Difference between revisions of "Part:BBa K1969007"

Latest revision as of 15:14, 27 October 2016

Nano-lantern(cAMP-1.6) regulated by ADH1 promoter

The sequence of Nano-lantern(cAMP-1.6) is cloned downstream the yeast ADH1 promoter and ended with an ADH1 terminator to generate a expression cassette in yeast Saccharomyces cerevisiae.

This composite part consists of the full length yeast ADH1 promoter BBa_K1969003, the cAMP reporter protein Nano-lantern (cAMP1.6) BBa_K1969000, the terminator BBa_K1486025 and two artificially designed scars, BBa_K1969008 and BBa_K1969009.

Fig.1 The picture of mechanism. Upon binding with extracellular glucose, the Gpa2 protein will disassociate from Gpr1 and activates adenylyl cyclase, yielding cAMp surge which can be detected by our composite part product.

Nano-lantern(cAMP-1.6) contains a portion of enhance YFP with 10 amino acids deleted at C terminus, denoted as Venus△C10, a portion of mutated Renilla luciferase with 3 amino acids deleted at N terminus, Rluc8, and a cAMP binding domain of EPAC1 (Exchange Protein Directly Activated by cAMP) flanked by two separate parts of Rluc8. Upon binding with the cAMP molecule, the catalytic activity of the split luciferase will increase as the separate parts are brought together because of the conformational change in EPAC, thus results in the change in luminescence via the BRET effect. Thus this part can tell us the relative concentration of extracellular ligands.

Fig.2 Characterization of expressed Nano-lantern(cAMP 1.6). (A) The fluorescent microscopy results together with the DIC result show the successful expression of NLc in transformants. Scale bar: 10μm; (B) The florescence of transformants was quantified using ImageJ, the value was the average difference between the yeast cell and background under three different fields and normalized with the fluorescence of pADH1-NLc. Data was analyzed using Student-t test, *** indicates p < 0.001.

As the cAMP surge in yeast triggered by GPCR sensing mechanism is incredibly fast and decays within 5 minutes, we monitor the glucose response in first 5 minutes. The results shows that the luminescence in engineered strain was obviously higher than control group (Fig.3). This part showed a good positive correlation between luminescence and the concentration of glucose, and we may need more experiment groups to improve the statistical power as the p values of one-way ANOVA was quite confusing in most of the experimental groups. As the results show, strain SMT-pAN has a good relationship between luminescence and glucose concentration at 0-1min (Fig.3).

Fig.3 The luminescence of transformants after glucose stimulation. (A) Dose dependent effects of glucose exposure on cAMP mediated luminescence measured simultaneously in 96-well plate format. Data was shown as average of three parallel groups. The pseudo-color scale indicates intensities of emitted luminescence signals; (B) P values calculated by One-way ANOVA at different time points within three parallel groups, the red line indicated the value of 0.05; (C) The R values calculated by linear regression of the average luminescence at different time points and the the logarithm of the glucose concentration (except 0mM) to base 10. Red line indicated the value of 0.9.

In order to detect the changes in glucose concentration in pathologic urine samples, we added corresponding amount of glucose into urine samples collected from a healthy individual to make mimic pathologic urine samples with glucose concentration of 0, 10, 50 mM. As the P value was the least at 30s in strain SMT-pAN, we compared the glucose response at 30s after adding mimic pathologic urine samples (Fig.4). The results showed postitive correlation between three strains. Although the significance between 10mM treatment groups and the control groups is not promising, we found our device capable of differentiate mimic pathologic urine samples containing 50mM glucose and the healthy urine.

Fig.4 Luminescence triggered by mimic pathologic urine samples with gradient glucose concentration. All data are presented as mean ± SD, n=3. Significance was analyzed using Student-t test.

Sequence and Features