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

Zone of inhibition

Plan and method

The growth of CEN.PK2-1C can be 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) Explore the influence of the growth of each strains cultured with different concentrations of α factor in the solid plate medium.

(2) Observe the growth situation of these strains and compare the differences among them. Analyze the functions of these strains cultured with different concentrations of α factor in the solid plate medium.

Results and discussion

(1) The strain CEN.PK2-1C stops growing at the phase G1 due to the effects of the α factor and zone of inhibition appears in the solid plate medium.

(2) The strain△sst2 is more sensitive to α factor. Meanwhile the larger zone of inhibition will appear when the concentration of α factor increased.

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

Conclusion： Knock out the gene sst2 can eliminate the inhibitory of the signal so that the strain △sst2 can amplify the signal when cultured with α factor. From the results above, the strain △far1+△sst2 didn’t stay at the phase G1, because the gene far1 knockout eliminates the growth arrest. Gene ste2 knockout makes the strain don’t response to the α factor.

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 growth of engineered CEN.PK2-1C strains were determined by measuring the initial rates of α factor at concentrations of 0-15 µM . We took 5ml cultivating liquid from each of them during 30h and correct them by the YPD solution which didn’t 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 was inhibited by α factor.

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

Conclusion：From the results above, we could see that in the α factor concentration range, the strain would be inhibited by α factor. With the increase of the concentration of the alpha factor, the inhibitory effect on the growth of the strain was enhanced. And more obviously. Knocking out the sst2 gene, making the strain of responding 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

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.

We used the synthetic genes T1R2, T1R3 to construct the gene circuits for receptors expression. The galactose-induced promoter, P_Gal1/P_Gal10was 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.

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.

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）.

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)

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

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.

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)

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.