cultivating E.coli</div>
 +
        <div class="article-content">Lab procedure:</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp; 1. Process the E.coli DH5α with Cacl2 to depolarize the
 +
            membrane of the cell and allow access of exterior DNA. </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. Extract 50ul of E.coli DH5α from stored glycerin. And
 +
            transfer into a 2ml Eppendorf tube.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Extract and add 5ul of T4 Polymerase (XynA) to the
 +
            Eppendorf tube.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Unfreeze the minus eighty degrees’ cells on ice for 30
 +
            minutes. (In order to allow DNAs to do free diffuse)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. Water bath the Eppendorf tube to heat shock the E.coli
 +
            for 90 seconds to let the plasmids absorb through the openings on the cell.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;6. Place the Eppendorf tube on ice for 30 minutes to
 +
            entirely close the openings on the cell and recovering the cell from competent.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;7. 895ul of LB liquid culture medium were added under the
 +
            sterile operating table as nutrients.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;8. The Eppendorf tube then put in a beaker and placed in a
 +
            shaking incubator for 30 minutes. (240rpm)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp; 9. Then the Eppendorf tube was centrifuged for 30 seconds.
 +
            (12000 times/min, centrifugal force: 13800)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;10. Solid LB culture medium was added into petri dish with
 +
            a final concentration of 100ug/ml Amp filtrated stock; 20ug/ml IPTG filtrated stock and 20ug/ml X-Gel
 +
            filtrated stock.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;11. Burned the micro pipette tips with the alcohol burner
 +
            and placed upside down till the top of tip smoothen.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;12. Extract 800ul of bacteria from the Eppendorf tube and
 +
            safe the rest of it.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;13. 50ul of droplets should gently drop in the center of
 +
            the petri dish and rub back and forth to coated the plate.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;14. Put the coated plate upside down into the incubator and
 +
            incubate overnight at 37℃.</div>
 +
        <div class="article-title">7.Plasmid extraction（PMD19-XynA) </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp; 1. Add E.coli DH5 α/PSIP403-PUS-XynA into 4ml LB solution
 +
            and put it into shaking incubator. (37℃, 240rmp, 16h) </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. Add 250ul buffer SP1 and blow and mix well. (SP1:
 +
            Glucose- form a suspension with suspended bacteria; RNase- degrade RNA; Buffer- provide the environment)
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Add 250ul buffer SP2, reverse it 5 to 10 times. SP2:
 +
            NaOH–Cleavage cell wall, slightly clarified liquid, DNA&RNA release from the cell; metal chelating
 +
            agent–wipe off the metal ion</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Add 350ul buffer SP3, quickly invert and mix. SP3: SDS
 +
            (protein denaturant)–Let the protein denature; CH3COOK–Neutralize PH (→NaOH), K→ The metal ions are turned
 +
            into water-insoluble salts and precipitated</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. Centrifuged it in a centrifuge. (5min, 12000 times/min,
 +
            centrifugal force: 13800) Then we get: Sediment: Protein, metal ions (some of), genome (wrap around the
 +
            protein)/Supernatant liquid: plasmid DNA, metal ions (some of ), residual protein (some dissolve in the
 +
            water)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;6. Add 500ul buffer S on the adsorption column. Centrifuged
 +
            it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom.
 +
            (Column can be activated and balanced./ Wash the silicate fiber membrane.)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;7. Add 800ul supernatant liquid on the adsorption column.
 +
            Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at
 +
            the bottom. (Plasmid DNA can be attached to the membrane.)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;8. Add 500ul buffer DW1 on the adsorption column.
 +
            Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800)Pour out the waste liquid at
 +
            the bottom. (Remove residual protein)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp; 9. [Add 500ul wash buffer(C2H6O) on the adsorption column.
 +
            Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at
 +
            the bottom.] *2 （Cleaning residue, like salt, metal, ions...)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;10. Put it in a centrifuge without adding any liquid. (30s,
 +
            12000 times/min, centrifugal force: 13800) (Centrifuge the wash buffer)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;11. Move the column to a new 1.5ml tube, open the lid and
 +
            let stand for 1min. (C2H6O volatulization)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;12. Add 50ul ddH2O (50℃ hot water) (Elution buffer cannot
 +
            be used, Elution buffer--Tris-HCl×) Centrifuged it in a centrifuge (60s, 12000 times/min, centrifugal force:
 +
            13800) (Eluting plasmid DNA)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;13. Throw the column into the lab pollution bin.
 +
            (Remaining: plasmid DNA and ddH2O)</div>
 +
        <div class="article-title">8. Running SDS PAGE protein electrophoresis</div>
 +
        <div class="article-content">Make SDS PAGE gel</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp; 1. 2.7ml lower layer solution, 2.7ml lower layer buffer,
 +
            60ul improved coagulant. (Blow and mix. It will set in 5-10 minutes) </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. Slot: Add water and see if the liquid level drops. It is
 +
            set when the liquid level doesn’t drop.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3. Add 1000ul for each time (The distance between the
 +
            liquid level and the short glass plate is 0.5cm longer than the comb length.)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;4. Add some water to see if the liquid is set.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;5. 0.75ml upper glue solution, 0.75ml red upper buffer,
 +
            15ul coagulant, mix, pour in, and insert the comb.</div>
 +
        <div class="article-content">Running</div>
 +
        <div class="article-content">Mechanism: Spacer gel: superstratum, small concentration, large pore. It helps the
 +
            protein to press into a
 +
            line at the boundary between the upper and lower layers.
 +
            Separation gel: substratum, large concentration, small pore. From here on, the small ones swim faster than
 +
            the big ones.
 +
        </div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. Power up and start running（120v/30min)</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;2. We can stop when the blue line reaches ths bottom.</div>
 +
        <div class="article-content">Coomassie brilliant blue staining</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1. Pour CBB into a box to cover the gel in it. Rapid
 +
            dyeing: microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB.</div>
 +
        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp; 2. [Pour CBB distaining solution. Rapid wash the dye:
 +
            microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB distaining solution.] *3-4
 +
        </div>
 +
        <div class="article-title">9. Measuring the enzyme activity</div>
 +
        <div class="article-content">Xylan Liquid (50mL):</div>
 +
        <div class="article-content">0.25g of xylan (5g/L) </div>
 +
        <div class="article-content">Add ddH2O until 50mL</div>
 +
        <div class="article-content">Reaction Medium (1mL):</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/1/1a/T--Shanghai_City_United--Experiment02.jpg" alt="" />
 +
        </div>
 +
        <div class="article-content">·Water bath--37℃--15min, 90℃--5min, add DNS--2ml, 90℃--8min, add ddH2O 7ml (the
 +
            capacity to 10mL)</div>
 +
        <div class="article-content">·Let it cool to room temperature.</div>
 +
        <div class="article-content">·Measuring 540nm light absorption value（The procedure is the same as that for
 +
            OD600）
 +
        </div>
 +
    </div>
 +
    <footer class="footer">
 +
        <section class="footer-wrap">
 +
            <div class="footer-contact">CONTACT INFO</div>
 +
            <p class="margin-bottom-20">Email: igemteamceres@gmail.com</p>
 +
            <p><i style="color: #2d2d2d;">WeChat Official Account: iGEM Ceres</i></p>
 +
        </section>
 +
    </footer>
 +
</body>
 +
<script>
 +
    let liTags = document.querySelectorAll(".top-nav-bar > ul > li");
 +
    let len = liTags.length;
 +
    for (let i = 0; i < len; i++) {
 +
        liTags[i].onclick = function (e) {
 +
            //先移除所有的点击样式
 +
            for (let j = 0; j < len; j++) {
 +
                liTags[j].classList.remove("active");
 +
            }
 +
            //再添加点击样式
 +
            let li = e.currentTarget;
 +
            li.classList.add("active");
 +
        }
 +
    }
 +
    //展开项点击事件
 +
    let sections = document.querySelectorAll(".sub-content> .section-wrap > .item-title");
 +
    let sLen = sections.length;
 +
    for (let j = 0; j < sLen; j++) {
 +
        sections[j].onclick = function (e) {
 +
            //再添加点击样式
 +
            let sec = e.currentTarget;
 +
            let clsArr = Array.from(sec.classList);
 +
            if (clsArr.includes("in")) {
 +
                sec.classList.remove("in");
 +
                sec.classList.add("out");
 +
            } else {
 +
                sec.classList.remove("out");
 +
                sec.classList.add("in");
 +
            }
 +
        }
 +
    }
 +
</script>
  
 
</html>
 
</html>

Revision as of 17:35, 2 October 2021

Shanghai_City_United

EXPERIMENTS
1. Preparation of LB medium
Materials:
    Yeast extract 5g/L
    Tryptone 10g/L
    NaCl 10g/L
    Agar(only for solid medium)1.5%
    H2O (as solvent) 1000ml
    Totally 1000ml (600ml for liquid medium, 400ml for solid medium)
Steps:
    1. Weighing 5 gram yeast extract
    2. Preparing 1000ml, and pouring into a huge measuring cylinder
    3. Adding the yeast extract into water
    4. Weighing 10 gram Tryptone
    5. Adding the tryptone into water
    6. Weighing 10 gram sodium chlorine
    7. Adding sodium chlorine into water
    8. Stirring the solution
    9. Preparing 5 flasks 3 of them are 250ml, and 2 are 500ml
    10. Adding 150ml solution into the 250ml flasks, and adding 300ml solution into 500ml solution
    11. For solid medium, adding the agar into the bottom of the flask first, and than pouring the solution into it.
    12. Use the membrane to seal the flask( double filter, 4 pores for air recycle)
    13. Put the flask into the High-Pressure Steam Sterilization Pot for sterilization(0 is standard pressure 1atm)(condition: 0-0.4, 120 centigrade 30 minute)
2. PCR amplification
Materials:
    Primer-F 1μL
    Primer-R 1μL
    2X Prime Star Max(DNA polymerase ,Buffer, dNTP) 50μL dillute to 1/2 25μL
    Template 1μL
    ddH2O 22μL
In the PCR machine:
    98℃ 5m pre-denaturation (break the hydrogen bond)
    98℃ 15s break the hydrogen bond
    55℃ 15s primer connect to the brands
    72℃ 5s/1k bp extend the DNA strands(the temperature which the enzyme most active)
    Repeat these three steps 30 times
    72℃ 3m let the DNA strands completely extend sufficiently
    4℃ preserve the DNA
Process:
    1. Primer 5.00 n mol diluter in water
    2. 500μL water add into Primer-F
    3. Split 200μLPrimer-F
    4. Adding template into 900μLwater(dilute)
    5. Adding 900μLwater into template
    6. Adding 22μL ddH2O into the PCR tube
    7. Adding 25μL Primer Star Max into the PCR tube
    8. Adding 2 primers
    9. Adding template
    10. Marked the number (determine)
    11. Put in the PCR machine(shut the top cover avoid the liquid evaporate)
    12. Setting the program has written above.
    13. Waiting for the process finish
    14. Preserve the DNA.
3. Running nucleic acid electrophoresis
Mechanism: DNA is negative charge, so it will be attract to the positive direction. Also, different DNA contains different number of base pair and shape. The speed of DNA are different either. We can compare the model DNA(500bp) with ours product after PCR to determine whether our products are that one we need.
A. Nucleic Acid Electrophoresis Gel
Materials:
    1. Agarose 1%
    2. TAE buffer 100ml
    3. Nucleic acid dye 10μL
    4. Marker
    5. DNA from PCR
Process:
    1. Mix the agarose and 100ml TAE buffer
    2. Heated in microwave about 3 min until all the agarose melt.
    3. Add Nucleic acid dye into the solution.(double glove avoid the dye contact skin)
    4. Pour the heated solution into the mold tank and plate and wait it to solid
    5. Take the gel(use knife around the gel and pick it up)
    6. Put the gel in the bottom of the tank
    7. Pour TAE buffer into the tank until it cover the gel
    8. Add the marker into the first ole of the gel(do not penetrate the gel)
    9. Add Loading buffer into PCR tube(let the DNA get color)
    10. Add other DNA into the gel.
    11. Clean the surrounding of the tank and cover it
    12. Energized it with 120V(there are bubbles going out from the liquid)
    13. Waiting about 15 min
    14. Observe the traits of the DNA under the violet light.
4. Measuring OD600
Mechanism: The light absorption of bacteria is used to measure the concentration of bacterial culture medium, so as to estimate the growth of bacteria. It is usually used to refer to the density of bacterial cells.
Experimental Procedure
    1. To measure the concentration, we use Nanodrop by measuring light absorption value.
    2. Use 2ul Micro pipette with ddH2O to clean the water on the sampling table, wipe the sweat with dust-free paper, repeat three times.
    3. Drop 2ul DNA sample on the detection platform and the internal light penetrate the DNA sample.( We have 100% internal light, if the metering wall shows 20% light, then we will know that the DNA sample absorb 80% light.) (A260/A230→calculation of concentration, A260/A280→calculation of purity—1.8 is close to pure)
    4. After the experiment, the ddH2O cleaning process should be repeated three times again.
    5. Building Plasmid (Double Digest&T4 connection)
Lactobacillus’s route:
    1. Carry on processing the previous E. coli that carries XynA, select white strains that express lac ab enzyme.
    2. Injecting the selected strains into LB nutrient solution and Amp antibiotic solution.
    3. Extracting the plasmids from PMD19-T—XynA. (Which has been mentioned in detail later)
    4. Double digestion for different biological parts that eventually constructed the final plasmid:
First time:
        Using restriction enzymes to cut different parts that serves for the connections. Using NheI and HindIII to cut the part of XynA; Using BamHI and NheI to cut the part of PUS; Using BamHI and HindIII to cut the part of PSIP403. Three cutted parts need to run the nucleic acid electrophoresis, getting the target length around 500bp for XynA; 300bp for PUS and 6kbp of PSIP403. (Notice worthily, by using the formal strategies on double digestion, we failed on the step of transplanting the building plasmids into the Lactobacillus. Excluding the effect of erythromycin, we’ve concluded that two restriction sites on the parts may cut off the segment of the genetic expression for ori---a codon initiator for the functioning of this plasmid inside the Lactobacillus.)
Second time:
        By first using PCR techniques for amplifying the restriction site fragment, we’ve changed one of our restriction enzymes from BamHI to EcoRI. (Details for the solutions are provided bellow)
5. T4 connecting system
     (PSIP403：PUS : XynA=1:1:1)
    10X T4 Ligase buffer 1μL
    psiP403 0.5μL
    Pus 5.5μL
    XynA 2μL
    T4Ligase 1μL
Solutions above need to be mixed in a testing tube to react for at least 30 minutes under the surrounding temperature of 6 degrees.
6. Cultivating E.coli
Lab procedure:
     1. Process the E.coli DH5α with Cacl2 to depolarize the membrane of the cell and allow access of exterior DNA.
    2. Extract 50ul of E.coli DH5α from stored glycerin. And transfer into a 2ml Eppendorf tube.
    3. Extract and add 5ul of T4 Polymerase (XynA) to the Eppendorf tube.
    4. Unfreeze the minus eighty degrees’ cells on ice for 30 minutes. (In order to allow DNAs to do free diffuse)
    5. Water bath the Eppendorf tube to heat shock the E.coli for 90 seconds to let the plasmids absorb through the openings on the cell.
    6. Place the Eppendorf tube on ice for 30 minutes to entirely close the openings on the cell and recovering the cell from competent.
    7. 895ul of LB liquid culture medium were added under the sterile operating table as nutrients.
    8. The Eppendorf tube then put in a beaker and placed in a shaking incubator for 30 minutes. (240rpm)
     9. Then the Eppendorf tube was centrifuged for 30 seconds. (12000 times/min, centrifugal force: 13800)
    10. Solid LB culture medium was added into petri dish with a final concentration of 100ug/ml Amp filtrated stock; 20ug/ml IPTG filtrated stock and 20ug/ml X-Gel filtrated stock.
    11. Burned the micro pipette tips with the alcohol burner and placed upside down till the top of tip smoothen.
    12. Extract 800ul of bacteria from the Eppendorf tube and safe the rest of it.
    13. 50ul of droplets should gently drop in the center of the petri dish and rub back and forth to coated the plate.
    14. Put the coated plate upside down into the incubator and incubate overnight at 37℃.
7.Plasmid extraction（PMD19-XynA)
     1. Add E.coli DH5 α/PSIP403-PUS-XynA into 4ml LB solution and put it into shaking incubator. (37℃, 240rmp, 16h)
    2. Add 250ul buffer SP1 and blow and mix well. (SP1: Glucose- form a suspension with suspended bacteria; RNase- degrade RNA; Buffer- provide the environment)
    3. Add 250ul buffer SP2, reverse it 5 to 10 times. SP2: NaOH–Cleavage cell wall, slightly clarified liquid, DNA&RNA release from the cell; metal chelating agent–wipe off the metal ion
    4. Add 350ul buffer SP3, quickly invert and mix. SP3: SDS (protein denaturant)–Let the protein denature; CH3COOK–Neutralize PH (→NaOH), K→ The metal ions are turned into water-insoluble salts and precipitated
    5. Centrifuged it in a centrifuge. (5min, 12000 times/min, centrifugal force: 13800) Then we get: Sediment: Protein, metal ions (some of), genome (wrap around the protein)/Supernatant liquid: plasmid DNA, metal ions (some of ), residual protein (some dissolve in the water)
    6. Add 500ul buffer S on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom. (Column can be activated and balanced./ Wash the silicate fiber membrane.)
    7. Add 800ul supernatant liquid on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom. (Plasmid DNA can be attached to the membrane.)
    8. Add 500ul buffer DW1 on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800)Pour out the waste liquid at the bottom. (Remove residual protein)
     9. [Add 500ul wash buffer(C2H6O) on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom.] *2 （Cleaning residue, like salt, metal, ions...)
    10. Put it in a centrifuge without adding any liquid. (30s, 12000 times/min, centrifugal force: 13800) (Centrifuge the wash buffer)
    11. Move the column to a new 1.5ml tube, open the lid and let stand for 1min. (C2H6O volatulization)
    12. Add 50ul ddH2O (50℃ hot water) (Elution buffer cannot be used, Elution buffer--Tris-HCl×) Centrifuged it in a centrifuge (60s, 12000 times/min, centrifugal force: 13800) (Eluting plasmid DNA)
    13. Throw the column into the lab pollution bin. (Remaining: plasmid DNA and ddH2O)
8. Running SDS PAGE protein electrophoresis
Make SDS PAGE gel
     1. 2.7ml lower layer solution, 2.7ml lower layer buffer, 60ul improved coagulant. (Blow and mix. It will set in 5-10 minutes)
    2. Slot: Add water and see if the liquid level drops. It is set when the liquid level doesn’t drop.
    3. Add 1000ul for each time (The distance between the liquid level and the short glass plate is 0.5cm longer than the comb length.)
    4. Add some water to see if the liquid is set.
    5. 0.75ml upper glue solution, 0.75ml red upper buffer, 15ul coagulant, mix, pour in, and insert the comb.
Running
Mechanism: Spacer gel: superstratum, small concentration, large pore. It helps the protein to press into a line at the boundary between the upper and lower layers. Separation gel: substratum, large concentration, small pore. From here on, the small ones swim faster than the big ones.
    1. Power up and start running（120v/30min)
    2. We can stop when the blue line reaches ths bottom.
Coomassie brilliant blue staining
    1. Pour CBB into a box to cover the gel in it. Rapid dyeing: microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB.
     2. [Pour CBB distaining solution. Rapid wash the dye: microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB distaining solution.] *3-4
9. Measuring the enzyme activity
Xylan Liquid (50mL):
0.25g of xylan (5g/L)
Add ddH2O until 50mL
Reaction Medium (1mL):
·Water bath--37℃--15min, 90℃--5min, add DNS--2ml, 90℃--8min, add ddH2O 7ml (the capacity to 10mL)
·Let it cool to room temperature.
·Measuring 540nm light absorption value（The procedure is the same as that for OD600）
CONTACT INFO

Email: igemteamceres@gmail.com

WeChat Official Account: iGEM Ceres