Team:Tianjin/Demonstrate
/* OVERRIDE IGEM SETTINGS */
Demonstrate
Mating-type Switch and Mating Switcher
SCRaMbLE to Improve the Resistance to Heavy Metals
Biosensor for Cu Ion's Detection
OVERVIEW
After communication with professors, teachers and factory leaders, our HPers summarized that It is also difficult to monitor the copper concentration of the solution in real-time. Solving this problem by biosensor seems like a good idea.
The inspiration came from promoters found in nature, which can be induced by different metal ions. Ligating this kind of promoters with reporter gene like RFP is a common idea to monitor the concentration of metals. Take Cu ions for example, we first search for promoters induced by Cu in parts, and we found both promoters in E.coli and S.cerevisiae. Actually, The resistance to Cu ions in E.coli and S.cerevisiae differs from each other. The maximum resistance to Cu in E.coli is 9 mM, which is less than that in S.cerevisiae. The resistance in S.cerevisiae is over 15 mM. Considering the response rage, the budding yeast is a much better host for Cu detection.
We built a biosensor based on CUP1 promoter and yEmRFP to detect Cu ion’s concentration. The response range of this biosensor were characterized with fluorescent microplate reader. To improve the sensitivity of biosensor, and enlarge the response intensity when it is induced, we used error-prone PCR to obtain a large amount of promoter mutants and characterized them.
CONSTRUCTION
The biosensor consists of two main parts. One is the Cu-induced promoter CUP1p, the other is yEmRFP, which is modified from a mCherry mRFP to adapt to the transcription environment in yeast. The promoter was synthesized without RFC sites (XbaI) and the RFP was amplified by PCR. We used overlap PCR to combine the two parts and added two restriction sites on the ends. By digestion and ligation, we constructed this biosensor on the plasmid pRS416 which contains a selective marker URA3. After that, we sequenced this part with M13F and M13R as primers. The sequencing result showed that this construction was successful, so we can take the next step – characterization.
We used Cyan fluorescence protein (CFP) as a real-time approach to monitor protein expression quantity by detecting the fluorescence signal in the plate-reader. The emission wavelength of the CFP is 479nm and the absorption wavelength is 435nm. This is the real-time detection of emission wavelength at 479nm. We detected the fluorescence absorption for 12 hours before adding PET film substrate.
