Team:Colombia Uniandes/Parts

Glucocorticoid sensor

Here are the parts sent to the registry:

You can see the confirmation gels of the parts below:

PCR confirmation: E1GF-E1GF(2)-E2GF-E2GF(2)-WM-E3GF-E3GF(2)-E4GF-E4GF(2)

Co-transformation: WM-E1GF2-E2GF-E3GF1-E3GF2-E4GF-WM- E3GF3-E4GF2-E2GF2

PCR confirmation of the parts cloned in the pSB1C3 backbone using the specifically designed primers 15 and 31 (see primers)

Characterization of reporters

To assess the strength of our promoters when induced with Dexamethasone, we performed a fluorometric assay using mCherry as our reporter.Since we don't have our transactivating protein ready yet, we co-transformed our parts into the E. coli DH10B strain with the pAT7002 vector (Aoyama and Chua, 1997), which contains a well characterized Glucocorticoid Responsive Element that also uses the GAL4 DNA binding domain.

Unsaturated curve where we see how the mCherry reporter appears after the addition of 10 uM dexamethasone, a glucocorticoid. Emmision intensity was measured at 607nm after excitation at 586nm

Saturated curve for fluorescence. After three hours of the addition of 10 uM dexamethasone the reporter (mCherry) reaches its highest signal. We can see the different strengths depending on the number of UAS boxes. Emmision intensity was measured at 607nm after excitation at 586nm

We also decided to visually inspect our induced transformants. Here two of the images taken using a epifluorescent microscopy with a TRITC filter.

Cells with the E2GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC

Cells with the E4GF part (BBa_K1144008) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC

References

Aoyama, T. & Chua N. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. The Plant Journal, 11(3): 605-612.

Nickel Removal System

Here are the parts sent to the registry:

Prcna-HoxN and HoxN-RFP