IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.2

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(<span style="font-size: 140%;">[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP'''] </span><span style="font-size: 80%;">(100ng/ul)</span>)
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===<span style="font-size: 140%;">[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP''']  </span><span style="font-size: 80%;">(100ng/&mu;l)</span>===
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= ''In vitro'' Testing of pTet-LuxR-pLux-GFP Construct=
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<br>
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====Testing at 25 <sup>o</sup>C ====
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<hr>
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Today we tested pLux in vitro using 3 different combinations of cell extract:
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<br>
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# pLux with Commercial(CM) S30 cell extract and CM Premix
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# pLux with Homemade(HM) S30 cell extract and HM Premix
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# pLux with HM S30 cell extract and CM Premix
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<br>
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The idea behind the first combination is to see if pLux works in vitro since we already know our commercial extract is working with pTet. If pLux does work with the commercial extract but proves to be reduntant in the second combination, which utilises only homemade cell extract & premix, then we know that our homemade extract inefficient. Lastly, the third combination is to test whether our homemade cell extract will work better with the commercial premix rather than the homemade one.
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The experiment was carried out over a period of nearly 6:30 hours, taking measuremets every half hour. Each combination had 3 repeats and 1 control. The average value of fluorescence for each combination is on the graph below:  
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__NOTOC__
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<br><br>
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==Aims==
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[[ Image:IC2007 Experimental Design PLux vitro 6hours.PNG|thumb|left|500px| Results of in vitro testing of pLux over a 6 hour period at 25<sup>o</sup>C ]]
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To determine if the following constructs work in vitro:
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<br>
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*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP'''
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'''The graph on the right dispays the following.'''
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<div style="color:Green">Fluorescence of the diluted GFP solution (+ve control).</div>
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To determine the consistency and efficacy of using different combinations of cell extract:
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<div style="color:#F88017">The average fluorescence of the pLux samples(3) in CM cell extract + CM premix</div>
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*pLux with Commercial(CM) S30 cell extract and CM Premix
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<div style="color:lightBlue">The average fluorescence of the pLux samples(3) in HM cell extract + HM premix</div>
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*pLux with Homemade(HM) S30 cell extract and HM Premix
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<div style="color:Black">The average fluorescence of the pLux samples(3) in HM cell extract + CM premix</div>
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*pLux with HM S30 cell extract and CM Premix
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<div style="color:#C45AEC">The fluoresence of a solution containing only S30 cell extract and nuclease free water <br>(-ve control) .</div>
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<br>
<br>
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From the graph we notice that pLux does work in vitro (with CM cell extract) and quite well since by the end of the 6.5 hours, its fluorescence has intersected with our positive control (diluted GFP). Unfortunately however, our homemade cell extract did not match the commercial one in performance despite both extracts having the same amounts of pLux DNA in them (20 ul).  
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Since it has been ascertained that the commercial extract works with pTet, the pLux construct should work in the first combination. This is to be compared with that of the second combination to test for the efficiency of the HM extract. The third combination investigates the possibility of combining our homemade cell extract with the commercial premix rather than the homemade one.
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Tested on  [[IGEM:IMPERIAL/2007/Notebook/2007-8-24 | 24-08-2007]]
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== Materials and Methods ==
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Refer to protocols page.
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== Results ==
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===[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP''']===
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====<font color=darkblue>''Test: 24-08-2007''</font>====
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'''''In vitro'' Testing of pTet-LuxR-pLux-GFP at 25&deg;C'''
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{|align="left"
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| width="450px"| [[ Image:IC2007 Experimental Design PLux vitro 6hours.PNG|thumb|left|450px| Fig.1: GFP expression of pLux ''in vitro'' over 6 hours at 25&deg;C ]]
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| width="50px"|
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|width="300px"| From Fig.1, it seems that there is a very strong fluorescent signal with that of pLux construct,
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we notice that pLux does work in vitro (with CM cell extract) and quite well since by the end of the 6.5 hours, its fluorescence has intersected with our positive control (diluted GFP). Unfortunately however, our homemade cell extract did not match the commercial one in performance despite both extracts having the same amounts of pLux DNA in them (20 ul).  
Our homemade commercial cell extract peaked at 5000 fluorescence units after just 1.5 hour from induction whereas the commercial one increased continuously for the 6.5 hours reaching a fluorescence of 15000 (x3 the efficiency).
Our homemade commercial cell extract peaked at 5000 fluorescence units after just 1.5 hour from induction whereas the commercial one increased continuously for the 6.5 hours reaching a fluorescence of 15000 (x3 the efficiency).
Finally, our homemade cell extract proved not to be compatible with the commercial premix since their combination resulted in the lowest performance, a basal level comparable with our negative control.  
Finally, our homemade cell extract proved not to be compatible with the commercial premix since their combination resulted in the lowest performance, a basal level comparable with our negative control.  
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|}
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<br clear="all">
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== Discussion ==
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== Conclusion ==
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<br><br>
<br><br>
Concluding, pLux is shown to work in a vitro system although the commercial extract (and commercial premix) should be preferrred over our homemade extract to obtain maximum perfomance for this construct.
Concluding, pLux is shown to work in a vitro system although the commercial extract (and commercial premix) should be preferrred over our homemade extract to obtain maximum perfomance for this construct.
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===[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP''']===

Revision as of 09:41, 14 October 2007

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

Aims

To determine if the following constructs work in vitro:

To determine the consistency and efficacy of using different combinations of cell extract:

  • pLux with Commercial(CM) S30 cell extract and CM Premix
  • pLux with Homemade(HM) S30 cell extract and HM Premix
  • pLux with HM S30 cell extract and CM Premix


Since it has been ascertained that the commercial extract works with pTet, the pLux construct should work in the first combination. This is to be compared with that of the second combination to test for the efficiency of the HM extract. The third combination investigates the possibility of combining our homemade cell extract with the commercial premix rather than the homemade one.

Tested on 24-08-2007


Materials and Methods

Refer to protocols page.


Results

pTet-LuxR-pLux-GFP

Test: 24-08-2007

In vitro Testing of pTet-LuxR-pLux-GFP at 25°C

Fig.1: GFP expression of pLux in vitro over 6 hours at 25°C
Fig.1: GFP expression of pLux in vitro over 6 hours at 25°C
From Fig.1, it seems that there is a very strong fluorescent signal with that of pLux construct,


we notice that pLux does work in vitro (with CM cell extract) and quite well since by the end of the 6.5 hours, its fluorescence has intersected with our positive control (diluted GFP). Unfortunately however, our homemade cell extract did not match the commercial one in performance despite both extracts having the same amounts of pLux DNA in them (20 ul). 

Our homemade commercial cell extract peaked at 5000 fluorescence units after just 1.5 hour from induction whereas the commercial one increased continuously for the 6.5 hours reaching a fluorescence of 15000 (x3 the efficiency). Finally, our homemade cell extract proved not to be compatible with the commercial premix since their combination resulted in the lowest performance, a basal level comparable with our negative control.




Discussion

Conclusion



Concluding, pLux is shown to work in a vitro system although the commercial extract (and commercial premix) should be preferrred over our homemade extract to obtain maximum perfomance for this construct.


pTet-LuxR-pLux-GFP

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