IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 1.2: Difference between revisions

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<br><span style="font-size: 180%;"> In Vivo Tetsing'''</span>
= ''In vivo'' Testing of pTet-LuxR-pLux-GFP Construct=
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__NOTOC__
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==Aims==
==Aims==
To Determine if the [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''Tet-LuxR-pLux-GFP'''] construct works ''in vivo''
To determine if the [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP'''] construct works ''in vivo'' after induction of 1mM AHL concentration
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Tested on  [[IGEM:IMPERIAL/2007/Notebook/2007-8-16 | Tested 17-08-2007]]
Tested on  [[IGEM:IMPERIAL/2007/Notebook/2007-8-16 | 17-08-2007]]
 
 
==Materials and Methods==
Refer to protocols page.


==Results==
==Results==
===[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 pTet-LuxR-pLux-GFP]===
====<font color=darkblue>''Test: 17-08-2007''</font>====
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{|align="left"
The contruct was successfully tested by flooding it with a concentration of ??? nM of AHL. Since we just wanted to simply test whether the construct works or not, we did not want AHL to be a constraint and that is why a large concentration was used. Three repeated measurements were performed on three different samples and compared to a diluted concentration of GFP used as a positive control.
| width="200px"|<br>[[Image:IC2007 Experimental Design Phase 1 protocol1-2-pLux-vivo.PNG|thumb|300px|Fig.1: Total Fluorescence of pTet-LuxR-pLux-GFP ''in vivo'' over 7 hours]]
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|width="400px"| Upon induction, the pTet-LuxR-pLux-GFP construct showed a significant fluorescent signal when compared to the negative control. In addition, the fluorescence levels were comparable to that of the positive control, indicating strong expression of GFP with the construct.
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[[Image:IC2007 Experimental Design Phase 1 protocol1-2-pLux-vivo.PNG|thumb|left|400px| Results of testing pLux in vivo with a consntant AHL concentration]]
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'''Controls:'''
On the graph on the right you can see the fluorescence reading of the pLux assembly 7 hours after it has been induced with AHL. The readings of pLux are comparable to the diluted GFP solution and leads us to conclude that GFP has been expressed.
*Positive control - diluted GFP solution of equal volume
*Negative control - LB media of equal volume


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==Discussion==
==Discussion==
The construct was found to work in vivo. Hence we will now go into its characterization, as well as to look at its alternative construct for the application, which is pLux-GFP. Further tests will need to be conducted to reveal its sensitivity to AHL.
===[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP''']===
Fig.1 showed that the pTet-LuxR-pLux-GFP construct gave a good amount of expression of GFP (~83000), indicating that the construct is functioning well ''in vivo''. The drawback of this construct, however, is that LuxR levels within the cells are inconsistent, to which this variation would complicate the kinetics of GFP expression. Thus it will be unfair to relate the increase in expression of fluorescence solely due to the increase in induction and strength of the pLux promoter.
 
There was also significant variability in the results across the different samples, which could be attributed either to experimental methodology, or the intrinsic nature of variability of expression in the ''in vivo'' chassis.
 
 
==Conclusion==
To conclude,
* pTet-LuxR-pLux-GFP construct gave a strong fluorescent signal, indicating good expression of GFP ''in vivo''.
* LuxR levels are inconsistent throughout experiment.
* Significant variability was found in all ''in vivo'' fluorometer experiments.

Latest revision as of 05:26, 14 October 2007

In vivo Testing of pTet-LuxR-pLux-GFP Construct

Aims

To determine if the pTet-LuxR-pLux-GFP construct works in vivo after induction of 1mM AHL concentration

Tested on 17-08-2007


Materials and Methods

Refer to protocols page.

Results

Test: 17-08-2007


Fig.1: Total Fluorescence of pTet-LuxR-pLux-GFP in vivo over 7 hours
 Upon induction, the pTet-LuxR-pLux-GFP construct showed a significant fluorescent signal when compared to the negative control. In addition, the fluorescence levels were comparable to that of the positive control, indicating strong expression of GFP with the construct.


Controls:

  • Positive control - diluted GFP solution of equal volume
  • Negative control - LB media of equal volume

Discussion

pTet-LuxR-pLux-GFP

Fig.1 showed that the pTet-LuxR-pLux-GFP construct gave a good amount of expression of GFP (~83000), indicating that the construct is functioning well in vivo. The drawback of this construct, however, is that LuxR levels within the cells are inconsistent, to which this variation would complicate the kinetics of GFP expression. Thus it will be unfair to relate the increase in expression of fluorescence solely due to the increase in induction and strength of the pLux promoter.

There was also significant variability in the results across the different samples, which could be attributed either to experimental methodology, or the intrinsic nature of variability of expression in the in vivo chassis.


Conclusion

To conclude,

  • pTet-LuxR-pLux-GFP construct gave a strong fluorescent signal, indicating good expression of GFP in vivo.
  • LuxR levels are inconsistent throughout experiment.
  • Significant variability was found in all in vivo fluorometer experiments.
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