Difference between revisions of "Part:BBa J37032:Experience"

Latest revision as of 12:02, 10 September 2009

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Applications of BBa_J37032

User Reviews

UNIQdd34825e15933dab-partinfo-00000000-QINU

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Aberdeen_Scotland 2009

Experimental Design
The BBa_J37032 BioBrick expresses GFP under control of a LuxR responsive promoter. The BBa_J37032 part was provided by iGEM-HQ, was succesfully mini-preppred and transformed into E.coli. BBa_J37032 was used to test our constitutive LuxR/LuxI operon (BBa_K182200). For that purpose SCS1 E.coli was transformed either with BBa_K182200 alone (referred to as pIR in figure below), BBa_J37032 alone, or a combination of both plasmids. The single transformants acted as negative controls.

Results
It would be expected that the BBa_K182200 transformant would not fluoresce, since it does not express GFP. This was confirmed (top panel, figure below, showing light microscope pictures on the left, and fluorescent images on the right). Similarly, the single BBa_J37032 transformant was not expected to exhibit much fluorescence, since there is no endogenous expression of LuxR or LuxI in E.coli. A combination of BBa_K182200 and BBa_J37032 would be expected to produce high amounts of fluorescence at high cell densities as quorum sensing triggers the Lux promoter of BBa_J37032. However, in overnight stationary phase cultures at high density, it was found that the BBa_J37032 construct expresses the same high amount of GFP fluorescence with or without the presence of LuxR (compare middle panel [J37032 single transformant] with bottom panel [J37032/pIR double transformant])

Conclusion
We conclude that BBa_J37032 is either not responsive to LuxR, or that the pLux promoter is extremely leaky, driving significant GFP expression in the absence of LuxR. It therefore cannot easily be used to test quorum sensing due to this high background level of GFP expression.

UNIQdd34825e15933dab-partinfo-0000000F-QINU

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-Experimental Design<br>
+<b>Experimental Design</b><br>
-The J37032 BioBrick expresses GFP under control of a LuxR responsive promoter. The J37032 part was provided by iGEM-HQ, was succesfully mini-preppred and transformed into E.coli.  J37032 was used to test our constitutive LuxR/LuxI operon (K182200). For that purpose SCS1 E.coli was transformed either with K182200 alone (referred to as pIR in figure below),  J37032 alone, or a combination of both plasmids. The single transformants acted as negative controls.
+The <partinfo>BBa_J37032</partinfo> BioBrick expresses GFP under control of a LuxR responsive promoter. The <partinfo>BBa_J37032</partinfo> part was provided by iGEM-HQ, was succesfully mini-preppred and transformed into E.coli.  <partinfo>BBa_J37032</partinfo> was used to test our constitutive LuxR/LuxI operon (<partinfo>BBa_K182200</partinfo>). For that purpose SCS1 E.coli was transformed either with <partinfo>BBa_K182200</partinfo> alone (referred to as pIR in figure below),  <partinfo>BBa_J37032</partinfo> alone, or a combination of both plasmids. The single transformants acted as negative controls.
-Results<br>
+<b>Results</b><br>
-It would be expected that the K182200 transformant would not fluoresce, since it does not express GFP. This was confirmed (top panel, figure below, showing light microscope pictures on the left, and fluorescent images on the right).  Similarly, the single J37032 transformant was not expected to exhibit much fluorescence, since there is no endogenous expression of LuxR or LuxI in E.coli. A combination of K182200 and J37032 would be expected to produce high amounts of fluorescence at high cell densities as quorum sensing triggers the Lux promoter of J37032. However, in overnight stationary phase cultures at high density, it was found that the J37032 construct expresses the same high amount of GFP fluorescence with or without the presence of LuxR (compare middle panel[J37032 single transformant] with bottom panel [J37032/pIR double transformant])
+It would be expected that the <partinfo>BBa_K182200</partinfo> transformant would not fluoresce, since it does not express GFP. This was confirmed (top panel, figure below, showing light microscope pictures on the left, and fluorescent images on the right).  Similarly, the single <partinfo>BBa_J37032</partinfo> transformant was not expected to exhibit much fluorescence, since there is no endogenous expression of LuxR or LuxI in E.coli. A combination of <partinfo>BBa_K182200</partinfo> and <partinfo>BBa_J37032</partinfo> would be expected to produce high amounts of fluorescence at high cell densities as quorum sensing triggers the Lux promoter of <partinfo>BBa_J37032</partinfo>. However, in overnight stationary phase cultures at high density, it was found that the <partinfo>BBa_J37032</partinfo> construct expresses the same high amount of GFP fluorescence with or without the presence of LuxR (compare middle panel [J37032 single transformant] with bottom panel [J37032/pIR double transformant])
-Conclusion<br>
+<b>Conclusion</b><br>
-We conclude that J37032 is either not responsive to LuxR, or that the pLux promoter is extremely leaky, driving significant GFP expression in the absence of LuxR. It therefore cannot easily be used to test quorum sensing due to this high background level of GFP expression.
+We conclude that <partinfo>BBa_J37032</partinfo> is either not responsive to LuxR, or that the pLux promoter is extremely leaky, driving significant GFP expression in the absence of LuxR. It therefore cannot easily be used to test quorum sensing due to this high background level of GFP expression.
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 [[Image:J37TEST_figure.jpg|center|400 px]]