Team:BIT-China/Results
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.
Antibacterial circle
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 antibacterial circle appears in the panel.
(2)△sst2 strain (sst2 is knocked out successfully) and the sensitivity of △sst2 strain to α factor will be increased. Meanwhile the larger antibacterial circle will appear when the amount of α factor is reduced.
(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 antibacterial circle will not be shown either.
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 Antibacterial circle 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.
(2)the growth curve of △sst2 is bit lower than CEN.PK2-1C. It’s strongly inhibited by α factor.
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
With the increase of the concentration of α factor, the fluorescence intensity increased.
With the increase of the concentration of α factor, the fluorescence intensity increased.
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.
