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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">DNA gel extraction</a> | + | <a href="#section-1">Core-part</a> |
| </li> | | </li> |
| <li> | | <li> |
− | <a href="#section-2">Electrophoresis</a> | + | <a href="#section-2">System</a> |
| </li> | | </li> |
| <li> | | <li> |
− | <a href="#section-3">Gibson assenbly</a> | + | <a href="#section-3">Pathway</a> |
| </li> | | </li> |
| <li> | | <li> |
− | <a href="#section-4">HPLC</a> | + | <a href="#section-4">Product</a> |
| </li> | | </li> |
| <li> | | <li> |
− | <a href="#section-5">Knock out genes of Ecoli</a> | + | <a href="#section-5">Conclusion</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;">DNA gel extraction</h2> | + | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">1. Core-part:the activity of rate limiting enzyme ceaS2 has been improved</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;">Electrophoresis</h2> | + | <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">2.System:S. cerevisiae is more suitable for chassis cells than E. coli</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;">Gibson assembly</h2> | + | <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">3.Pathway:Successfully build a new acrylic acid synthesis pathway and increase acrylic acid production</h2> |
| <h4> | | <h4> |
| 1. Set up the reaction. | | 1. Set up the reaction. |
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| <h4> </h4> | | <h4> </h4> |
| <h4> </h4> | | <h4> </h4> |
− | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">HPLC</h2> | + | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">4.Product:Multi - Conditional Optimization of Acrylic Cell Factory Catalytic Reaction Process</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;">Knock out genes of E ▪ coli (MG1655)</h2> | + | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">5.Conclusion </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. |
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| <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> |