Difference between revisions of "Part:BBa K1144005"
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[[File:E4GF.jpg|450px|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|450px|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==== | ====References==== | ||
Aoyama, T. & Chua N. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. ''The Plant Journal, 11''(3): 605-612. | Aoyama, T. & Chua N. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. ''The Plant Journal, 11''(3): 605-612. |
Revision as of 04:37, 28 September 2013
GAL4 activation reporter
The level of expression from the GAL1 promoter (Saccharomyces cerevisiae) is enhanced due to the presence of UAS sites. We fused pGAL1 with a KOZAC sequence, mCherry and the HHO1 yeast terminator to asses the level of expression due to the activacion of GAL4 binding domain
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 84
- 1000COMPATIBLE WITH RFC[1000]
Characterization
To assess the strength of our GAL4 responsive promoter set (BBa_K1144001, BBa_K1144002, BBa_K1144003 and BBa_K1144004) 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.