Difference between revisions of "Team:SZU-China/Results"

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                         <h3 class="uppercase color-primary mb40 " style="margin-bottom: 40px;font-size:50px"><center>Results</center> </h3>
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                         <h3 class="uppercase color-primary mb40 " style="margin-bottom: 40px;font-size:50px"><center>COLLABORATIONS</center> </h3>
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                        <p class="lead mb40" style="width:60%;margin:0 auto">After transferring the device plasmid, we finally equipped WB800 bacteria with 3 functional gene modules, which have functions of secreting Carbonic anhydrase, accelerating germination and surviving in alkaline environment respectively. We also conducted experiment based on these 3 aspects.</p>
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                            <center>Carbonic anhydrase</center>
 
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                            <p class="lead" style="color:black;">We verified the translation of Carbonic anhydrase(CA) by SDS-PAGE protein electrophoresis and Coomassie blue staining. As you can see, the band near 35kDa was α-CA protein which only exist in the transformed bacteria but not in the WB800. We can conclude that the protein was translated successfully.</p><br/>
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                        <center>SSTi-SZGD</center> <br/>
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                        <center>SMS-Shenzhen</center> <br/>
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                        <center>Shenzhen-SFLS</center><h3>
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                            <center style="padding-top:5px;color:#2f2f2f;font-size:14px;width:50%;margin:0 auto">Fig.1 The intracellular expression of Carbonic anhydrase.  This western blot result shows that we have successfully transformed gene CA into B.subtilis WB800. From the left lane to the right, there is marker, two α-CA proteins and the whole intracellular proteins of B.subtilis of WB800.</center>
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                          <p class="lead" style="color:black">We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.</p>
 
  
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                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 2. The difference in the hydration of CO2 by Carbonic anhydrase(CA). The  graph illustrate the ability of the hydration of CO2  by different carbonic anhydrase(CA). Through the decrease in the pH per second. We verify that α-CA has the most efficient way in the hydration of CO2 , for it has the highest rate in catalyzing the production of hydrogen ion so pH can decrease.</center>
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                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px">Fig 3. The enzyme activity of different carbonic anhydrase. The column graph shows the enzyme activity of carbonic anhydrase, by calculating the production of hydrogen ions produced in average time.</center>
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                            <p class="lead" style="color:black">Since the fact that CA can also catalyze the hydration reaction of ester,which can be used to assay the activity of CA, we conducted another measurement on CA according to a method from Verpoorte JA(1967).</p><br/>
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                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px;width:90%;margin:0 auto">Fig 4. The difference in the hydration of CO2 by Carbonic anhydrase(CA). The  graph illustrate the ability of the hydration of CO2  by different carbonic anhydrase(CA). Through the decrease in the pH per second. We verify that α-CA has the most efficient way in the hydration of CO2 , for it has the highest rate in catalyzing the production of hydrogen ion so pH can decrease.</center>
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                                <center style="padding-top:5px;color:#2f2f2f;font-size:14px;width:90%;margin:0 auto">Fig 5. The enzyme activity of different carbonic anhydrase. The column graph shows the enzyme activity of carbonic anhydrase, by calculating the production of hydrogen ions produced in average time.</center>
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                            <p class="lead" style="color:black">From the above results, we may draw the conclusion that transferred strains range a higher level in CA activity.</p>
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                            <p class="lead" style="color:black;">To verify the gene gerAa for promoting spore germination, we divide the germination detection into two groups: control group (B. subtilis WB800) and gerAa group (B.subtilis WB800 that has gerAa gene with promotor pssPb). Theoretically, through the Germination detection, gerAa group should exhibit higher germination proportion and germination rate than control group, as gerAa is overexpressed. As shown in Fig.1, the germination rate and proportion varied between control group and gerAa group. gerAa group shows a high proportion in germination and rapid rate in germination. Therefore, gene gerAa is verified to be in good condition and can work efficiently.</p><br />
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                            <center style="padding-top:5px;color:#2f2f2f;font-size:14px;width:50%;margin:0 auto">Fig 6. The germination result of different groups. The line graph demonstrate the germination status in WB800 group(control group) and gerA group(B. subtilis WB800 that has been transformed with gene gerA). As the OD600 decrease, the endospores are germinating. The rate of decrease can descript the ability of germinate, which is mediated by germination gene.</center>
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                            <p class="lead" style="color:black">We determined CA activity according a method modified from Brownell et al. (1991). The CA activity was measured by the different rates of decrease in pH in the system involving 5ml CO2-saturated water, 10ml buffer and 500ul CA samples or Dl water. We calculated the activity according to the formula U= (T0 -Ts )/ T0 ,where T0and Ts represent time for pH change with blank and samples, respectively. By testing different kinds of CA each for several times and taking the average ,we got the CA activity per milliliter of bacteria solution.</p>
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                            <center>Alkali Resistance</center>
 
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                            <p class="lead" style="color:black;margin-bottom:5px">To verify the function of TupA for alkaline resistance in our modified baceria, we test the WB800 strain by the following steps:</p><br/>
+
                        <center>SSTi-SZGD</center> <br/>
−
                            <p class="lead" style="color:black;">At first, we conduct a gene transformation experiment of origin strain WB800, by transforming an extraordinary gene TupA. Then we collect the mono bacteria from the petri dish to culture. We also have a series of assays following up: extracting the plasmids, digesting the plasmids with endonuclease and running through DNA electrophoresis just to verify we have successfully transformed gene TupA into the B.subtilis WB800 (as shown in Fig.7).</p>
+
                        <center>SMS-Shenzhen</center> <br/>
 +
                        <center>Shenzhen-SFLS</center><h3>
  
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                            <div style="text-align:center;">
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                            </div>
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                            <center style="padding-top:5px;color:#2f2f2f;font-size:14px;width:50%;margin:0 auto">Fig 7. The result of DNA electrophoresis. In the right lane places the marker. And the DNA sample extracted from the digested plasmid in a transformed WB800 is on its left.</center>
 
 
                              
 
                              
 +
                            <div class="col-sm-6">
 +
                                <p class="lead mb40" style="color:black;">This year we made a long-term collaboration with SSTi-SZGD. We helped them to build a team and figure out how to participate in this competition, including team management, details of competition, and experiment protocols. In return, they helped us test enzyme activity of CA and plot standard curves using two different methods.</p>
 +
                                <p class="lead mb40" style="color:black">
 +
                                    You can see the <a href="#" style="color: rgb(75, 151, 165);font-weight:bold;font-size:20px">results </a>here.
 +
                                   
 +
                                </p>
  
−
                            <br/><br/>
 
−
                          <p class="lead" style="color:black">Furthermore, we want to know if the WB800 we transformed can express gene TupA. So we have designed the way to verify:</p>
 
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                            <p class="lead" style="color:#2f2f2f;">1. We divide the bacteria into two groups: one is the control group (B.subtilis WB800), and another is the experiment group (B.subtilis WB800 that includes gene TupA). And culture these two groups with liquid medium.</p>
 
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                            <p class="lead" style="color: #2f2f2f;  ">2. Prepare four types of solid medium with the pH of 9,10 and 11 respectively .</p>
 
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                            <p class="lead" style="color: #2f2f2f; ">3. When the concentration of the B.subtilis from two groups are nearly equal, take 5μL of sample and drop it on the plate(each plate has already been divided into 4 even zones) at the correct zone.(Also we have take 10μL of the sample to repeat). Culture the B.subtilis on the solid plate.</p>
 
−
                            <p class="lead" style="color: #2f2f2f; ">4. After 48hours, observe and verify the plate by dyeing with crystal violet before wash the plate with ddH₂O.</p>
 
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                                <img src="https://static.igem.org/mediawiki/2017/0/01/T--SZU-China--N4.jpg" style="" />
 
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                                <p style="padding: 5px 1px 2px 1px; color: #2f2f2f; font-size: 14px">Fig 8. The alkaline resistance result. By culturing the modified WB800(with a tupA gene) and WB800 in different pH.  They shows the different alkaline resistance, with the stained plaque indicating the survival bacteria.</p>
 
 
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                                <img class="cast-shadow" src="https://static.igem.org/mediawiki/2017/2/25/T--SZU-China--collab1.jpg" style="width:90%;float:left;margin:20px" />
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                            </div>
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                        <div class="row text-center" style="width:80%;margin:0 auto;clear:both">
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                            <div class="col-sm-5">
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                                <img class="cast-shadow" src="https://static.igem.org/mediawiki/2017/9/99/T--SZU-China--collab2.jpg" style="width:90%;float:left;margin:20px" />
 +
                            </div><br/><br/>
 +
                            <div class="col-sm-6">
 +
                                <p class="lead mb40" style="color:black;">In summer，we shared our laboratories with a high school iGEM team SMS-Shenzhen for they could not get into their campus during summer vacation. Furthermore, we also gave them some directions on experiments, involving transformation, ultrasonication and so on. Reciprocally, they helped us with regulating lab and data processing. We also helped team Shenzhen-SFLS with project model.</p>                             
 +
                          </div>
 
                              
 
                              
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                            <p></p><br /><br />
 
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                            <p class="lead" style="color:black">So we have come to the conclusion: gene TupA can regulate the alkaline resistance of B.subtilis.</p>
 
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                         <h3 class="uppercase color-primary mb40 mb-xs-24" style="margin-bottom: 40px;">
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                             <center>Conclusion</center>
+
                             <center>Bit</center>
 
                         </h3>
 
                         </h3>
  
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                             <p class="lead" style="color:black;margin-bottom:5px">Based on upstream experiment on genetic engineering level, our genetic modified WB800 successfully achieved the 3 functions needed for concrete self-repairing sysyem. Next, we will measure their true performance when embedded in the concrete as form of micro-capsule. See the <a href="https://2017.igem.org/Team:SZU-China/Demonstrate"class="color-primary" style="font-size:15px">DEMONSTRATE</a> page for our downstream results.</p><br />
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                             <div class="col-sm-6">
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                                <p class="lead mb40" style="color:black;">Before the semester began, we were invited to BIT campus for a deep bilateral communication and we interchanged not only the designs of our projects but also our experience on the iGEM competition, especially in members selection and management of the team. BIT-China is a large team with more than 30 iGEMers, while there are only 8 members in our team, but we firmly agreed that attitude is everything. At the same time, we gained available feedbacks from them. Their project this year were highly admired by 2 medical students in our team.</p>
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                            </div>
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                            <div class="col-sm-5">
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                                <img class="cast-shadow" src="https://static.igem.org/mediawiki/2017/a/aa/T--SZU-China--colla3.jpg" style="width:90%;float:left;margin:20px" />
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                            </div>
 +
                            <p class="lead mb40" style="color:black;clear:both;">
 +
                                &nbsp;&nbsp;&nbsp;&nbsp;After the CCiC, we interchanged our protocols with SCU-WestChina, and helped them with transformations.
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                    <span style="font-size: 3px;color: #4B97A5;font-size:27px"><center>REFERENCE</center></span>
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                <div style="width:90%;margin:0 auto;font-size:16px">
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[1] Krulwich T A, Ito M, Guffanti A A. The Na(+)-dependence of alkaliphily in Bacillus.[J]. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2001, 1505(1):158-168. </p>
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[2] Khalifah R G. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C[J]. Journal of Biological Chemistry, 1971, 246(8):2561. </p>
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[3] Cabreramartinez R M, Tovarrojo F, Vepachedu V R, et al. Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis.[J]. Journal of Bacteriology, 2003, 185(8):2457-64.</p>
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                            <div class="col-sm-6">
 +
                                <p class="lead mb40" style="color:black;">This year we made a long-term collaboration with SSTi-SZGD. We helped them to build a team and figure out how to participate in this competition, including team management, details of competition, and experiment protocols. In return, they helped us test enzyme activity of CA and plot standard curves using two different methods.</p>
 +
                                <p class="lead mb40" style="color:black">
 +
                                    You can see the <a href="#" style="color: rgb(75, 151, 165);font-weight:bold;font-size:20px">results </a>here.
 +
                                   
 +
                                </p>
 +
 +
                            </div>
 +
                            <div class="col-sm-5">
 +
                                <img class="cast-shadow" src="https://static.igem.org/mediawiki/2017/2/25/T--SZU-China--collab1.jpg" style="width:90%;float:left;margin:20px" />
 +
                            </div>
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                        </div>
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                            <div class="col-sm-5">
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                                <img class="cast-shadow" src="https://static.igem.org/mediawiki/2017/9/99/T--SZU-China--collab2.jpg" style="width:90%;float:left;margin:20px" />
 +
                            </div><br/><br/>
 +
                            <div class="col-sm-6">
 +
                                <p class="lead mb40" style="color:black;">In summer，we shared our laboratories with a high school iGEM team SMS-Shenzhen for they could not get into their campus during summer vacation. Furthermore, we also gave them some directions on experiments, involving transformation, ultrasonication and so on. Reciprocally, they helped us with regulating lab and data processing. We also helped team Shenzhen-SFLS with project model.</p>                             
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                          </div>
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                        </div>
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            </div>
 
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                            <center>Bit</center>
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                        </h3>
  
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                            <div class="col-sm-6">
 +
                                <p class="lead mb40" style="color:black;">Before the semester began, we were invited to BIT campus for a deep bilateral communication and we interchanged not only the designs of our projects but also our experience on the iGEM competition, especially in members selection and management of the team. BIT-China is a large team with more than 30 iGEMers, while there are only 8 members in our team, but we firmly agreed that attitude is everything. At the same time, we gained available feedbacks from them. Their project this year were highly admired by 2 medical students in our team.</p>
 +
                            </div>
 +
                            <div class="col-sm-5">
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                                <img class="cast-shadow" src="https://static.igem.org/mediawiki/2017/a/aa/T--SZU-China--colla3.jpg" style="width:90%;float:left;margin:20px" />
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                            </div>
 +
                            <p class="lead mb40" style="color:black;clear:both;">
 +
                                &nbsp;&nbsp;&nbsp;&nbsp;After the CCiC, we interchanged our protocols with SCU-WestChina, and helped them with transformations.
 +
                            </p>
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Revision as of 17:43, 1 November 2017


Collaboration

COLLABORATIONS

SSTi-SZGD

SMS-Shenzhen

Shenzhen-SFLS

Collaboration

COLLABORATIONS

SSTi-SZGD

SMS-Shenzhen

Shenzhen-SFLS



This year we made a long-term collaboration with SSTi-SZGD. We helped them to build a team and figure out how to participate in this competition, including team management, details of competition, and experiment protocols. In return, they helped us test enzyme activity of CA and plot standard curves using two different methods.

You can see the results here.



In summer，we shared our laboratories with a high school iGEM team SMS-Shenzhen for they could not get into their campus during summer vacation. Furthermore, we also gave them some directions on experiments, involving transformation, ultrasonication and so on. Reciprocally, they helped us with regulating lab and data processing. We also helped team Shenzhen-SFLS with project model.

Bit

<

Before the semester began, we were invited to BIT campus for a deep bilateral communication and we interchanged not only the designs of our projects but also our experience on the iGEM competition, especially in members selection and management of the team. BIT-China is a large team with more than 30 iGEMers, while there are only 8 members in our team, but we firmly agreed that attitude is everything. At the same time, we gained available feedbacks from them. Their project this year were highly admired by 2 medical students in our team.

    After the CCiC, we interchanged our protocols with SCU-WestChina, and helped them with transformations.


This year we made a long-term collaboration with SSTi-SZGD. We helped them to build a team and figure out how to participate in this competition, including team management, details of competition, and experiment protocols. In return, they helped us test enzyme activity of CA and plot standard curves using two different methods.

You can see the <a href="#" style="color: rgb(75, 151, 165);font-weight:bold;font-size:20px">results </a>here. 

                               <img class="cast-shadow" src="T--SZU-China--collab1.jpg" style="width:90%;float:left;margin:20px" />

                       </div>

                               <img class="cast-shadow" src="T--SZU-China--collab2.jpg" style="width:90%;float:left;margin:20px" />


In summer，we shared our laboratories with a high school iGEM team SMS-Shenzhen for they could not get into their campus during summer vacation. Furthermore, we also gave them some directions on experiments, involving transformation, ultrasonication and so on. Reciprocally, they helped us with regulating lab and data processing. We also helped team Shenzhen-SFLS with project model.

                   </div>
               </div>
             

           </div>
       </section>

       <section class="cd-section" style="padding:0;background-color:white">

Bit

<

Before the semester began, we were invited to BIT campus for a deep bilateral communication and we interchanged not only the designs of our projects but also our experience on the iGEM competition, especially in members selection and management of the team. BIT-China is a large team with more than 30 iGEMers, while there are only 8 members in our team, but we firmly agreed that attitude is everything. At the same time, we gained available feedbacks from them. Their project this year were highly admired by 2 medical students in our team.

                               <img class="cast-shadow" src="T--SZU-China--colla3.jpg" style="width:90%;float:left;margin:20px" />

    After the CCiC, we interchanged our protocols with SCU-WestChina, and helped them with transformations. 



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