IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.1

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==Results==
==Results==
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===[http://parts.mit.edu/registry/index.php/Part:BBa_I13522 '''pTet-GFP'''](100ng/μl)===
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===[http://parts.mit.edu/registry/index.php/Part:BBa_I13522 '''pTet-GFP'''] (100ng/μl)===
====<font color=darkblue>''Test: 21-08-2007''</font>====
====<font color=darkblue>''Test: 21-08-2007''</font>====
''' ''In vitro'' testing at 37&deg;C'''
''' ''In vitro'' testing at 37&deg;C'''
{|align="left"
{|align="left"
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| width="200px"|[[Image:IC2007 Experimental Design phase 1 protocol 2-1PTet-vitro-37deg.PNG|thumb|300px|Fig.1: GFP Expression of pTet-GFP ''in vitro'']]
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| width="300px"|[[Image:IC2007 Experimental Design phase 1 protocol 2-1PTet-vitro-37deg.PNG|thumb|300px|Fig.1: GFP Expression of pTet-GFP ''in vitro'']]
| width="50px"|
| width="50px"|
|width="400px"| The pTet-GFP contruct was tested ''in vitro'' using commercial S30 Cell Extract at 37 &deg;C. As shown in Fig.1, pTet showed a marked increase in the first hour before levelling, reaching a slow but steady increase throughout a period of 4 hours.
|width="400px"| The pTet-GFP contruct was tested ''in vitro'' using commercial S30 Cell Extract at 37 &deg;C. As shown in Fig.1, pTet showed a marked increase in the first hour before levelling, reaching a slow but steady increase throughout a period of 4 hours.
|}
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<br><br>
 
====<font color=darkblue>''Test: 21-08-2007''</font> to <font color=darkblue>''Test: 23-08-2007''</font>====
====<font color=darkblue>''Test: 21-08-2007''</font> to <font color=darkblue>''Test: 23-08-2007''</font>====
'''pTet-GFP ''in vitro'' GFP expression at 37&deg;C'''
'''pTet-GFP ''in vitro'' GFP expression at 37&deg;C'''
{|align="left"
{|align="left"
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| width=200px"|[[Image:IC2007 experimental design phase 1 protocol1-2 PTet-vitro-37deg-56hours.PNG|thumb|left|300px| Results of in vitro testing of pTet over a 56 hour period ]]
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| width=300px"|[[Image:IC2007 experimental design phase 1 protocol1-2 PTet-vitro-37deg-56hours.PNG|thumb|left|300px| Fig.2: GFP Expression of pTet-GFP ''in vitro'' at 37&deg;C over 56 hours ]]
| width="50px"|
| width="50px"|
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|width="400px"| The pTet-GFP in vitro samples were left and restested over three days to test to see what a typical expression curve looks like. The results of the average fluorescence of the samples and the positive control are shown to the left.
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|width="400px"| Fig.2 shows the incomplete curve of fluorescence levels over a staggered time period. As expected, the fluorescence levels of the pTet construct increase marked during the first few hours. This fluorescence level was higher, albeit reducing, over the middle portion of the graph, where it decreased to less than half the maximum level measured at the end of the time course. Interestingly, the supposed positive control of diluted GFP solution showed similar patterns in terms of fluorescence levels.
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The graph on the left shows the following data:
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<div style="color:Green">Fluorescence of the diluted GFP solution. (+ve control)</div>
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<div style="color:#F88017">The average fluorescence of the pTet samples(3)</div>
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|}
|}
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<br><br>
 
====<font color=darkblue>''Test: 22-08-2007''</font>====
====<font color=darkblue>''Test: 22-08-2007''</font>====
'''pTet-GFP ''in vitro'' expression at 10&deg;C'''
'''pTet-GFP ''in vitro'' expression at 10&deg;C'''
{|align="left"
{|align="left"
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| width="200px"|[[Image:IC2007 ExperimentalDesign Phase1 PTet vitro 10degrees.PNG|thumb|300px|<font color=blue>22-08-2007</font>- Results of pTet at 10&deg;C]]
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| width="300px"|[[Image:IC2007 ExperimentalDesign Phase1 PTet vitro 10degrees.PNG|thumb|300px|Fig.3: GFP Expression of pTet-GFP ''in vitro'' at 10&deg;C over 4 hours]]
| width="50px"|
| width="50px"|
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|width="400px"| We tested pTet-GFP in vitro in commercial S30 cell extract at 10&deg;C for 4 hours, sampling every 30 minutes. The results show us that the pTet-GFP does not work at 10 &deg;C. The fluorescence of the samples remains nearly constant to that of the negative control. However, the negative control and the sample show slight variation around the basal level. The positive control shows a decay over the four hours, however because of the short sampling time we cannot tell whether this decay is expotential.  
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|width="400px"| There appears to be no significant increase in fluorescent levels of the pTet construct over the measured time course. Also shown in Fig.3 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.
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The graph the the right corresponds to the following:
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<div style="color:Green">Positive Control - Fluorescence of the diluted GFP solution</div>
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<div style="color:#F88017">The average fluorescence of the pTet samples(3)</div>
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<div style="color:#C45AEC">Negative Control - The fluoresence of a solution containing only S30 cell extract</div>
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|}
|}
<br clear=all>
<br clear=all>
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<br><br>
 
====<font color=darkblue>''Test: 22-08-2007''</font>====
====<font color=darkblue>''Test: 22-08-2007''</font>====
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{|align="left"
{|align="left"
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| width="200px"|[[Image:IC2007 Experimental Design PTet vitro 45degrees-4hours.PNG|thumb|300px|<font color=blue>22-08-2007</font>- Results of pTet at 45&deg;C]]
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| width="200px"|[[Image:IC2007 Experimental Design PTet vitro 45degrees-4hours.PNG|thumb|300px|Fig.4: GFP Expression of pTet-GFP ''in vitro'' at 45&deg;C over 4 hours]]
| width="50px"|
| width="50px"|
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|width="400px"|We tested pTet-GFP in vitro in commcercial S30 extract at 45&deg;C for 4 hours, sampleing every 30minutes.
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|width="400px"| There appears to be no significant increase in fluorescent levels of the pTet construct over the measured time course. Also shown in Fig.4 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints. 
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The results show that there minimal expression of GFP at 45&deg;C. The sample only slightly increases above the negative control showing minimal expression. The positive control shows decay, we cannot tell if this decay is expotential because of the short sampling time. Interestingly there is even greater variation within the results, giving very unpreditable trend lines.
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The results to the left show the followig
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<div style="color:Green">Positive Control - Fluorescence of the diluted GFP solution</div>
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<div style="color:#F88017"> The average fluorescence of the pTet samples(3)</div>
+
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<div style="color:#C45AEC">Negative Control - The fluoresence of a solution containing only S30 cell extract</div>
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|}
|}
<br clear=all>
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<br><br>
 
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<span style="font-size: 120%;">[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 '''pT7-GFP In Vitro''']
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</span><span style="font-size: 80%;">(100ng/&mu;l)</span>
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<br>
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 +
 
 +
===[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 '''pTet-GFP'''] (100ng/&mu;l)===
 +
 
====<font color=darkblue>''Test: 21-08-2007 to 22-08-2007''</font>====
====<font color=darkblue>''Test: 21-08-2007 to 22-08-2007''</font>====
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'''37 Degrees'''
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''' ''In vitro'' testing at 37&deg;C'''
{|align="left"
{|align="left"
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| width="200px"|[[Image:Ic2007_Experimental_Design_Phase1_protocol_2-1-pT7_vitro.PNG|thumb|300px|<font color=blue>21-08-2007</font>- Results of pT7 at 37&deg;C]]
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| width="200px"|[[Image:Ic2007_Experimental_Design_Phase1_protocol_2-1-pT7_vitro.PNG|thumb|300px|Fig.5: GFP Expression of pTet-GFP ''in vitro'']]
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| width="50px"|
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| width="50px"|  
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|width="400px"|The pT7 was tested in vitro in commercial S30 Cell extract at 37<sup>o</sup>C for 4 hours. The samples initially showed a higher fluorescence than that of the control, however, both control and samples decreased. The positive control showed a decay over 4 hours of measuring.
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|width="400px"|There appears to be no significant increase in fluorescent levels of the pT7 construct over the measured time course. Also shown in Fig.5 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.
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'''The graph on the left dispays the following.'''
+
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<div style="color:Green">Fluorescence of the diluted GFP solution. (+ve control)</div>
+
-
<div style="color:#F88017">The average fluorescence of the pT7 samples(3)</div>
+
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<div style="color:#C45AEC">The fluoresence of a solution containing only S30 cell extract (-ve control).</div>
+
|}
|}
<br clear=all>
<br clear=all>
-
 
+
'''pT7-GFP ''in vitro'' GFP expression at 37&deg;C'''
<br>
<br>
{|align="left"
{|align="left"
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| width="200px"|[[Image:IC2007 PT7-vitro-37deg-29 hours.PNG|thumb|left|300px| Results of in vitro testing of pT7 over a 29 hour period at 37<sup>o</sup>C]]
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| width="200px"|[[Image:IC2007 PT7-vitro-37deg-29 hours.PNG|thumb|left|300px|Fig.6: GFP Expression of pT7-GFP ''in vitro'' at 37&deg;C over 29 hours]]
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| width="50px"|
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| width="50px"|  
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|width="400px"|
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|width="400px"|The plate containing the samples was stored in a 37<sup>o</sup>C incubator overnight. It was re-tested the next morning to see whether GFP had been expressed,22 hours after of induction. The results were joined with the initial testing done over the first 4 hours of induction and are shown below.
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The plate containing the samples was stored in a 37<sup>o</sup>C incubator overnight. It was re-tested the next morning to see whether GFP had been expressed,22 hours after of induction. The results were joined with the initial testing done over the first 4 hours of induction and are shown below.
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The graph legend is the same as the one of the graph above.  
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Fig.6 shows that fluorescence levels of the sample increased minimally over the 2 days. This pattern of increase is also apparent in the negative control. Overall fluorescence levels also decreased in the positive control.  
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The fluorexcence of the sample did increase over the 2 days, however, so did teh fluorescence of the negative control which leads us to think that thsi increase in fluorescence is due to the methodology. The positive control shows an decrease in fluorescence due to teh decay of GFP.
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[[Media:PT7_in_vitro_37oC.xls |Complete set of results and raw data ]]
[[Media:PT7_in_vitro_37oC.xls |Complete set of results and raw data ]]

Revision as of 07:25, 14 October 2007

In vitro Testing of pTet-GFP and pT7-GFP Constructs

Aims

To Determine if the following constructs work in vitro:

To test the operating range of the constructs at 10°C, 37°C and 45°C over a staggered 24 hour period.

To identify problems in experimental methodology.


The testing was comprised of several tests:


Materials and Methods

Refer to protocols page.


Results

pTet-GFP (100ng/μl)

Test: 21-08-2007

In vitro testing at 37°C

Fig.1: GFP Expression of pTet-GFP in vitro
Fig.1: GFP Expression of pTet-GFP in vitro
The pTet-GFP contruct was tested in vitro using commercial S30 Cell Extract at 37 °C. As shown in Fig.1, pTet showed a marked increase in the first hour before levelling, reaching a slow but steady increase throughout a period of 4 hours.


Test: 21-08-2007 to Test: 23-08-2007

pTet-GFP in vitro GFP expression at 37°C

Fig.2: GFP Expression of pTet-GFP in vitro at 37°C over 56 hours
Fig.2: GFP Expression of pTet-GFP in vitro at 37°C over 56 hours
Fig.2 shows the incomplete curve of fluorescence levels over a staggered time period. As expected, the fluorescence levels of the pTet construct increase marked during the first few hours. This fluorescence level was higher, albeit reducing, over the middle portion of the graph, where it decreased to less than half the maximum level measured at the end of the time course. Interestingly, the supposed positive control of diluted GFP solution showed similar patterns in terms of fluorescence levels.


Test: 22-08-2007

pTet-GFP in vitro expression at 10°C

Fig.3: GFP Expression of pTet-GFP in vitro at 10°C over 4 hours
Fig.3: GFP Expression of pTet-GFP in vitro at 10°C over 4 hours
There appears to be no significant increase in fluorescent levels of the pTet construct over the measured time course. Also shown in Fig.3 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.


Test: 22-08-2007

pTet-GFP in vitro GFP expression at 45°C

Fig.4: GFP Expression of pTet-GFP in vitro at 45°C over 4 hours
Fig.4: GFP Expression of pTet-GFP in vitro at 45°C over 4 hours
There appears to be no significant increase in fluorescent levels of the pTet construct over the measured time course. Also shown in Fig.4 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.




pTet-GFP (100ng/μl)

Test: 21-08-2007 to 22-08-2007

In vitro testing at 37°C

Fig.5: GFP Expression of pTet-GFP in vitro
Fig.5: GFP Expression of pTet-GFP in vitro
There appears to be no significant increase in fluorescent levels of the pT7 construct over the measured time course. Also shown in Fig.5 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.


pT7-GFP in vitro GFP expression at 37°C

Fig.6: GFP Expression of pT7-GFP in vitro at 37°C over 29 hours
Fig.6: GFP Expression of pT7-GFP in vitro at 37°C over 29 hours
The plate containing the samples was stored in a 37oC incubator overnight. It was re-tested the next morning to see whether GFP had been expressed,22 hours after of induction. The results were joined with the initial testing done over the first 4 hours of induction and are shown below.

Fig.6 shows that fluorescence levels of the sample increased minimally over the 2 days. This pattern of increase is also apparent in the negative control. Overall fluorescence levels also decreased in the positive control.



Complete set of results and raw data

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