Lab chronology

Week 1.

After intense discussion about the viability of the Project, we have decided propose for 2012 IGEM a Carbon dioxide (CO2 ) biosensor. The carbon dioxide (CO2 ) is a inert gas, odorless and tasteless is using in sodas, vines and other beverages, the CO2 is one of the greenhouse gases and its excess and high concentrations could represent a risk for human health. The design of our constuction was as follow. We sent the secuences to synthetize to Gen Script and waited to arrival



Week 2.

After 5 weeks waiting and multi customs problems, finally we have our plasmid from Gen Script (Thanks guys). We prepared competent cels (DH5α) for our transformations. For the induction we developed 3 different experiments with different times and concentrations. Inductor 1. 5 Mm NaHCO3, Time 10, 30 and 60 min. Inductor 2. 10 Mm NaHCO3, Time 10, 30 and 60 min. Inductor 3. 25 Mm NaHCO3, Time 10, 30 and 60 min.



Week 3

Transformation, we used the 3 stocks of sodium bicarbonate as inductor to see if significant florescence is produced. We realized induction as follow. Once geting our solutions proceeded to induce the E.coli transformants using 50 µl transformant cells and 50 µl of solution and immediately we made observations on fluorescence microscopy. Results. Florescence was too low for significant obsevation, the conclusion was that the inductor needs to be complemented with several other elements like MgCl2 and ATP for optimal cAMP production.

Week 4

Induction refinement . We utilized new inductor with the following concentrations. Strong inductor. 40 mM NaHCO3- , 10 mM MgCl2 and 5 mM ATP Medium inductor, 25 mM NaHCO3-, 10 mM MgCl2 and 5 mM ATP No inductor. 0 mM NaHCO3- 10 mM MgCl2 and 5 mM ATP Basal contol. Only water Induction method was the same we used last week (50 0 µl transformant cells and 50 µl of solution) observations was made 30 minutes after induction to ensure induction. Results. GFP expression was higher in the strong inductor samples, followed by medium inductor. No inductor and basal presents some flouresense, so there is something in media used that activates GFP production.



Week 5

Basal production determination. To exclude basal GFP production of our measurements we make some measurements of transformed cells and without induction and some DH5α untransformed cells to compare the basal florescence. Results. Basal fluorescence was presented in both set of samples, but in samples containing untransformed cells fluorescence is minor that fluorescence in samples with transformed cells. Final results. Induction of GFP by HCO3-. The cells express different quantities of GFP dependent to the media they are added to induction, from mayor to minor fluorescence in samples was as followed: Strong inductor, medium inductor, no inductor, basal control (only transformed cells) and untransformed cells