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=
<hr>
 
__NOTOC__
__NOTOC__
==Aims==
==Aims==
To Determine if the [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''Tet-LuxR-pLux-GFP'''] construct works ''in vivo'' after inducing at 1mM AHL concentration
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
<br>
<br>
<br>
<br>
Tested on  [[IGEM:IMPERIAL/2007/Notebook/2007-8-16 | Tested 17-08-2007]]
Tested on  [[IGEM:IMPERIAL/2007/Notebook/2007-8-16 | Tested 17-08-2007]]
==Materials and Methods==
Refer to protocols page.


==Results==
==Results==
====<font color=darkblue>''Test: 17-08-2007''</font>====
====<font color=darkblue>''Test: 17-08-2007''</font>====
{|align="left"
{|align="left"
| width="200px"|<br>[[Image:IC2007 Experimental Design Phase 1 protocol1-2-pLux-vivo.PNG|thumb|300px|<font color=blue>17-08-2007</font>- Results of pLux]]
| 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]]
|width="50px"|  
|width="50px"|  
|width="400px"|The results show that the pLux construct works ''in vivo''. Three repeated measurements were performed on three different samples and compared to a diluted concentration of GFP used as a positive control. 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 those of the positive control, diluted GFP solution, and so we can conclude that GFP has been expressed.
|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.
|}
|}


<br clear=all>
<br clear=all>
'''Controls:'''
*Positive control - diluted GFP solution of equal volume
*Negaitve control - LB media of equal volume


==Discussion==
==Discussion==
The construct has been found to work in vivo. The results show a strong response after induction of the pLux promoter. In addition there was a lot of variability within the results, this was a problem for the pTet construct and so we will investigate the fluorometer settings to improve upon this variability 
===[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP''']===
==Conlusion==
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.  
We will now test to see if pLux can express in vitro
 
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.

Revision as of 04:26, 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 Tested 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
  • Negaitve 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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