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

Revision as of 11:17, 10 September 2009

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Applications of BBa_J37032

User Reviews

UNIQa0227c91a283b52d-partinfo-00000000-QINU

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Aberdeen_Scotland 2009

Experimental Design The J37032 BioBrick expresses GFP under control of a LuxR responsive promoter. 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.

Results 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. 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])

Conclusion 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.

UNIQa0227c91a283b52d-partinfo-00000002-QINU

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 <I> Aberdeen_Scotland 2009 </I>
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-This part was used to test our LuxR/LuxI operon (K182200). For that purpose J37032 has been transformed in SCS1 E.coli cells as negative control and furthermore SCS1 cells have been transformed with J37032 and K182200 (called pIR in below figure). A second control was the transformance of SCS1 cells with K182200 only. It was found that this testing construct produces the same amount of GFP with or without the presence of LuxR. J37032 is hence not responsive to LuxR or the pLux promoter is extremely leaky and thereby should not be used to test quorum sensing. The Figure below shows the negative controls (J37032 alone and pIR alone) as well as both biobricks together under a light and fluorescence microscope.
+Experimental Design
+The J37032 BioBrick expresses GFP under control of a LuxR responsive promoter. 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.
+Results
+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. 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])
+Conclusion
+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.
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 [[Image:J37TEST_figure.jpg|center|400 px]]