Difference between revisions of "Team:BIT-China/Project/Results"

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       <h4 class="title-h5">2. 2.Display the location of the receptor by immunofluorescence</h4>
 
       <h4 class="title-h5">2. 2.Display the location of the receptor by immunofluorescence</h4>
       <p class="my-content-p">In order to confirm that T1R2, T1R3 can be located on the membrane of <i>Saccharomyces Cerevisiae</i>, we fused antigen tags MYC and 6xHIS at the N-terminals of T1R2, T1R3 respectively. The gene circuits for displaying the location of human receptors were constructed as Fig.4. </p>
+
       <p class="my-content-p">In order to confirm that T1R2, T1R3 can be located on the membrane of <i>Saccharomyces Cerevisiae</i>, we fused antigen tags MYC and 6xHIS at the N-terminals of T1R2, T1R3 respectively. The gene circuits for displaying the location of human receptors were constructed as Fig.11. </p>
  
 
       <div class="my-content-box">
 
       <div class="my-content-box">
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       <h4 class="title-h5">3. Function test of T1R2 and T1R3 by adding sweeteners</h4>
 
       <h4 class="title-h5">3. Function test of T1R2 and T1R3 by adding sweeteners</h4>
 
       <p class="my-content-p">Although we had simulated the function of T1R2 and T1R3 in modeling, there was still a problem whether T1R2, T1R3 in yeast cells can sense the sweeteners. In order to prove the function of this receptor in yeast cell, another plasmid with our reporter device, pRS42K-mRFP-<i>CYC1t</i>, was transformed into <i>CEN.PK2-1C</i>, More details about this part of work will be displayed in host website <a href="https://2017.igem.org/Team:BIT-China/Project/Transduction">transduction</a> and <a href="https://2017.igem.org/Team:BIT-China/Project/Detection">detection</a>.  </p>
 
       <p class="my-content-p">Although we had simulated the function of T1R2 and T1R3 in modeling, there was still a problem whether T1R2, T1R3 in yeast cells can sense the sweeteners. In order to prove the function of this receptor in yeast cell, another plasmid with our reporter device, pRS42K-mRFP-<i>CYC1t</i>, was transformed into <i>CEN.PK2-1C</i>, More details about this part of work will be displayed in host website <a href="https://2017.igem.org/Team:BIT-China/Project/Transduction">transduction</a> and <a href="https://2017.igem.org/Team:BIT-China/Project/Detection">detection</a>.  </p>
       <p class="my-content-p">The sense device, BBa_2368007 and BBa_2368008, was expressed successfully in our host yeast. And if the receptor can detect the sweetener, the mRFP which is the signal output will be expressed then we can detect the signal. (Fig. 6)</p>
+
       <p class="my-content-p">The sense device, BBa_2368007 and BBa_2368008, was expressed successfully in the host yeast. Once the receptor can respond the presence of sweeteners, the <i>mRFP</i> will be expressed as output. (Fig. 13)</p>
  
 
       <div class="my-content-box">
 
       <div class="my-content-box">
 
               <img style="width: 50%; height: auto;" src="https://static.igem.org/mediawiki/2017/c/c8/T--BIT-China--2017result_pic6.png" />
 
               <img style="width: 50%; height: auto;" src="https://static.igem.org/mediawiki/2017/c/c8/T--BIT-China--2017result_pic6.png" />
               <span>Fig. 13 A simplified model of the αpheromone pathway with T1R2/T1R3</span>
+
               <span>Fig. 14 A simplified model of the αpheromone pathway with T1R2/T1R3</span>
 
       </div>
 
       </div>
 
+
  <p class="my-content-p">Following the above, we separately added a list of sweeteners like fructose, sucrose, glucose into the culture to detect the red fluorescence intensity based on whether its regulatory interaction with sweet taste receptor T1R2-T1R3. </p>
      <p class="my-content-p">So when we adding sweeteners like fructose, sucrose, glucose and so on into the culture to detect the red fluorescence intensity was a way to prove that T1R2 and T1R3 feature. </p>
+
       <p class="my-content-p">So we selected two sweeteners, fructose and sucrose, for the first testing. </p>
       <p class="my-content-p">So we selected two sweeteners, fructose and sucrose, for first testing. </p>
+
       <p class="my-content-p">Firstly, we selected a single cell clone of <i>CEN.PK2-1C</i> with the circuits mentioned previously and cultured this clone in SD medium with 2% glucose and corresponding concentration of antibiotic G418 at 30℃ overnight. </p>
       <p class="my-content-p">Firstly, we selected a single cell clone of <i>CEN.PK2-1C</i> with our circuits mentioned previously and cultured this clone in SD medium with 2% glucose and corresponding concentration of antibiotic G418 at 30℃ overnight. </p>
+
       <p class="my-content-p">After harvesting cells, we centrifuged the cells and added new SD medium with 2% galactose and antibiotic G418 for inducing the expression of T1R2 and T1R3. Incubated at 30℃ for 12 hours. </p>
       <p class="my-content-p">After harvesting cells, we centrifuged the cells and added new SD medium with 2% galactose and antibiotic G418 for inducing expression of <i>t1r2</i> and <i>t1r3</i>. Incubated at 30℃ for 12 hours. </p>
+
 
       <p class="my-content-p">Then added sweeteners into culture according to Table 1</p>
 
       <p class="my-content-p">Then added sweeteners into culture according to Table 1</p>
 
       <div class="my-img-box" style="justify-content: flex-start;">
 
       <div class="my-img-box" style="justify-content: flex-start;">
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       <p class="my-content-p">After adding sweeteners, we used plate reader to measure the fluorescence intensity and OD600, calculated the value of fluorescence intensity/ OD600 as the red fluorescence intensity at a single cell level. The red fluorescence intensity is showed in Fig.14. </p>
+
       <p class="my-content-p">After adding sweeteners, we used plate reader to measure the fluorescence intensity and OD<sub>600</sub>, calculated the value of fluorescence intensity/ OD<sub>600</sub> as the mean fluorescence intensity at the single cell level. The mean fluorescence intensity is showed in Fig.15. </p>
  
 
       <div class="my-content-box">
 
       <div class="my-content-box">
 
               <img style="width: 50%; height: auto;" src="https://static.igem.org/mediawiki/2017/b/b8/T--BIT-China--2017result_pic7.png" />
 
               <img style="width: 50%; height: auto;" src="https://static.igem.org/mediawiki/2017/b/b8/T--BIT-China--2017result_pic7.png" />
               <span>Fig. 14 The red fluorescence intensity of experimental group and control group after 12, 16 hours</span>
+
               <span>Fig. 15 The mean fluorescence intensity of experimental group and control group after 12, 16 hours</span>
 
       </div>
 
       </div>
  
  
       <p class="my-content-p">As the picture shows, the fructose group and sucrose group established obvious increasing compared to control group from 12 hour to 16 hour. </p>
+
       <p class="my-content-p">As the picture shows, the fructose group and sucrose group established obvious increase compared to control group from 12h to 16h. </p>
       <p class="my-content-p">The red fluorescence intensity at a single cell level all increased, but the sucrose group and fructose group increased faster than control group in single level, which meant adding sweetener, indeed accelerated the fluorescence protein production in yeast cells on our system.</p>
+
       <p class="my-content-p">The red fluorescence intensity at the single cell level all increased, but the sucrose group and fructose group increased faster than control group, which meant adding sweeteners indeed accelerated the fluorescence protein production in yeast cells in our system.</p>
       <p class="my-content-p">In other words, our system can be sensitive to sweeteners. Besides, the red fluorescence intensity increasing rate of fructose group was higher than sucrose group’s, which means that the signal of fructose is stronger than sucrose. This result also conforms to the result measured through people tastes. </p>
+
       <p class="my-content-p">In other words, our system can be sensitive to sweeteners. Besides, the red fluorescence intensity increasing rate of fructose group was higher than sucrose group’s, which means that the sweetness of fructose is higher than sucrose’s. This result also conforms to the result measured through people tastes. </p>
       <p class="my-content-p">In order to confirm this result, we repeated this experiment three times.</p>
+
       <p class="my-content-p">In order to confirm this result, we repeated this experiment three times.  
      <p class="my-content-p">The all result shows the similar tendency. These result demonstrate that our system can work as we expectation.</p>
+
The all results showed the similar tendency. These results demonstrate that the system can work as our expectation.</p>
       <p class="my-content-p">Through the fluorescence intensity is still at a low level, the distinction between each group can be detected. The reason may is that natural signal pathway from yeast can’t adapt well with the human being sweetness receptor. The improving work is continuing. (Fig. 15)</p>
+
       <p class="my-content-p">Though the fluorescence intensity is still at a low level, the distinction between each group can be observed obviously. The reason may be that natural signal pathway in yeast can’t adapt well with the human sweet taste receptor. The improvements of this work are continuing. (Fig. 16)</p>
  
 
       <div class="my-content-box">
 
       <div class="my-content-box">
 
               <img style="width: 80%; height: auto;" src="https://static.igem.org/mediawiki/2017/9/9a/T--BIT-China--2017result_pic8.png" />
 
               <img style="width: 80%; height: auto;" src="https://static.igem.org/mediawiki/2017/9/9a/T--BIT-China--2017result_pic8.png" />
               <span>Fig. 15 The schematic diagram of G mutation  </span>
+
               <span>Fig. 16 The schematic diagram of G<sub>α</sub> mutation  </span>
 
       </div>
 
       </div>
 
       <h4 class="title-h4">Summary </h4>
 
       <h4 class="title-h4">Summary </h4>
         <li class="my-content-li2">1. We had optimized codon and synthesized human receptor gene <i>t1r2</i>, t1r3 using OE-PCR successfully.</li>
+
         <li class="my-content-li2">1. We had the optimized codon and synthesized human receptor gene T1R2,T1R3 successfully by using OE-PCR.</li>
         <li class="my-content-li2">2. We constructed the gene into shuttle vector and expressed this heterologous receptor in yeast successfully confirmed by immunofluorescence</li>
+
         <li class="my-content-li2">2.The heterologous receptor was expressed and located on the cell membrane successfully in yeast, which has been confirmed by immunofluorescence.</li>
         <li class="my-content-li2">3. Our system can sense sucrose, fructose. And our system can work as we expectation. </li>
+
         <li class="my-content-li2">3.Our system can sense sucrose, fructose as our expectation. </li>
  
  

Revision as of 10:13, 28 October 2017

BIT-CHINA

Result

The strategy for the function of engineered CEN.PK2-1C stains lacks △far1 +△sst2, △sst and △far1+△sst2+△ste2 were divided into the following parts;

(1). Testing the inhibitive effects on the growth of engineered CEN.PK2-1C, whose cellular period can be stopped at the G1 stage by α factor.

(2). Expression and identification of Pfus+RFP+cyc1t+PRS42K in engineered CEN.PK2-1C strains △far1+△sst2, △far1+△sst2+△ste2.

Zone of inhibition

Plan and method

The growth of CEN.PK2-1C is inhibited by α factor,We want to test the function of △sst2, △far1+△sst2 and △far1+△sst2+△ste2 by comparing with CEN.PK2-1C .

(1)The influence of the growth of bacteria caused by different amount of α factor in the solid plate.

(2)The observed difference among the function of engineered CEN.PK2-1C strains, △far1+△sst2, △far1+△sst2+△ste2 acted on the growth of CEN.PK2-1C, △sst2, △far1+△sst2, △far1+△sst2+△ste2 impaired by different amount of α factor.

Results and discussion

(1)The bacteria strain of CEN.PK2-1C stops growing in G1 due to the effects acted by the α factor and Zone of inhibition appears in the panel.



Fig.1 The diagrammatic sketch of zone of inhibition.

(2)△sst2 strain (sst2 is knocked out successfully) and the sensitivity of △sst2 strain to α factor will be increased. Meanwhile the larger Zone of inhibition will appear when the amount of α factor is reduced.

Fig.2 The diagrammatic sketch of zone of inhibition.

(3)The far1 of △far1+△sst2, △far1+△sst2+△ste2 is knocked out successfully, and this strain will still keep growing under the effects acted by α factor and Zone of inhibition will not be shown either.

Fig.3 The diagrammatic sketch of zone of inhibition.

Conclusion: Because far1 can make cellular period stop at the G1 stage by α factor and sst2 can reduce the intensity of the transmitted signal. So we want to knock out of far1 and sst2.From the above results we can see that in the α factor concentration range, the strain will be inhibited by α factor. With the increase of the concentration of the alpha factor,the radius of Zone of inhibition increases and the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking out of the sst2 gene, making the strain respond to α factor enhanced.

Growth curve

The impacts acted by the various concentration of α factor to the growth of the bacteria immersed into liquid.

The research of the impacts acted by various concentration of α factor to the growth of Cen.PK2-1C, △sst2 of CEN.PK2-1C.

Plan and method

The gowth of engineered CEN.PK2-1C strains were determined by measuring the initial rates of α factor at concentrations of 0-15 µM . We take out 5ml cultivating bacteria liquid of each of them during 30h and correct them by the YPD solution which not inoculates yeasts after shake culture. We obtain the value of OD600 at position of wavelength of 600nm by UV-Vis spectrophotometer.

The results and discuss

(1)With the increasing concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.



Fig.4 The diagrammatic sketch of growth curve.

(2)the growth curve of △sst2 is bit lower than CEN.PK2-1C. It’s strongly inhibited by α factor.



Fig.5 The diagrammatic sketch of growth curve.

Conclusion:From the above results we can see that in the α factor concentration range, the strain will be inhibited by α factor. With the increase of the concentration of the alpha factor, the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking down the sst2 gene, making the strain respond to α factor enhanced.

Fluorescence

Test the expression of Pfus+mRFP+cyc1t+pRS42K in engineered CEN.PK2-1C strains, △far1+△sst2, △far1+△sst2+△ste2, △sst2.

We research the impacts acted by various concentration of α factor to the amount of expression of fluorescence as well as the the growth of engineered CEN.PK2-1C strains, △far1+△sst2、△far1+△sst2+△ste2,△sst2 containing the examined circuit.

Plan and method

We set the concentration of alpha factor as 0umol/L,1 umol/L, 2.5 umol/L, 5 umol/L,10 umol/L,15 umol/L.We take out 5ml cultivating bacteria liquid of each of them after 0、4、8、10、12、14、16、18、20、22、24、26、28、30h respectively and correct them by the YPD solution which not inoculated yeasts after shake culture. Afterwards,the intensity of triggered fluorescence is measured in microplate reader(584/607).We create the coordianate system of the expression of fluorescence of engeneering yeast by setting the value of that intensity of triggered fluorescence/OD600 as Y-axis and time taken as X- axis.

The results and discuss



Fig.6 The diagrammatic sketch of fluorescence curve.



Fig.7 The diagrammatic sketch of fluorescence curve.



Fig.8 The diagrammatic sketch of fluorescence curve.

The strongest expression of fluorescence belongs to △far1+△sst2, while that of △far1+△sst2+△ste2 and CEN.PK2-1C are similar.

Conclusion:From the above results we can see that in the α factor concentration range, the expression of mRFP was induced. With the increase of the concentration of the alpha factor, the fluorescence expression was enhanced. And more obvious. Knocking down the sst2 and far1gene, making the strain respond to α factor enhanced.

Sensing

The expression of human sweet taste receptor in yeast

In the sensing part, the G protein coupled receptor (GPCR) T1R2 and T1R3 were heterologously expressed in Saccharomyces Cerevisiae due to its ability to regulate a wide range of cellular processes such as the senses of taste and smell. Moreover, we constructed a mRFP-based assay to achieve proper detection of sweetness responses.

So we had to achieve two goals:

  • 1. To express T1R2 and T1R3 in Saccharomyces Cerevisiae.
  • 2. Construct a response signal circuit to confirm that our receptors can detect the sweeteners.
  • Results:

  • 1. Synthesized the gene of human receptors T1R2 and T1R3
  • 2. Expressed the heterologous GPCR in Saccharomyces Cerevisiae.
  • 3. The engineered yeast with T1R2 and T1R3 can sense the sweeteners.
  • Details of the results:

    1. The gene synthesis of T1R2 and T1R3

    GPCR sequences are extracted from NCBI’s NR database. The sweet taste receptor T1R2-T1R3 (ID: 83756 and 80834) responds to diverse stimuli associated with the human sense of sweet taste, such as sucrose, d-tryptophan and aspartame. In reliable expression in Saccharomyces Cerevisiaei, we finished the codon optimization and reverse transcription of protein sequences into DNA sequences on the website http://54.235.254.95/gd/. The sequences of our receptors were established into the parts BBa_K2368003 and BBa_K2368004.

    We synthesized the human receptor genes T1R2 and T1R3 using building block which based on overlapping-extension PCR. The PCR result was showed as Fig.9.

    Fig. 9 The PCR result of synthetic genes T1R2 (2520 bp) and T1R3 (1989 bp).

    We used the synthetic genes T1R2, T1R3 to construct the gene circuits for receptors expression. The galactose-induced promoter, PGal1/PGal10was used to express the T1R2,T1R3. We cloned the circuits (BBa_2368007 and BBa_2368008) into the shuttle vector pESC-Leu in E.coli Top 10.

    Fig. 10 The gene circuits of T1R2 and T1R3 expression cassettes (BBa_2368007 and BBa_2368008).
    Fig. 11 The PCR result of T1R2 (3448 bp) and T1R3 (2814 bp) expression cassettes.

    2. 2.Display the location of the receptor by immunofluorescence

    In order to confirm that T1R2, T1R3 can be located on the membrane of Saccharomyces Cerevisiae, we fused antigen tags MYC and 6xHIS at the N-terminals of T1R2, T1R3 respectively. The gene circuits for displaying the location of human receptors were constructed as Fig.11.

    Fig. 12 The gene circuit of T1R2 and T1R3 expression cassettes with His and MYC tags

    The plasmid, pESC-Leu-T1R2-MYC-T1R3-HIS, was transformed into engineered yeast CEN.PK2-1C (sst2, ste2, far1). After transformation, the expression of T1R2,T1R3 was induced by adding 2% galactose. T1R2 and T1R3 were confirmed to e located on the membrane of yeast by immunofluorescence. (More details is showed in our protocols).

    Fig. 13 The immunofluorescence results about the T1R2 and T1R3 linked with MYC, HIS respectively.The scale bars represent 10µm.

    As picture showing, the red fluorescence could be observed around the cells by confocal microscopy. Based on this stained result, we can confirm that T1R2 and T1R3 was expressed and located on the membrane successfully.

    3. Function test of T1R2 and T1R3 by adding sweeteners

    Although we had simulated the function of T1R2 and T1R3 in modeling, there was still a problem whether T1R2, T1R3 in yeast cells can sense the sweeteners. In order to prove the function of this receptor in yeast cell, another plasmid with our reporter device, pRS42K-mRFP-CYC1t, was transformed into CEN.PK2-1C, More details about this part of work will be displayed in host website transduction and detection.

    The sense device, BBa_2368007 and BBa_2368008, was expressed successfully in the host yeast. Once the receptor can respond the presence of sweeteners, the mRFP will be expressed as output. (Fig. 13)

    Fig. 14 A simplified model of the αpheromone pathway with T1R2/T1R3

    Following the above, we separately added a list of sweeteners like fructose, sucrose, glucose into the culture to detect the red fluorescence intensity based on whether its regulatory interaction with sweet taste receptor T1R2-T1R3.

    So we selected two sweeteners, fructose and sucrose, for the first testing.

    Firstly, we selected a single cell clone of CEN.PK2-1C with the circuits mentioned previously and cultured this clone in SD medium with 2% glucose and corresponding concentration of antibiotic G418 at 30℃ overnight.

    After harvesting cells, we centrifuged the cells and added new SD medium with 2% galactose and antibiotic G418 for inducing the expression of T1R2 and T1R3. Incubated at 30℃ for 12 hours.

    Then added sweeteners into culture according to Table 1

    Table 1. The sweeteners added for detecting function of the system
    Sweetener and concentration (w %) Description
    2% Fructose Experimental group
    2% Sucrose Experimental group
    0% Control group

    After adding sweeteners, we used plate reader to measure the fluorescence intensity and OD600, calculated the value of fluorescence intensity/ OD600 as the mean fluorescence intensity at the single cell level. The mean fluorescence intensity is showed in Fig.15.

    Fig. 15 The mean fluorescence intensity of experimental group and control group after 12, 16 hours

    As the picture shows, the fructose group and sucrose group established obvious increase compared to control group from 12h to 16h.

    The red fluorescence intensity at the single cell level all increased, but the sucrose group and fructose group increased faster than control group, which meant adding sweeteners indeed accelerated the fluorescence protein production in yeast cells in our system.

    In other words, our system can be sensitive to sweeteners. Besides, the red fluorescence intensity increasing rate of fructose group was higher than sucrose group’s, which means that the sweetness of fructose is higher than sucrose’s. This result also conforms to the result measured through people tastes.

    In order to confirm this result, we repeated this experiment three times. The all results showed the similar tendency. These results demonstrate that the system can work as our expectation.

    Though the fluorescence intensity is still at a low level, the distinction between each group can be observed obviously. The reason may be that natural signal pathway in yeast can’t adapt well with the human sweet taste receptor. The improvements of this work are continuing. (Fig. 16)

    Fig. 16 The schematic diagram of Gα mutation

    Summary

  • 1. We had the optimized codon and synthesized human receptor gene T1R2,T1R3 successfully by using OE-PCR.
  • 2.The heterologous receptor was expressed and located on the cell membrane successfully in yeast, which has been confirmed by immunofluorescence.
  • 3.Our system can sense sucrose, fructose as our expectation.
  • TOP