Team:BCCS-Bristol/Wetlab/adding RFP

From 2010.igem.org

Implementing the Ratio Method

To solve the problem of not knowing whether a strong signal was due to many weakly fluorescing bacteria or a few strong ones we decided to implement a ratio method. This involved transforming MG1655 not only with our new biobrick BBa_K381001 but also BBa_J04450 - constitutive RFP production.

Thus rather than measuring only the amount of GFP, a farmer would instead measure the ratio of GFP/RFP. As RFP would scale up with population size, this counters the problem of differing concentrations of E. coli in our beads, it also partially eliminates problems of false positives (other things fluorescing in soil) and negatives (all bacteria in a bead dying).

We began with a double transformation of MG1655, following our standard transformation protocol as outlined below:

1. Add 2μL of BBa_K381001 (diluted 1:5 from a midiprep) and 2μL of BBa_J04450 to a chilled microfuge tube
2. Add 100μL ice cold competent MG1655
3. Incubate on ice for 30 minutes
4. Heat shock by placing at 42°C for 2 minutes
5. Add 0.5mL of LB and incubate at 37°C for 30 - 90 minutes
6. Pellet cells by spinning briefly (30s at 13000rpm) in microfuge. Discard supernatent
7. Resuspend pellet in 100mL of LB and plate out onto agar containing both Ampicillin and Kanamycin and incubate overnight at 37°C

This resulted in a plate with >50 colonies