Difference between revisions of "Team:NCKU Tainan/Results"

Result / Proof of concept

U-know Achiments

1. We constructed and submitted our Detection circuit and Safety circuit that produce the function of glucose sensor and suicide system.

2. We successfully differentiated diabetic urine sample with control with statistical significance and great sensitivity.

3. We characterized the relation between glucose concentration and RFP fluorescent intensity and derived the prediction formula for urine glucose level to quantify the amount of glucose.

4. We validated our bio-safety suicide system.

5.We built functional prototypes to let our U.coli to work in real world condition

Glucose Detection

Experiment Design

The goal for our Detection circuit to detect and indicate the presence of urine glucose with promoter PI. To measure the precise level of urine glucose , we will look at the fluroscence intensity of RFP.

We induce our U.coli detection circuit with urine sample containing different glucose level , and measure the fluorescent intensity both kinetically and at different timepoints

characterization of glucose induced RFP expression

To preliminary test the function of our Detection circuit , we induced our U coli for 12 hours with final glucose concentration of : 0, 5 ,15 ,30 , 60 mM/L

Fig2 result: PI_RBS_RFP_TT transformed in E.coli BL21 DE3 . After 12-hour induction with final glucose concentration of : 0, 5 ,15 ,30 , 60 mM/L , the fluroscence is observed under excitation ray of 470 nm and with the filter “LEE filter 019”

To further quantify the fluorescent intensity ,and simultaneously the growth curve of U coli , we used final glucose concentration of : 0, 5 ,15 ,30 mM/L to induce our U coli for 8 hours with total volume of 20 ml , and measure the fluorescent intensity at excitation/emission wavelength 562nm/599nm and OD600 for growth curve.

Fig3 result: above is the growth curve and fluorescent intensity , 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/L were add to induce the expression of RFP . The fluorescent intensity were measured every 2 hours , after total time exceeds 10 hours , experiments with 5 ,15 ,30 mM/L glucose has 2~4 fold induction compared to control 〈0 mM/L〉. 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/L does not show significant difference .

Proof of Concept : Differentiating diabetic urine with normal

To proof that our Ucoli do have the function of differentiating diabetic urine sample 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 urine sample surpass the upper limit of control , we can refer the urine sample “diabetic “ , and the diabetic test has sensitivety : / spreciticity :

Fig4 result: the 95% prediction interval of glucose positive 〈5mmol/L〉urine sample 〈green〉and glucose negative〈0.1mmol/L〉 urine 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 urine sample〈red〉as the “diabetic threshold “ , we performed a test to clarify whether our U coli can differentiate diabetes 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 our Ucoli do Differentiate diabetic urine with norma

To further elaborate our proof of concept ,we have a statistical calculation

Proof of Concept : Prediction and Quantifying the amount of urine glucose

After successfully differentiating diabetic urine sample with control ,we want determine the amount of glucose present in urine sample. Thus, we used final glucose concentration of : 0,1,3 5 ,15 ,30,45 mM/L to induce our U coli for 12 hours in 96 well and measure the fluorescent intensity at excitation/emission wavelength 562nm/599nm , the difference of fluorescent intensity between each and every group become significant at time T= 8hr.

Fig5 result : fluorescence intensity with 95% confidence interval after 8 hour induction with final glucose concentration 0,1,3 5 ,15 ,30,45 mM/L , all differences between each group shows statistical significance with P < 0.05 , proving that out U.coli do differentiate urine sample containing various amount of glucose , ranging from 0~45 mM/L.

To verify the formula , we performed a test to clarify whether our U coli can predict and quantify the concentration of urine glucose.

Reuslt : with n=63, the average recovery rate is 104% 〈95% ~114%〉. which proved that our Ucoli do predict and quantify the concentration of urine glucose

In conclution .by applying this prediction formula , proved that our Ucoli do predict and quantify the concentration of urine glucose.

Experiment Design

The goal for our Safety circuit to protect our U.coli user and the environment by lysing and killing the U.coli with promoter pBAD that can be activated by arabinose , which is present in our reading column 〈 Device.〉 To determine the efficacy of the Safety circuit , we measured the growth curve and performed CFU assay.

Growth Inhibition and Lysis activated by Arabinose

Promoter pBAD , is activated by arabinose and repressed by glucose . when activated the downstream lysis gene will lyse and kill the Ucoli , to preliminary test the function of our Safety circuit , we induced our U coli 3 hours with the following condition ,and kinetically measure the OD 600 as the indicator of bacteria concetration

Fig: expression of promoter pBAD in different arabinose and gucose concentration , the prescense of glucose will repress promoter pBAD even when arabionse is present

Fig7: result: pBAD-RBS-lysis-TT transformed in E.coli BL21 DE3 , was culture for 6 hr and transfferd〈1%〉to modified M9 meduim containing different concentration of arabionse and glucose , and OD600 was measured kinetically by 96well plate reader. Compared to control , arabinose significantly inhibited the growth of U.coli after time T>50 mins . In addition ,when glucose and arabinose are both present , glucose will repress the growth inhibitory effect of arabinose , making the cells continue to growth . Still , compared to control , the growth rate is slightly decreased by arabinose , even with the presence of glucose.

To further validate the effect of our Safety circuit , we performed a CFU assay .