Difference between revisions of "Part:BBa K1144002"
(→‎Characterization)
 
Line 19: Line 19:
 
===Characterization===
 
===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.  
+
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''']]  
Line 30: Line 30:
  
  
−
[[File:E1GF.jpg|400px|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''']]  
+
[[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''']]  
  
  
−
[[File:E2GF.jpg|400px|thumb|right|''' Cells with the E2GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']]
+
[[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''']]  
−
 
+
−
 
+
−
[[File:E3GF.jpg|400px|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''']]
+
−
 
+
−
[[File:E4GF.jpg|400px|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''']]  
+
  
 
<html>
 
<html>

Latest revision as of 05:15, 28 September 2013

pGAL1-3UAS-KOZAC

Modified GAL1 promoter with 2 additional UAS sites. Original TATA box was maintaned and KOZAC sequence was added

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

References

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