IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.2: Difference between revisions

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===<div style="font-size: 140%;">[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP'''] </div>===
= ''In vitro'' Testing of pTet-LuxR-pLux-GFP Construct=
 
__NOTOC__
==Aims==
To determine if the following constructs work in vitro:
*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP''']
 
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
<br>
<br>
====Testing at 25 <sup>o</sup>C ====
<hr>
Today we tested pLux in vitro using 3 different combinations of cell extract:
<br>
# 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
<br>
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.


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:  
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.
<br>
 
[[ 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 ]]
Tested on [[IGEM:IMPERIAL/2007/Notebook/2007-8-24 | 24-08-2007]]
 
 
== Materials and Methods ==
Refer to protocols page.
 
 
== Results ==
===[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP''']===
====<font color=darkblue>''Test: 24-08-2007''</font>====
'''''In vitro'' Testing of pTet-LuxR-pLux-GFP at 25&deg;C'''
{|align="left"
| 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 ]]
| width="50px"|
|width="300px"| From Fig.1, it seems that there is a very strong fluorescent signal observed from the pLux construct using the first combination as its fluorescence has intersected with the positive control after 6 hours. On the other hand, the HM cell extracts did not compare well in terms of fluorescence despite both extracts having the same amount of pLux DNA.
 
This performance discrepency of cell extracts is further accentuated in the thid combination, where the combination of HM cell extract and CM premix resulted in an even lower performance that was comparable to that of the negative control.
|}
 
<br clear="all">
'''Controls:'''
*Positive control - diluted GFP solution of equal volume
*Negative control - S30 cell extract of equal volume
 
 
== Discussion ==
Fig.1 indicates that there was a fair amount of expression of GFP with the pTet-LuxR-pLux-GFP construct, leading to an increase in fluoresence over time. To this, it can be concluded that the construct is working well ''in vitro''.


<br>
The optimization of cell extracts for commercial use is probably the reason for the discrepency in terms of coupled transcription/translation performance. Indeed while the homemade cell extract was found to be working, it was less than half as efficient as that of the commercial extract, presumably due to the fact that it was a crude attempt at emulating protocols obtained from various journals. Despite the reduction in efficiency, homemade cell extracts have the advantage of being more cost effective, and larges batches of homogenous cell extracts can be obtained as opposed to the reaction mixtures sold by commercial firms.  
'''The graph on the right dispays the following.'''


<div style="color:Green">Fluorescence of the diluted GFP solution (+ve control).</div>
The use of large batches of homogenous cell extracts is essential for the consistency of subsequent experiments as it was found that the quality control of commercial cell extracts depended on its minimum yield. This means that the variation across cell extracts can be significant, and different batches of cell extracts will yield different coupled transcription/translation performance. As such, it may be a wiser decision to maintain the homogeneity of cell extracts across each experiment.
<div style="color:#F88017">The average fluorescence of the pLux samples(3) in CM cell extract + CM premix</div>
<div style="color:lightBlue">The average fluorescence of the pLux samples(3) in HM cell extract + HM premix</div>
<div style="color:Black">The average fluorescence of the pLux samples(3) in HM cell extract + CM premix</div>
<div style="color:#C45AEC">The fluoresence of a solution containing only S30 cell extract and nuclease free water <br>(-ve control) .</div>
<br><br>
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).
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).


<br clear="all">
<br clear="all">


===Experiment 1===
 
*Concentration of [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 '''pTet-LuxR-pLux-GFP'''] = 100ng/ul
== Conclusion ==
===Experiment 2===
* pTet-LuxR-pLux-GFP construct works ''in vitro''.
* Commercial cell extracts performed better than our homemade cell extracts.
* It is integral to maintain the homogeneity of cell extracts across our experiments.

Latest revision as of 11:02, 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
 From Fig.1, it seems that there is a very strong fluorescent signal observed from the pLux construct using the first combination as its fluorescence has intersected with the positive control after 6 hours. On the other hand, the HM cell extracts did not compare well in terms of fluorescence despite both extracts having the same amount of pLux DNA.

This performance discrepency of cell extracts is further accentuated in the thid combination, where the combination of HM cell extract and CM premix resulted in an even lower performance that was comparable to that of the negative control.


Controls:

  • Positive control - diluted GFP solution of equal volume
  • Negative control - S30 cell extract of equal volume


Discussion

Fig.1 indicates that there was a fair amount of expression of GFP with the pTet-LuxR-pLux-GFP construct, leading to an increase in fluoresence over time. To this, it can be concluded that the construct is working well in vitro.

The optimization of cell extracts for commercial use is probably the reason for the discrepency in terms of coupled transcription/translation performance. Indeed while the homemade cell extract was found to be working, it was less than half as efficient as that of the commercial extract, presumably due to the fact that it was a crude attempt at emulating protocols obtained from various journals. Despite the reduction in efficiency, homemade cell extracts have the advantage of being more cost effective, and larges batches of homogenous cell extracts can be obtained as opposed to the reaction mixtures sold by commercial firms.

The use of large batches of homogenous cell extracts is essential for the consistency of subsequent experiments as it was found that the quality control of commercial cell extracts depended on its minimum yield. This means that the variation across cell extracts can be significant, and different batches of cell extracts will yield different coupled transcription/translation performance. As such, it may be a wiser decision to maintain the homogeneity of cell extracts across each experiment.



Conclusion

  • pTet-LuxR-pLux-GFP construct works in vitro.
  • Commercial cell extracts performed better than our homemade cell extracts.
  • It is integral to maintain the homogeneity of cell extracts across our experiments.
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