Team:NCKU Tainan/Demonstrate
Demonstrate
After months of hard work and effort, iGEM NCKU Tainan 2019 is proud to reveal that ‘Oh My Gut’ is now complete! Our team has spared no effort in giving their all to ensure that our project goes smoothly. With this project, we hope to achieve our vision of providing a comprehensive solution to chronic kidney disease, and also spreading awareness on the significance of p-Cresol on human health.
Blood p-Cresol Reader
Our device, CreSense consists of three major parts: a centrifugal platform for blood plasma separation, a fluorescence intensity reader and a WiFi-module to monitor the fluorescence intensity online. In this section, we will demonstrate how our blood p-Cresol reader works.
Whole blood is injected into our microfluidic chip. All the user needs to do is to press a button to start the blood centrifugation. After approximately 15 minutes, the blood will be separated into two layers. The blood plasma will flow into the reaction chamber and react with our p-Cresol sensing bacteria. Then, the reader will show the real time fluorescence intensity reading on the LCD screen and simultaneously send it to an online database.
This device is meant to be placed in the diagnostic center and other healthcare service providers. However, this device is not limited to just patients who take our live therapeutic. Patients who are concerned about their health or is at a high risk of developing chronic kidney disease may also use this device as a preventative measure.
To demonstrate our engineered system works, we have made a video to explain each part of CreSense and the operating process.
Alternative Tyrosine Fermentation Pathway
iGEM NCKU Tainan 2019 engineered E. coli Nissle and introduced an alternative pathway for tyrosine in the gut by adding Tyrosine Ammonia Lyase. It converts tyrosine into a beneficial product, p-Coumaric acid. We improved the p-Coumaric acid production by changing the ribosome binding sites by from Native to B0034, the conversion of tyrosine into p-Coumaric acid increase by 1.73-fold.
We then further improved the conversion of tyrosine into p-Coumaric acid by adding a tyrosine transporter (BBa_K2997000). As seen in Fig 7., when BBa_K2997000 is added, the production of p-Coumaric acid is significantly higher than when BBa_K2998000 is not added. When tyrosine transporter is introduced into E.coli Nissle containing BBa_K2997009 and BBa_K2997010, conversion of tyrosine into p-Coumaric acid is increased by 1.44-fold and 1.31-fold respectively.
By culturing E. coli Nissle with dual plasmids containing TAL (BBa_K2997010) and tyrP (BBa_K2997009) in LB medium with different concentrations of tyrosine, we are able to prove that the TAL enzyme can specifically use tyrosine as its substrate. As seen in Fig. 8, although no significance in p-Couramic acid production in culture supplemented with 0.5mM and 1.0mM tyrosine, there was however an increasing trend. Furthermore, there was a significant increase when comparing culture supplemented with 1.0mM and 2.0mM tyrosine. We speculate that there was indeed a dose-dependent effect, but due to the limit of n-octanol extraction method was apparent.
Biosafety: can Gene Knock Out
To confirm the biosafety measurement of E.coli Nissle, we did a phenotype test by streaking out the can gene mutant bacteria on different plates and placing them in different conditions.
E.coli Nissle and other strains were streaked onto agar plates and placed in (A) 0.04% CO2; (B) 5% CO2; (C) 5% CO2 and contains Chloramphenicol (Cm) for phenotyping.
As seen in Fig 9., Δcan::CmR and Δcan::FRT requires a higher CO2 level to survive. When streaked on Cm plates, Δcan::CmR is still able to grow while Δcan::FRT is unable to grow, thus proving that we have succeeded in knocking out the can gene and replacing it with CmR cassette and FRT sites respectively.