Difference between revisions of "Team:Mingdao/Basic Part"
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| − | + | <a href="https://2017.igem.org/Team:Mingdao" class="w3-bar-item w3-button w3-padding-large w3-white">Home</a> | |
| − | + | <a href="#" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-blue w3-hover-white" style="text-decoration: none; color: white">Achievements</a> | |
| − | + | <a href="https://2017.igem.org/Team:Mingdao/Demonstrate" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-hover-white" style="text-decoration: none; color: white">Project</a> | |
| − | + | <a href="https://2017.igem.org/Team:Mingdao/HP/Silver" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-hover-white" style="text-decoration: none; color: white">Human-Practice</a> | |
| − | + | <a href="#" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-hover-white" style="text-decoration: none; color: white">Notebook</a> | |
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| − | <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem;"> <i class="fa fa-bullseye fa-fw"></i> | + | <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem;"> <i class="fa fa-bullseye fa-fw"></i> Basic</button> |
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| − | <form action="https://2017.igem.org/Team:Mingdao/ | + | <form action="https://2017.igem.org/Team:Mingdao/Composite_Part" style="margin: 0 15px -10px 0;"> |
| − | <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem"> <i class="fa fa-bullseye fa-fw"></i> | + | <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem"> <i class="fa fa-bullseye fa-fw"></i> Composite</button> |
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| − | <form action="https://2017.igem.org/Team:Mingdao/ | + | <form action="https://2017.igem.org/Team:Mingdao/Improve" style="margin: 0 15px -10px 0;"> |
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| − | <form action="https://2017.igem.org/Team:Mingdao/ | + | <form action="https://2017.igem.org/Team:Mingdao/Part_Collection" style="margin: 0 15px -20px 0;"> |
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<h1 class="display-3"> </h1> | <h1 class="display-3"> </h1> | ||
| − | <h1 class="display-3"> | + | <h1 class="display-3 text-white"> Basic Part </h1> |
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| − | + | <div class="card-header text-center" style="color: white; letter-spacing: 0.3rem;font-size:1.8rem; background-color:#004480"> | |
| − | + | Basic Part | |
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| − | + | <!-- PART start --> | |
| − | + | <img src="https://static.igem.org/mediawiki/2017/a/ad/Mingdaochuck1003-76.jpeg" width="50%"> | |
| − | + | <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No. :</b> BBa_K2230022</h3> | |
| − | + | <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Type: </b>Basic part</h3> | |
| − | + | <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning: </b></h3> | |
| − | + | <h4> | |
| − | + | The STM1128 gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3. This part has been sequenced. | |
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| − | + | <span style="padding: 0 8rem 0 8rem">Gene Cloning – glucose transporter genes </span> | |
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| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/5/53/Mingdaochuck1003-78.jpeg" alt="Card image cap" style="display: block; margin: auto"> | ||
| + | </div> | ||
| + | <!-- second --> | ||
| + | <div class="card"> | ||
| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/9/9a/Mingdaochuck1003-79.jpeg" alt="Card image cap" style="display: block; margin: auto"> | ||
| + | </div> | ||
</div> | </div> | ||
| + | <div class="card-group" style="margin-right: 5rem; margin-left: 5rem"> | ||
| + | <!-- third --> | ||
| + | <div class="card"> | ||
| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/f/fd/Mingdaochuck1003-80.jpeg" alt="Card image cap" style="display: block; margin: auto"> | ||
| + | </div> | ||
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| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/9/98/Mingdaochuck1003-81.jpeg" alt="Card image cap" style="display: block; margin: auto"> | ||
| + | </div> | ||
| + | <!-- fourth END --> | ||
| + | </div> | ||
| + | <div class="card-group" style="margin-right:5rem; margin-left: 5rem"> | ||
| + | <!-- fifth --> | ||
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| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/4/49/Mingdaochuck1003-82.jpeg" alt="Card image cap" style="display: block; margin: auto"> | ||
| + | </div> | ||
| + | <!-- sixth --> | ||
| + | <div class="card"> | ||
| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/6/68/Mingdaochuck1003-83.jpeg" alt="Card image cap" style="display: block; margin: auto"> | ||
| + | </div> | ||
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| + | <button class="btn text-white" style="letter-spacing: 0.3rem;background-color: #004480; margin-top: 3rem"> | ||
| + | <i class="fa fa-flask" aria-hidden="true"></i> | ||
| + | Functional assay | ||
| + | <span class="sr-only"></span> | ||
| + | </button> | ||
| + | <!-- PART start --> | ||
| + | <div style="margin-top: 3rem; margin-bottom: 3rem"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/e6/Mingdaochuck1003-93.jpeg" width="60%"> | ||
</div> | </div> | ||
| − | + | <h4> | |
| + | In order to express the genes in E. coli for demonstration and in probiotics for proof-of-concept in a real world. We chose promoter CP29 that is a strong constitutive promoter working well in both E. coli and Lactobacillus spp1. The biobrick part, CP29-RBS-aeBlue (BBa_K1033280) was used and to be assembled with the transporter genes. | ||
| + | </h4> | ||
| + | |||
| + | <!-- PART end --> | ||
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| + | </div> | ||
| + | </div> | ||
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</div> | </div> | ||
| − | <!-- | + | </div> |
| + | <!-- Recombination end --> | ||
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| + | <!-- CSMU --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-10 col-sm-10" style="display: block; margin: auto; margin-top: 3rem"> | ||
| + | <div class="card"> | ||
| + | <h4 class="card-header text-center" style="letter-spacing: 0.3rem; font-size:1.8rem; background-color: #004480; color: white">Experiment & Result</h4> | ||
| + | <div class="card-body"> | ||
| + | <!-- TEST --> | ||
| + | <div id="test1019" style="padding: 3rem 3rem 3rem 3rem "> | ||
| + | <!-- Box --> | ||
| + | <div class="box" style="background-color: rgba(255, 255, 255, 0); margin-right: 50px"> | ||
| + | <h4 style="font-color: #a6a6a6;">To measure glucose uptake by the engineered E. coli expressing PTS system or Na+/glucose cotransporter, the bacteria were culture in LB broth supplemented with 34μg/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD<sub>600</sub> = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.</h4> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-6 col-sm-12"> | ||
| + | <br><br> | ||
| + | <img src=" https://static.igem.org/mediawiki/2017/7/76/Mingdaophil1019-6.png" width="100%" > | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <h4 style="text-align: center; padding-top: 2rem;"></h4> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="col-md-6 col-sm-12"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/6/69/Mingdaophil1019-9.png" width="70%" style="display: block; margin: auto;"> | ||
| + | </div> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <h4>Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.</h4> | ||
| + | </div> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/a/a7/Mingdaophil1019-8.png" width="55%" style="display: block; margin: auto;"> | ||
| + | </div> | ||
| + | |||
| + | <!-- Result Section --> | ||
| + | <div class="col-md-12 col-sm-12" style="margin-top: 5rem"> | ||
| + | <span class="w3-center w3-padding-small w3-black w3-xlarge w3-wide w3-animate-opacity"> <span class="w3-hide-small"></span> </span> | ||
| + | <span class="w3-center w3-padding-large w3-wide w3-xlarge w3-animate-opacity">Result</span> | ||
| + | <br/><br/> | ||
| + | </div> | ||
| + | |||
| + | <div class="col-md-12 col-sm-12" style="padding-top: 3rem; padding-bottom: 3rem"> | ||
| + | <h4> | ||
| + | Fig. 3 represented that the cell growth of E. coli expressing the Na+/Glucose transporter was comparable and even slightly higher than the control group. The glucose began to be absorbed at the 3<sup>rd</sup> hour. The glucose uptake efficiency was greater in Na+/Glu group than in control group with 1.2 times difference. | ||
| + | </h4> | ||
| + | <h4> | ||
| + | In our result, glucose absorption efficiency was just slightly enhanced in Na+/glucose transporter expressing bacteria compared to general E. coli. To further increase glucose uptake, it is necessary to think about the glucose metabolism or conversion to other materials when entering into the cell. | ||
| + | </h4> | ||
| + | </div> | ||
| + | <!-- Fig 4. ROW --> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/5/5e/Mingdaophil1019-3.png" width="70%" style="display: block; margin: auto;"> | ||
| + | </div> | ||
| + | |||
| + | <!-- Fig 4. ROW END --> | ||
| + | <!-- caption of fig --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <strong style="padding-left: 6rem">Fig 3. The cell growth overnight and glucose uptake of E. coli expressing the Na+/Glu transporter at different time point</strong> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- caption of fig END --> | ||
| + | <!-- Result Section --> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- TEST --> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- CSMU END --> | ||
| + | </section> | ||
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| + | {{Team:Mingdao}} | ||
Latest revision as of 03:55, 31 October 2017
Basic Part
Basic Part
Part No. : BBa_K2230022
Type: Basic part
Cloning:
The STM1128 gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3. This part has been sequenced.
In order to express the genes in E. coli for demonstration and in probiotics for proof-of-concept in a real world. We chose promoter CP29 that is a strong constitutive promoter working well in both E. coli and Lactobacillus spp1. The biobrick part, CP29-RBS-aeBlue (BBa_K1033280) was used and to be assembled with the transporter genes.
Experiment & Result
To measure glucose uptake by the engineered E. coli expressing PTS system or Na+/glucose cotransporter, the bacteria were culture in LB broth supplemented with 34μg/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD600 = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.
Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.
Result
Fig. 3 represented that the cell growth of E. coli expressing the Na+/Glucose transporter was comparable and even slightly higher than the control group. The glucose began to be absorbed at the 3rd hour. The glucose uptake efficiency was greater in Na+/Glu group than in control group with 1.2 times difference.
In our result, glucose absorption efficiency was just slightly enhanced in Na+/glucose transporter expressing bacteria compared to general E. coli. To further increase glucose uptake, it is necessary to think about the glucose metabolism or conversion to other materials when entering into the cell.
Fig 3. The cell growth overnight and glucose uptake of E. coli expressing the Na+/Glu transporter at different time point
© iGEM Mingdao 2017. Design: Kevin Li. All rights reserved.
