Difference between revisions of "Part:BBa K1144005"

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<partinfo>BBa_K1144005 short</partinfo>
 
<partinfo>BBa_K1144005 short</partinfo>
  
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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  
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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  
  
 
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===Characterization===
 
===Characterization===
  
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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.  
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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, using our reporter set of parts (BBa_K1144005, BBa_K1144006, BBa_K1144007 and BBa_K1144008) .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:E1GF.jpg|450px|thumb|left|''' Cells with the E1GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']]  
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[[File:E1GF.jpg|700px|thumb|center|''' Cells with the E1GF part (BBa_K1144005) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']]  
  
  
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[[File:E2GF.jpg|450px|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''']]  
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[[File:E3GF.jpg|700px|thumb|center|''' Cells with the E3GF part (BBa_K1144007) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']]  
  
  
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[[File:E3GF.jpg|450px|thumb|left|''' Cells with the E3GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']]
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'''Characterization in ''Saccharomyces cerevisiae'' '''
  
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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''']]
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To test if our constructs work in ''S. cerevisiae'' we performed a fluorometric assay (as described before). Experiments were conducted at 30C with constant agitation.
  
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[[File:ResponseYeast.jpg|700px|thumb|center|''' Induction of pGAL1-like promoters. After 100 min 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''']]
  
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We used epifluorescence microscopy to inspected our induced Yeast transformants. Here ''Saccharomyces cerevisiae'' trasformed with the BBa_K1144008. The picture was taken using the DIC path and the TRITC filter.
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[[File:E4GFYeast.jpg|700px|thumb|center|''' Yeast 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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[[File:E4GFYeastTRITC.jpg|700px|thumb|center|''' Cells with the E4GF part (BBa_K1144004)''']]
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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.

Latest revision as of 21:56, 5 October 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 84
  • 1000
    COMPATIBLE 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, using our reporter set of parts (BBa_K1144005, BBa_K1144006, BBa_K1144007 and BBa_K1144008) .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 E1GF part (BBa_K1144005) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC


Cells with the E3GF part (BBa_K1144007) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC


Characterization in Saccharomyces cerevisiae

To test if our constructs work in S. cerevisiae we performed a fluorometric assay (as described before). Experiments were conducted at 30C with constant agitation.

Induction of pGAL1-like promoters. After 100 min 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 used epifluorescence microscopy to inspected our induced Yeast transformants. Here Saccharomyces cerevisiae trasformed with the BBa_K1144008. The picture was taken using the DIC path and the TRITC filter.

Yeast cells with the E4GF part (BBa_K1144008) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC
Cells with the E4GF part (BBa_K1144004)


References

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