Difference between revisions of "Team:BIT-China/Project/Results"
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<h4 class="title-h4">Results:</h4> | <h4 class="title-h4">Results:</h4> | ||
<li class="my-content-li2">1. Synthesized the gene of human receptors <i>t1r2</i> and <i>t1r3</i>.</li> | <li class="my-content-li2">1. Synthesized the gene of human receptors <i>t1r2</i> and <i>t1r3</i>.</li> | ||
| − | <li class="my-content-li2">2. Expressed the heterologous GPCR in Saccharomyces Cerevisiae.</li> | + | <li class="my-content-li2">2. Expressed the heterologous GPCR in <i>Saccharomyces Cerevisiae</i>.</li> |
<li class="my-content-li2">3. The engineered yeast with T1R2 and T1R3 can sense the sweeteners.</li> | <li class="my-content-li2">3. The engineered yeast with T1R2 and T1R3 can sense the sweeteners.</li> | ||
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<h4 class="title-h5">2. Displayed the receptor location by immunofluorescence</h4> | <h4 class="title-h5">2. Displayed the receptor location by immunofluorescence</h4> | ||
| − | <p class="my-content-p">In order to confirm that T1R2, T1R3 can be located on the membrane of Saccharomyces Cerevisiae, we fused antigen tag 6xHIS and MYC at the N-terminals of T1R2, T1R3. The gene circuits for displaying the opposite of human receptors were constructed as Fig.12. </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 tag 6xHIS and MYC at the N-terminals of T1R2, T1R3. The gene circuits for displaying the opposite of human receptors were constructed as Fig.12. </p> |
<div class="my-content-box"> | <div class="my-content-box"> | ||
Revision as of 03:55, 28 October 2017
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
The 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.
The 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.
The 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.
The 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.
The 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 main work we needed to do was the expression of heterologous G protein coupled receptor (GPCR) T1R2 and T1R3 in Saccharomyces Cerevisiae. In this part, we used human sweet taste receptor as a sensing part in our system which can detect the sweetness and produce initial signals. So we had to achieve two goals:
Results:
Results in detail:
1. The gene synthesis of t1r2 and t1r3
Search the proteins information of T1R2 and T1R3 in NCBI. The ID numbers of these two proteins were 83756 (T1R2) and 80834 (T1R3). 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, was 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. Displayed the receptor location by immunofluorescence
In order to confirm that T1R2, T1R3 can be located on the membrane of Saccharomyces Cerevisiae, we fused antigen tag 6xHIS and MYC at the N-terminals of T1R2, T1R3. The gene circuits for displaying the opposite of human receptors were constructed as Fig.12.
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 after the genes sst2, ste2, far1 had been knocked out. After transformation, the expression of t1r2, t1r3 was induced by adding 2% galactose. T1R2 and T1R3 were confirmed to be displayed on the membrane of yeast by immunofluorescence. (More details is showed in our protocols)
The scale bars represent 10µm
As picture showing, the red fluorescence showed up (or appeared) around the cells and no red fluorescence on the other structures of the cells was observed 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 detecting sweeteners
Although we had simulated the function of T1R2 and T1R3 in modeling, there was no direct method to solve the 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, in which all the interference genes was knocked out. More detail 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 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)
Fig. 13 A simplified model of the αpheromone pathway with T1R2/T1R3
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.
So we selected two sweeteners, fructose and sucrose, for first testing.
Firstly, we selected a single cell clone of CEN.PK2-1C with our 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 expression of t1r2 and t1r3. Incubated at 30℃ for 12 hours.
Then added sweeteners into culture according to Table 1
| 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 red fluorescence intensity at a single cell level. The red fluorescence intensity is showed in Fig.14.
Fig. 14 The red fluorescence intensity of experimental group and control group after 12, 16 hours
As the picture shows, the fructose group and sucrose group established obvious increasing compared to control group from 12 hour to 16 hour.
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.
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.
In order to confirm this result, we repeated this experiment three times.
The all result shows the similar tendency. These result demonstrate that our system can work as we expectation.
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)
Fig. 15 The schematic diagram of G mutation
Summary
