Difference between revisions of "Team:SMS Shenzhen/Experiments/PriliminaryExperiments"

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        <h1 style="text-align:center">Priliminary Experiments Report(the characterization of glucose, dextran and sucrose)</h1>
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    <h1>I Abstract</h1>
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        <h1> </h1>
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    <p style="font-size: 18px"> The goal of this experiment was to measure the physical properties of dextran, sucrose and glucose. Measuring the physical properties of these three sugars is fundamental in our experiment. we assume that glucose will be stained by our stains. Then, dextran and sucrose will not decompose. We set the three sugars into different concentrations and then put them in a water bath. Finally, the absorbance of solution is finally measured by the microplate reader. </p>
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        <h1>I Abstract</h1>
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        <p style="font-size:18px">The goal of this experiment is to measure the physical properties, especially stability, of dextran, sucrose and glucose as the substrates and the products of the reaction. </br>In the experiment, a standard curve in relation between glucose concentration and absorbance will be determined under neutral pH condition in room temperature. Then, the stabilities of sugars will be measured by DNS chromogenic method, after being water bathing in different realistic temperatures and pH conditions simulating the oral cavity.</p>
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        <h1>II Background</h1>
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    <h1> II Background and Hypothesis</h1>
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        <p style="font-size:18px"> Seq 1: K2224001</p>
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    <p style="font-size: 18px"> 1. Our colorant is called DNS which will react with the reducing sugar in a 100 ℃ water bath;  </p>
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        <p style="font-size:18px">Promoter+RBS+Lecanicillium lecanii strain 432 endochitinase (chit1)+Terminator</p>
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    <p style="font-size: 18px"> 2. Glucose is found in our literature to be a reducing sugar. We assume that DNS colorant reaction, the higher the concentration, the higher the absorbance of the solution after the reaction (OD540); </p>
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        <!--图片放在这里-->
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    <p style="font-size: 18px"> 3. Dextran and sucrose are not reducing sugars, DNS does not react with them, so their concentrations are not related to absorbance. </p>
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<img src="https://static.igem.org/mediawiki/2017/f/ff/EnzymeA1-SMS_shenzhen.jpg  " width="100%" >
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        <h1> </h1>
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        <p style="font-size:18px"> Seq 2: K2224002</p>
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        <p style="font-size:18px">Promoter+RBS+Lecanicillium lecanii strain CA-12 cuticle-degrading proteinase (Pr1)+Terminator</p>
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        <!--图片放在这里-->
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<img src=" https://static.igem.org/mediawiki/2017/a/a9/EnzymeA2-SMS_shenzhen.jpg " width="100%" >
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        <h1> </h1>
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        <h2> Enzyme Activity Definition:</h2>
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        <p style="font-size:18px">The enzyme activity is defined by the amount of N- acetyl glucosamine that produced by Chitinase' s hydrolysis to chitin divided by the number of cells (or bacteria concentration). Since the product, N- acetyl glucosamine, can lead to light absorbtion peak at 585nm and the light absorption can be detected by Spectrophotometer, we can figure out the relative activity of enzyme DQ412944.</p>
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        <h1>III Objective</h1>
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    <h1> III Purpose</h1>
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        <p style="font-size:18px"></p>
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    <p style="font-size: 18px"> 1. Construct the standard curve between the concentration of glucose and its absorbance.  </p>
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        <p style="font-size:18px"></p>
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    <p style="font-size: 18px"> 2. Considering sucrose and dextran, in the polymer state, are not the reducing sugar and do not react with DNS. But their monsters will react with them. So we measured two other factors that could cause their decomposition, pH and temperature. Taking into account the effects of the external environment on Dextran and sucrose in the real reaction, we control the variables separately and design experiments on the pH and hydration of dextran under thermal conditions.  </p>
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        <h1>IV Hypothesis</h1>
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    <h1>IV Experiment Protocol</h1>
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        <p style="font-size:18px">There is a linear relationship between Glucose concentration and the absorbance in 550nm.</p>
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    <h1> </h1>
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    <h2>Experiment: glucose standard curve</h2>
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    <p style="font-size: 18px"> 1. Configure 1 mg/ml standard glucose solution, converse it to 250 mg/250 ml (adding 0.25 g glucose with distilled water to the constant volume of the 250ml volumetric flask) </p>
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        <h1>V Procedure</h1>
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    <p style="font-size: 18px"> 2. Use the solution in the first step as stock liquor, take 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml from it into the 15 ml test tube adding distilled water to 1ml. </p>
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        <h1> </h1>
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    <p style="font-size: 18px"> 3. Add 2ml DNS into each tube. </p>
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        <h3>1. Construction of the standard curve between the concentration of glucose and its absorbance on 550nm.</h3>
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    <p style="font-size: 18px"> 4. After adequate mixing, put each tube in 100 °C water bath for two minutes.  </p>
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        <p style="font-size:18px">(1) Materials:</p>
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    <p style="font-size: 18px">  5. After cooling, add distilled water to the solution to 15ml. </p>
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        <p style="font-size:18px">a) TOP10 of Escherichia coli and LB solid medium materials (yeast extract, tryptone, sodium chloride, agar powder, DD water) LB liquid culture medium material (yeast extract, tryptone, sodium chloride, DD water)</p>
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    <p style="font-size: 18px"> 6. Take 100ul from each tube into the labelling board, note down the positions of solutions in different concentrations.   </p>
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        <p style="font-size:18px">b) inoculation ring, antibiotic (chloramphenicol), Petri dish, adjustable transfer gun, constant temperature shaking incubator, LB medium, coating rod, alcohol lamp, EP tube</p>
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    <p style="font-size: 18px"> 7. Use microplate reader to measure solutions’ absorbance at 540 nm wave length and use distilled water as the control group.   </p>
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        <p style="font-size:18px">c) balance, water bath, centrifuge, visible spectrophotometer, 1 mL glass cuvette, distilled water</p>
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    <p style="font-size: 18px"> 8. The resulted data were photographed and kept as excel in USB flash disk. </p>
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        <p style="font-size:18px">d) chitinase activity detection kit:</p>
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    <h1> </h1>
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        <p style="font-size:18px">Extract: liquid 100mL x 1 bottles, stored at 4℃.</p>
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        <p style="font-size:18px">Reagent 1: liquid 20mL x 1 bottles, stored at 4℃.</p>
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        <p style="font-size:18px">Reagent 2: liquid 10mL * 1 bottles, stored at 4℃.</p>
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        <p style="font-size:18px">Reagent 3: liquid 10mL * 1 bottles, stored at 4℃.</p>
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        <p style="font-size:18px">Reagent 4: liquid 20mL * 1 bottles, stored at 4℃ away from light.</p>
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        <h1> </h1>
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        <p style="font-size:18px">(2) Method:</p>
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        <p style="font-size:18px">a) Glucose Standard Curve Determination</p>
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        <p style="font-size:18px">Configure 1 mg/ml standard glucose solution, converse it to 250 mg/250 ml (adding 0.25 g glucose with distilled water to the constant volume of the 250ml volumetric flask)</p>
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        <p style="font-size:18px">Use the solution in the first step as stock liquor, take 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml from it into the 15 ml test tube (the yellow shagg tube), adding distilled water to 1ml.</p>
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        <p style="font-size:18px">Add 2ml DNS into each tube.</p>
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        <p style="font-size:18px">After adequate mixing, put each tube in 100 °C water bath for two minutes. After cooling, add distilled water to the solution to 15ml.</p>
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        <p style="font-size:18px">Take 100ul from each tube into the labelling board, note down the positions of solutions in different concentrations. 7. Use micro plate spectrophotometer to measure solutions’ absorbance at 540 nm wave length and use distilled water as the control group. </p>
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        <p style="font-size:18px">The resulted data were photographed and kept as excel in USB flash disk.</p>
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        <p style="font-size:18px">b) Preparation of the Measurement of Dextran Thermal Stability and Acid Stability: </p>
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        <blockquote><p style="font-size: 18px">
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            Acid stability preparation:
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        </p></blockquote>
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        <p style="font-size: 18px">Take 1g dextran and dissolve it into 12.5ml Tris-HCl buffer and 12.5ml DD water.</p>
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        <p style="font-size: 18px">Divide the solution into three parts and mark clearly (acid/ alkaline/ neutral +dex).</p>
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        <p style="font-size: 18px">A small beaker can be used to add three drops of concentrated sulphuric acid into the portion marked "acid" and two grains sodium hydroxide into the portion marked “alkali”. Mix each portion uniformly and leave the “neutral” portion unchanged. </p>
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        <p style="font-size: 18px">Take 100ul from each portion into a 2 ml eppendorf tube and mark it (acid/ alkaline/ neutral+dex). Let them stand on the finger tube frame.</p>
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        <blockquote><p style="font-size: 18px">
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    <h1> </h1>
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            Thermal stability preparation:
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    <h2>Experiment: preparation of the measurement of dextran thermal stability and acid stability</h2>
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        </p></blockquote>
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    <h3>· Acid stability preparation</h3>
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        <p style="font-size:18px">c)Take the remaining substrate marked “neutral" in the preparation for acid stability and put them into three 2 ml finger tubes, each 100ul, and mark them clearly(45, 35, 25+dex)</p>
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    <p style="font-size: 18px">1. Take 1g dextran and dissolve it into 12.5ml Tris-HCl buffer and 12.5ml DD water.   </p>
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        <p style="font-size:18px">c)React 10 min at the corresponding temperature, then put the eppendorf tube into the previous finger tube frame. </p>
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    <p style="font-size: 18px">2. Divide the solution into three parts and mark clearly (acid/ alkaline/ neutral +dex).</p>
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        <h1> </h1>
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    <p style="font-size: 18px"> 3. A small beaker can be used to add three drops of concentrated sulphuric acid into the portion marked "acid" and two grains sodium hydroxide into the portion marked “alkali”. Mix each portion uniformly and leave the “neutral” portion unchanged. </p>
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        <h2>2. Measurement of decomposition of Dextran and Sucrose under realistic thermal and pH condition</h2>
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    <p style="font-size: 18px"> 4. Take 100ul from each portion into a 2 ml tube and mark it (acid/ alkaline/ neutral+dex). Let them stand on the finger tube frame. </p>
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        <h1> </h1>
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    <h1> </h1>
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        <h3>(1) Materials:</h3>
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        <p style="font-size:18px">a) visible spectrophotometer, water bath pot, magnetic stirrer, adjustable transfer gun, 1mL glass cuvette, 1.5 mL EP tube and distilled water. Chitinase activity detection kit:</p>
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        <p style="font-size:18px">b) reagent 1: liquid, stored at 4℃</p>
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        <p style="font-size:18px">Reagent 2: powder, stored at 4℃. Dissolve with 10 mL distilled water before use.</p>
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        <p style="font-size:18px">Reagent 3: powder, store at 4℃ away from light. Before adding 10 mL reagent, boiling water dissolves.</p>
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        <p style="font-size:18px">Reagent 4: powder, stored at 4 degrees. Dissolve with 50 mL distilled water before use.</p>
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        <p style="font-size:18px">Reagent 5: liquid, stored at 4 ℃.</p>
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        <p style="font-size:18px">Standard: liquid, 0.25 mol/mL standard tyrosine solution, stored at 4 c..</p>
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        <p style="font-size:18px">c) inoculation ring, antibiotic (chloramphenicol), Petri dish, adjustable transfer gun, constant temperature shaking incubator, LB medium, coating rod, alcohol lamp, EP tube</p>
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        <h1> </h1>
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        <h3>(2) Method:</h3>
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        <p style="font-size:18px">a) Draw Standard Calibration Curve for Bacterial Concentration.</p>
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        <p style="font-size:18px">b) Glycerol Bacteria Recovery.</p>
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        <p style="font-size:18px">c) Crude Enzyme Extraction: according to the number of cells (104): reagent volume (mL) ratio of 500~1000:1 (5 million cells with 1mL reagent), ultrasonic cell crushing ice bath (ultrasonic power 300W, 3 seconds, 7 seconds between the total time, 3min and 8000g); 4, C, centrifugal 10min, Pick supernatant and place on ice to for further measuring. (Note: get the number of bacteria by measuring the ABS value.)</p>
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        <h1>VI Data</h1>
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    <h2>Experiment: preparation for measurement of sucrose thermal stability and acid stability
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        <h1> </h1>
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</h2>
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        <p style="font-size:18px">1. Standard Calibration Curve for Bacterial Concentration:</p>
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    <h3>· Acid stability preparation</h3>
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        <h1> </h1>
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    <p style="font-size: 18px">1. Take 1g sucrose and dissolve it into 12.5ml Tris-HCl buffer and 12.5ml DD water.</p>
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        <!--图片放在这里-->
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    <p style="font-size: 18px"> 2. Divide them into three parts and mark clearly (acid/ alkaline/ neutral +dex).  </p>
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<img src=" https://static.igem.org/mediawiki/2017/5/55/EnzymeA3-SMS_shenzhen.jpg  " width="100%" >
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    <p style="font-size: 18px"> 3. A small beaker can be used to add three drops of concentrated sulphuric acid into the portion marked "acid" and two grains sodium hydroxide into the portion marked “alkali”. Mix each portion uniformly and leave the “neutral” portion unchanged.   </p>
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        <h1> </h1>
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    <p style="font-size: 18px"> 4. Take 100ul from each portion into a 2 ml tube and mark it (acid/ alkaline/ neutral+dex). Let them stand on the finger tube frame.  </p>
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        <!--图片放在这里-->
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<img src=" https://static.igem.org/mediawiki/2017/5/5d/EnzymeA4-SMS_shenzhen.jpg " width="100%" >
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        <h1> </h1>
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        <p style="font-size:18px">2. Chitinase Activity Measurement</p>
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        <h1> </h1>
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        <!--图片放在这里-->
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<img src=" https://static.igem.org/mediawiki/2017/4/47/EnzymeA5-SMS_shenzhen.jpg " width="100%" >
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        <h1> </h1>
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        <!--图片放在这里-->
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<img src=" https://static.igem.org/mediawiki/2017/a/a4/EnzymeA6-SMS_shenzhen.jpg " width="100%" >
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        <h1>VII Analysis</h1>
 
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        <h1> </h1>
 
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        <p style="font-size:18px">From the first set of data, we can clearly discover the linear relationship between sample bacteria concentration and the absorbance. Data suggest that as the concentration of bacteria fluid becomes greater, the relative absorbance increase proportionately. This result justified our assumption.</p>
 
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        <p style="font-size:18px">From the second set of data we can reach an agreement that our TOP10 Expression System is suitable for the expression of plasmid K2224001, which CDS is the modified chitinase. However, the chitinase activity curve with insufficient data only shows an uncertain relationship with time. We can define this relationship as a linear relationship but since it is weak and uncertain, further research and experiment should be taken, until a confirmation can be done.</p>
 
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        <p style="font-size:18px">Unfortunately, the expected protease activity measurement is not successfully done since the measurement kit is invalid by inappropriate preservation operation.</p>
 
 
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         <h1> Reference</h1>
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         <h1>V Result</h1>
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        <h1> </h1>
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    <p style="font-size: 18px"> To view our experiment result, please<a href="https://2018.igem.org/Team:SMS_Shenzhen/Results"><font color="green"><u>click here for more information.</font></u></a></p>
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        <p style="font-size:18px"><a href = "http://parts.igem.org/File:Chitinase_activity_measurement_kit.pdf">Chitinase activity measurement_kit</a></p>
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        <p style="font-size:18px"><a href = "http://parts.igem.org/File:A_research_for_Chitinase_in_Verticillium_lecanii.pdf">A research for Chitinase in Verticillium_lecanii.pdf</a></p>
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        <p style="font-size:18px"><a href = "http://parts.igem.org/File:A_simple_method_to_test_the_concentration_of_bacteria_fluid.pdf">A simple method to test the concentration of bacteria fluid.pdf</a></p>
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        <p style="font-size:18px"><a href = "http://http://parts.igem.org/File:Esterase_consortium_Process_Biochem_2016_%EF%BC%88marked.pdf">Esterase consortium Process Biochem 2016</a></p>
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        <p style="font-size:18px"><a href = "http://parts.igem.org/File:The_function_of_protease_and_chitinase_in_the_process_of_Verticillium_lecanii_invasion.pdf">The function of protease and chitinase in the process of Verticillium_lecanii invasion</a></p>
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        <p style="font-size:18px"><a href = "http://parts.igem.org/File:Chitinase_activity_measurement_kit.pdf">Chitinase_activity_measurement_kit</a></p>
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        <p style="font-size:18px"><a href = "http://parts.igem.org/File:Lab_opertaion_recording.docx">iGEM Lab_opertaion_recording for activity measurement</a></p>
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Revision as of 23:55, 17 October 2018

Title

Title

I Abstract

The goal of this experiment was to measure the physical properties of dextran, sucrose and glucose. Measuring the physical properties of these three sugars is fundamental in our experiment. we assume that glucose will be stained by our stains. Then, dextran and sucrose will not decompose. We set the three sugars into different concentrations and then put them in a water bath. Finally, the absorbance of solution is finally measured by the microplate reader.

II Background and Hypothesis

1. Our colorant is called DNS which will react with the reducing sugar in a 100 ℃ water bath;

2. Glucose is found in our literature to be a reducing sugar. We assume that DNS colorant reaction, the higher the concentration, the higher the absorbance of the solution after the reaction (OD540);

3. Dextran and sucrose are not reducing sugars, DNS does not react with them, so their concentrations are not related to absorbance.

III Purpose

1. Construct the standard curve between the concentration of glucose and its absorbance.

2. Considering sucrose and dextran, in the polymer state, are not the reducing sugar and do not react with DNS. But their monsters will react with them. So we measured two other factors that could cause their decomposition, pH and temperature. Taking into account the effects of the external environment on Dextran and sucrose in the real reaction, we control the variables separately and design experiments on the pH and hydration of dextran under thermal conditions.

IV Experiment Protocol

Experiment: glucose standard curve

1. Configure 1 mg/ml standard glucose solution, converse it to 250 mg/250 ml (adding 0.25 g glucose with distilled water to the constant volume of the 250ml volumetric flask)

2. Use the solution in the first step as stock liquor, take 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml from it into the 15 ml test tube adding distilled water to 1ml.

3. Add 2ml DNS into each tube.

4. After adequate mixing, put each tube in 100 °C water bath for two minutes.

5. After cooling, add distilled water to the solution to 15ml.

6. Take 100ul from each tube into the labelling board, note down the positions of solutions in different concentrations.

7. Use microplate reader to measure solutions’ absorbance at 540 nm wave length and use distilled water as the control group.

8. The resulted data were photographed and kept as excel in USB flash disk.

Experiment: preparation of the measurement of dextran thermal stability and acid stability

· Acid stability preparation

1. Take 1g dextran and dissolve it into 12.5ml Tris-HCl buffer and 12.5ml DD water.

2. Divide the solution into three parts and mark clearly (acid/ alkaline/ neutral +dex).

3. A small beaker can be used to add three drops of concentrated sulphuric acid into the portion marked "acid" and two grains sodium hydroxide into the portion marked “alkali”. Mix each portion uniformly and leave the “neutral” portion unchanged.

4. Take 100ul from each portion into a 2 ml tube and mark it (acid/ alkaline/ neutral+dex). Let them stand on the finger tube frame.

Experiment: preparation for measurement of sucrose thermal stability and acid stability

· Acid stability preparation

1. Take 1g sucrose and dissolve it into 12.5ml Tris-HCl buffer and 12.5ml DD water.

2. Divide them into three parts and mark clearly (acid/ alkaline/ neutral +dex).

3. A small beaker can be used to add three drops of concentrated sulphuric acid into the portion marked "acid" and two grains sodium hydroxide into the portion marked “alkali”. Mix each portion uniformly and leave the “neutral” portion unchanged.

4. Take 100ul from each portion into a 2 ml tube and mark it (acid/ alkaline/ neutral+dex). Let them stand on the finger tube frame.

V Result

To view our experiment result, pleaseclick here for more information.