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+ | <div> | ||
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+ | <header class="align-center"> | ||
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+ | <h2>Inserting expression vector </h2> | ||
+ | </header> | ||
+ | <header class="align-center"> | ||
+ | |||
+ | <h2>Choosing prokaryotic expression vector (pET-28a)</h2> | ||
+ | </header> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <h2>Designing primers and restriction sites </h2> | ||
+ | </header> | ||
+ | <p style="color:black;font-size:35px;"> | ||
+ | Chitin deacetylase 1 p5 GGATCCATGGCAAGAGTAAGATGCGG<br> | ||
+ | Chitin deacetylase 1 p3 AAGCTTTTAGAATCCCTCGCCC<br> | ||
+ | <br> | ||
+ | LOC108681649 p5 GGATCCATGCTGATGCATGTAGCCATC<br> | ||
+ | LOC108681649 p3 AAGCTTTTATATAATTTCCACGCCATCC<br> | ||
+ | <br> | ||
+ | LOC 108681651 p5 GGATCCATGTCTAGATTAAGATTCGTCC<br> | ||
+ | LOC 108681651 p3 AAGCTTTTAGAATAGTCCATCACCATTGG<br> | ||
+ | <br> | ||
+ | Verm p5 CGCGGATCCATGCTGACCGTTGAT<br> | ||
+ | Verm p3 GATTCGTGGTAGCTGAAGTTGCACTGGTC<br> | ||
+ | <br> | ||
+ | ChinB p5 CTTCAGCTACACCACGAATCCGGGTGTTAGC<br> | ||
+ | ChinB p3 CCGGAATTCCTACTGGAGTTGCCAC<br> | ||
+ | <br> | ||
+ | The restriction enzyme cutting sites of our experiment are BamH I (198) and Hind III (173)<br> | ||
+ | </p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <h2>Transfection </h2> | ||
+ | </header> | ||
+ | <p style="color:black;font-size:35px;"> | ||
+ | The plasmid was diluted 1000-fold and 10000-fold, and the competent bacteria BL-21 was added for 30 minutes in an ice bath. Then we heated the bacteria at 42 degrees for one minute, and ice-bathed for five more minutes. After that, we added 400 ml of LB without Kana antibiotic and shook the bacterial fluid. Finally, the bacteria were sprayed on a petri dish with kana antibiotics. | ||
+ | <header class="align-center"> | ||
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+ | <h2>Bacterial Fluid PCR to filter positive clones</h2> | ||
+ | </header> | ||
+ | <p style="color:black;font-size:35px;">To insure the plasmids were successfully transfected, we conducted bacterial fluid PCR. | ||
+ | Our groupmates take ten 1.5 ml EP tubes, add 200 μl of kana resistant medium separately, directly draw a single clone with a small pipette tip and push the TIP directly into an EP tube. We covered the EP tube and shake the bacteria at 37 degrees for 2-3 hours. Then 1μl of the bacterial solution was used as a template to identify the PCR positive clone. DNA gel electrophoresis was used to see if there was a target strip to judge. | ||
+ | </p> | ||
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+ | <h2>Inducing Expression </h2> | ||
+ | </header> | ||
+ | <p style="color:black;font-size:35px;"> | ||
+ | We set up various conditions to discover the most effecient condition. In total, we induced the sample in 180 different conditions. The samples are divided into different groups- the experiment group and the control group | ||
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+ | <!-- One --> | ||
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+ | <img src="https://static.igem.org/mediawiki/2018/e/ed/T--SDSZ_China--6.jpeg" class="rounded mx-auto d-block" alt="..." width="100%" height="100%" style="Padding:0px"> | ||
+ | <!--<h2>MODULES</h2>--> | ||
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+ | <!--<h2>MODULES</h2>--> | ||
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+ | <h2>Detecting Ability </h2> | ||
+ | </header> | ||
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+ | <header class="align-center"> | ||
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+ | <h2>Page electrophoresis </h2> | ||
+ | </header> | ||
+ | <p style="color:black;font-size:35px;"> | ||
+ | |||
+ | When the bacteria were induced, the page electrophoresis is necessary to identify whether the protein could be expressed successfully or not. | ||
+ | </p> | ||
+ | <header class="align-center"> | ||
+ | |||
+ | <h2>Measuring Enzyme Abilities </h2> | ||
+ | </header> | ||
+ | <p style="color:black;font-size:35px;"> | ||
+ | We add 1 mL 200 m g / L of substrate to a clean test tube, 3mL 0.05 mol / L phosphate buffer , 3 min 50 ° C water bath temperature protection Min, and add 1 mL enzyme solution, mix and heat in 50 degrees water 15 min. After that, we used boiling water to terminate the enzymatic reaction, and add water to a volume of 10 mL. Evenly, the solution was centrifuged at 3000r /min for 10 min. Measure the OD value of the upper clear. | ||
+ | Definition of enzyme unit: The amount of enzyme required to produce 1 gram of p-nitroaniline per hour under the above reaction conditions is defined as 1 enzyme activity ( U /mL ). | ||
+ | </p> | ||
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+ | <h2>References </h2> | ||
+ | </header> | ||
+ | <p style="color:black;font-size:35px;"> | ||
+ | |||
+ | Ding Guowei. Molecular characteristics and functional analysis of chitin deacetylase genes inOxya chinensis(Orthoptera:Acrididae). MS thesis. university of Shan Xi, 2015. | ||
+ | Tokuyasu, Ken, Mayumi Ohnishi-Kameyama, and Kiyoshi Hayashi. "Purification and characterization of extracellular chitin deacetylase from Colletotrichum lindemuthianum." Bioscience, biotechnology, and biochemistry 60.10 (1996): 1598-1603. | ||
+ | ElMekawy, Ahmed, et al. "Kinetic properties and role of bacterial chitin deacetylase in the bioconversion of chitin to chitosan." Recent patents on biotechnology 7.3 (2013): 234-241. | ||
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+ | </p> | ||
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Latest revision as of 18:56, 17 October 2018