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</nav> | </nav> | ||
<div class="batu" style="background: url('https://static.igem.org/mediawiki/2017/f/fe/Npu-background.png') no-repeat fixed; overflow: hidden;"> | <div class="batu" style="background: url('https://static.igem.org/mediawiki/2017/f/fe/Npu-background.png') no-repeat fixed; overflow: hidden;"> | ||
− | |||
<!-- Page Content --> | <!-- Page Content --> | ||
<div class="container"> | <div class="container"> | ||
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<ul class="nav nav-pills nav-stacked" data-spy="affix" style="width:250px; position:fixed;"> | <ul class="nav nav-pills nav-stacked" data-spy="affix" style="width:250px; position:fixed;"> | ||
<li class="active"> | <li class="active"> | ||
− | <a href="#section-1"> | + | <a href="#section-1">DNA gel extraction</a> |
</li> | </li> | ||
<li> | <li> | ||
− | <a href="#section-2"> | + | <a href="#section-2">Electrophoresis</a> |
</li> | </li> | ||
<li> | <li> | ||
− | <a href="#section-3"> | + | <a href="#section-3">Gibson assembly</a> |
</li> | </li> | ||
<li> | <li> | ||
− | <a href="#section-4"> | + | <a href="#section-4">HPLC</a> |
</li> | </li> | ||
<li> | <li> | ||
− | <a href="#section-5"> | + | <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> | </li> | ||
</ul> | </ul> | ||
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<div class="col-md-9"> | <div class="col-md-9"> | ||
− | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;"> | + | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">DNA gel extraction</h2> |
<h4> | <h4> | ||
1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube. | 1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube. | ||
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<br /> | <br /> | ||
</h4> | </h4> | ||
− | <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;"> | + | <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 | <h4>Agarose-Electrophoresis is used in order to see if the PCR product is correct and seperate DNA by the number of | ||
base pairs. | base pairs. | ||
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<br /> | <br /> | ||
</h4> | </h4> | ||
− | <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;"> | + | <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">Gibson assembly</h2> |
<h4> | <h4> | ||
1. Set up the reaction. | 1. Set up the reaction. | ||
Line 221: | Line 247: | ||
<h4> </h4> | <h4> </h4> | ||
<h4> </h4> | <h4> </h4> | ||
− | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;"> | + | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">HPLC</h2> |
<h4> | <h4> | ||
for acrylic acid | for acrylic acid | ||
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<br /> | <br /> | ||
</h4> | </h4> | ||
− | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;"> | + | <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 | <h4>1. Pre-chill 1.5ml and 2ml microcentrifuge tubes, deionized water, 10% glycerol. Add 1ml LB to 2ml microcentrifuge | ||
tubes. | tubes. | ||
Line 265: | Line 291: | ||
<br /> | <br /> | ||
</h4> | </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> | </div> |
Latest revision as of 02:38, 2 November 2017