Part:BBa_K1920001:Experience

Design

The goal of BBa_ K1920001 is to detect and indicate the presence of glucose with promoter PI.
To measure the precise level, we will look at the fluroscence intensity of RFP .
We induce our E.coli with glucose solution sample containing different glucose level and measure the fluorescent intensity both kinetically and at certain end point.

Result

We quantified the fluorescent intensity ,and simultaneously the growth curve of E. coli BL21 DE3 .
we used final glucose concentration of : 0, 5 ,15 ,30 mM to induce our U coli for 10 hours after 2-hour incubation with total volume of 20 ml , and measure the fluorescent intensity at excitation/emission wavelength 562nm/599nm and OD600 for growth curve .

Fig.1a Growth curve with different Glucose concentration.

Fig.1b Fluorescent intensity with different Glucose concentration.

Fig.1 are the growth curve(a) and fluorescent intensity(b), the experiments were done in E.coli BL21 DE3 in modified M9 medium , the cell were cultured in modified M9 medium for 2 hours and glucose of final concentration  : 0, 5 ,15 ,30 mM were add to induce the expression of RFP . The fluorescent intensity were measured every 2 hours , after total time exceeds 12 hours, experiments with 5 ,15 ,30 mM glucose has 2~4 fold induction compared to control 〈0 mM〉and the difference of fluorescent intensity between each group all shows statistical significance. The presence of glucose also affects the growth curve of our U.coli compared to control , but the growth curves of 5 ,15 ,30 mM does not show significant difference .

To further proof that BBa_ K1920001 do have the function of differentiating glucose solution with control , we calculated the average prediction interval of fluorescent intensity corresponding to each glucose concentration and we used the upper limit of control as the threshold of “positive” , that is , if the fluorescent intensity of a glucose solution sample surpass the upper limit of control , we can refer the glucose solution sample “positive “ , and the test has sensitivety : 95%

Fig.2

Fig2:the 95% prediction interval of glucose positive 〈5mM〉sample sample 〈green〉and glucose negative〈0mM〉 sample〈red〉，the two intervals can be separated after T> 101 mins . To verify the function , by applying the upper limit of the 95% prediction interval of glucose negative sample〈red〉as the “threshold “ , we performed a test to determine whether BBa_ K1920001 can differentiate positive from control . At time T= 101mis with n= 84 , our result shows sensitivity= 95% and if we delay the testing time to T=120mins , the sensitivity will be 100%.〈n=84〉, which proved that BBa_ K1920001 do Differentiate glucose solution with control

Discussion

The promoter PpI is a promoter designed for the E.coli which is slightly modified from a glucose induced promoter BBa_K861171. Monomeric RFP, BBa_E1010, is a highly engineered mutant of red fluorescent protein from Discosoma striata (coral). Combining them together become the indicator for glucose, also the major part in our project. In the normal condition, without glucose, cAMP binds to the cAMP receptor protein, which in its bound form, is able to bind tightly to the promoter and repress the gene downstream. On the contrary, high glucose concentration will result in the expression of the monomeric RFP.

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