Safety
1. What is your chassis organism?
E. coli (lab strains that are not harmful to humans) & Yeast (Saccharomyces)
E. coli that we use for vector cloning are DH5-Alpha and DH10-Beta.
E. coli that we use for toehold switch RNA expression is BL21 DE3.
Yeast that we use for CXCR1 protein expression is S288C & W303.
2. Do you plan to experiment with any other organisms, besides your chassis?
Our experiment goal wants to make a biosensor which can detect overexpressed mRNA and protein in human saliva for early diagnosis of oral cancer. Our designed constructs are all finished in E. coli and yeast. However, to make a preliminary verification for our constructs, we use human THP1 cell line. We lysed THP1 cell line and used it for positive control in Western Blot whose primary antibody is anti-CXCR1 to confirm if our new biobrick part expressed.
3. How do the project work?
Our experiment is divided into two parts. First, our E. coil express synthetic toehold switch gene regulators as RNA sensors to detect specific mRNA biomarkers expressed in oral squamous cell carcinoma (OSCC). They sense specific mRNA transcripts as biomarkers. The other, our yeast express Interleukin-8 receptor to sense Interleukin-8 protein in saliva, which can act as OSCC biomarker.
4. Any safety concern? What risks does your project pose at the laboratory stage? What actions are you taking to reduce those risks?
We comply with general safety including appropriate clothing in lab, lab environment, usage of safety equipments, emergency procedures of lab, the regulation of public lab equipments and biological lab waste management and so on. Besides, before and after experiment, we will spray 70% alcohol to make sure bench remain clean. And when we culture cell line in culture room, we will also turn on the UV light to avoid any possible contamination. And for further safety, we are not testing our system with OSCC cells in our validation experiment. We are going to test our prototype by synthetic nucleic acid from IDT and recombinant protein to mimic the microenvironment of saliva in OSCC patients. Because THP1 cell line is a pathogen-free cell line, it is safer for our prototype validation than using OSCC cell line directly.
5. How would your project be used in the real world?
Our RNA sensor and yeast will be developed into a kit to detect biomarkers (RNA and protein) in OSCC patients' saliva. It will be a simple, non-invasive and objective bio-sensing chip for diagnosis oral cancer at the early stage. Besides this application, if our synthetic toehold switch sensor and engineered yeast detection system are fully developed, we can replace any detection targets people desired in our detecting platform.
6. What risks might your project pose, if it were fully developed into a real product that real people could use? What future work might you do to reduce those risks?
If our toehold switch RNA and yeast detecting system are fully developed into a real product that real people can use, they will all have two concerns. One is it may infect to humans, the other is it may cause environmental contamination. Therefore, for our E. coli detecting system, we will develop it into a cell free system. We will just purify its toehold switch on our bio-sensing chip, so it will not have alive E.coli in our final product, and we can eliminate concerns about infection and environmental contamination. On the other hand, for our yeast detecting system because we have replaced its G alpha mating system with our detecting system through homologous recombination, so it will have no ability to mate with other yeast strains in human body and environment, and we can eliminate concerns about infection and environmental contamination too.
