Team:Colombia Uniandes/Parts
From 2013.igem.org
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==='''Characterization of reporters'''=== | ==='''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 | + | 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. |
[[File:Response1hr.jpg|700px|thumb|center|''' 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''']] | [[File:Response1hr.jpg|700px|thumb|center|''' 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''']] | ||
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[[File:E2GF.jpg|370px|thumb|left|''' Cells with the E2GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']] | [[File:E2GF.jpg|370px|thumb|left|''' Cells with the E2GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']] | ||
- | [[File:E4GF.jpg|370px|thumb|right|''' Cells with the E4GF part (BBa_K1144008) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC | + | [[File:E4GF.jpg|370px|thumb|right|''' Cells with the E4GF part (BBa_K1144008) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']] |
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+ | ====References==== | ||
+ | Aoyama, T. & Chua N. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. ''The Plant Journal, 11''(3): 605-612. | ||
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Latest revision as of 03:40, 28 September 2013
Parts
Contents |
Glucocorticoid sensor
Here are the parts sent to the registry:
Part Name | Registry Number |
E1 | BBa_K1144001 |
E2 | BBa_K1144002 |
E3 | BBa_K1144003 |
E4 | BBa_K1144004 |
E1GF | BBa_K1144005 |
E2GF | BBa_K1144006 |
E3GF | BBa_K1144007 |
E4GF | BBa_K1144008 |
You can see the confirmation gels of the parts below:
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.
We also decided to visually inspect our induced transformants. Here two of the images taken using a epifluorescent microscopy with a TRITC filter.
References
Aoyama, T. & Chua N. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. The Plant Journal, 11(3): 605-612.