IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 1.1

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*pTet and pT7 ''in vivo'' - [[IGEM:IMPERIAL/2007/Notebook/2007-8-16 | Tested 15-08-2007]]
*pTet and pT7 ''in vivo'' - [[IGEM:IMPERIAL/2007/Notebook/2007-8-16 | Tested 15-08-2007]]
*Retesting pT7 ''in vivo'' - [[IGEM:IMPERIAL/2007/Notebook/2007-8-17 | Tested 17-08-2007]]
*Retesting pT7 ''in vivo'' - [[IGEM:IMPERIAL/2007/Notebook/2007-8-17 | Tested 17-08-2007]]
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Controls:
 
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*Positive control - diluted GFP solution of equal volume
 
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*Negaitve control - LB media of equal volume
 
==Results==
==Results==
====<font color=darkblue>''Test: 17-08-2007''</font>====
====<font color=darkblue>''Test: 17-08-2007''</font>====
{|align="left"
{|align="left"
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| width="200px"|[[Image:IC2007 Experiments Phase1 Protocol1-1Res.JPG|thumb|300px|Fig.1: Total Fluorescence of pTet and pT7 ''in vivo'' over 6 hours]]
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| width="200px"|[[Image:IC2007 Experiments Phase1 Protocol1-1Res.JPG|thumb|300px|Fig.1: Total Fluorescence of pTet-GFP and pT7-GFP ''in vivo'' over 6 hours]]
| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1.jpg|thumb|300px|Fig.2: pTet ''in vivo'' over 6 hours]]
| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1.jpg|thumb|300px|Fig.2: pTet ''in vivo'' over 6 hours]]
| width="50px"|
| width="50px"|
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|width="300px"| pTet and pT7 were both compared to a negative and positive control, where pTet showed significant fluorescence, while pT7 did not show any dramatic increase (Fig.1). When measured over intervals, pTet showed a steady increase in fluorescence (Fig.2). Fig.2 also suggests that there may be some variation across the samples that have been measured.
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|width="300px"| pTet-GFP and pT7-GFP were both compared to a negative and positive control, where pTet-GFP showed significant fluorescence, while pT7 did not show any dramatic increase (Fig.1). When measured over intervals, pTet showed a steady increase in fluorescence (Fig.2). Fig.2 also suggests that there may be some variation across the samples that have been measured.
|}
|}
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====<font color=darkblue>''Test: 21-08-2007</font>''====
====<font color=darkblue>''Test: 21-08-2007</font>''====
{|align="left"
{|align="left"
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| width="200px"| [[Image:IC2007 Experimental design protocol1-1res2 .PNG|thumb|280px| Fig.3 - Total Fluorescence of pT7 ''in vivo'' over 4 hours]]
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| width="200px"| [[Image:IC2007 Experimental design protocol1-1res2 .PNG|thumb|280px| Fig.3 - Total Fluorescence of pT7-GFP ''in vivo'' over 4 hours]]
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| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1Res1.PNG|thumb|310px|Fig.4 - pT7 ''in vivo'' over 4 hours]]
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| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1Res1.PNG|thumb|310px|Fig.4 - pT7-GFP ''in vivo'' over 4 hours]]
| width="50px"|
| width="50px"|
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|width="300px"| Fig.3 shows that the pT7 construct had significant fluorescence compared to the negative control, which seems to suggest that GFP has been expressed. This is however not the case, as Fig.4 indicates no increase in fluorescence over the time period.  
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|width="300px"| Fig.3 shows that the pT7-GFP construct had significant fluorescence compared to the negative control, which seems to suggest that GFP has been expressed. However, when fluorescence correlated over the time period, there was little or no increase in fluorescence (Fig.4), suggesting little or no expression of GFP over the time course.
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+
|}
|}
<br clear="all">
<br clear="all">
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 +
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'''Controls:'''
 +
*Positive control - diluted GFP solution of equal volume
 +
*Negaitve control - LB media of equal volume
 +
[http://openwetware.org/images/4/42/IC2007_Experiments_Phase_1_Results-16-08-2007.xls  Complete set of results and raw data ]
[http://openwetware.org/images/4/42/IC2007_Experiments_Phase_1_Results-16-08-2007.xls  Complete set of results and raw data ]
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==Discussion==
==Discussion==
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<br>
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===[http://parts.mit.edu/registry/index.php/Part:BBa_I13522 '''pTet-GFP''']===
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<br><span style="font-size: 140%;"> <font color=darkblue>''Test: 17-08-2007''</font>'''</span>
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Fig.1 and Fig.2 both strongly indicate that there was a good amount of expression of GFP (~54000) with the pTet-GFP construct, leading to an increase in fluoresence over time. This shows that the construct is functioning well ''in vivo''.
-
<br>
+
 
-
<br>
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Fig.2 however suggests that there may be significant variation in the measurement of our samples. This could be due to experimental methodology, or the intrinsic nature of variability of expression in the ''in vivo'' chassis.
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[http://parts.mit.edu/registry/index.php/Part:BBa_I13522 '''pTet-GFP''']<br>
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The pTet construct works in vivo, however there are massive amounts of variation which are likely to be due to the experimental methodology. The error could be due the intrinsic nature of variability of the in vivo chassis or due to variation with the protocol i.e. measurement errors.
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[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 '''pT7-GFP''']<br>
 
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Initial testing of the pT7 - GFP contruct in vivo failed. We think that this could be because it was not properly induced. We need to add IPTG to a suitable concentration(~1mM) and grow overnight.
 
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<br>
 
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<br>
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===[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 '''pT7-GFP''']===
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<br><span style="font-size: 140%;"> <font color=darkblue>''Test: 21-08-2007''</font>'''</span>
+
As our initial tests of the pT7-GFP construct did not yield expected results, it was postualated that the absence of fluorescence increase could be due to several reasons. Firstly, the cells might not be properly induced, to which IPTG of a suitable concentration(~1mM) needs to be added, and the culture grown overnight.  It could also be that the construct was not working at all, or that the culture was in stationary phase when it was tested, leading to little or not expression of GFP.
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<br>
+
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<br>
+
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[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 '''pT7-GFP''']<br>
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This allows us to conclude that the pT7-GFP contruct does not work as fast or powerful as pTet in vivo or it does not work at all. Two reasons were hypothesized:
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# The bacteria cells were in stationary phase  
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In the re-test, the culture had been properly induced with IPTG at growth phase. While absolute fluorescence levels indicate the presence of GFP in the culture, it was not as significant as that of pTet-GFP. In addition, there was little or no increase of fluorescence over the time period at which it was measured. This could be due to the fact that the pT7-GFP construct either did not express as quickly or strongly as pTet-GFP ''in vivo'', or that the construct was simply not working. Further investigation into the construct also revealed a weaker ribosome binding site compared to the pTet-GFP construct which could account for the weak fluorescent signal.  
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# The pT7 construct is not working.  
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We do not think it is the first reason because this is in the retest the protocol we followed ensures that the ''E.coli'' are in the groth phase.
 
==Conclusion==
==Conclusion==
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We investigated the possibility that it was due to the fact that the pT7 consrtuct was not working. After investigating into the construct we found out that it has a weak ribosome binding site, this could explain the weak signal for the construct. We are going to look into cloning out the promoter and building our own construct.
+
We investigated the possibility of using different constructs, and strength of different promoters for our projects. To conclude,
 +
* pTet-GFP construct gave a strong fluorescent signal, indicating good expression of GFP ''in vivo''.
 +
* pT7-GFP construct gave little or no fluorescent signal, to which several reasons had been suggested, and further experiments are required to corrobate them.

Revision as of 06:02, 14 October 2007


In Vivo Testing


Aim

To Determine if the following constructs work in vivo:

The testing was comprised of several tests:

Results

Test: 17-08-2007

Fig.1: Total Fluorescence of pTet-GFP and pT7-GFP in vivo over 6 hours
Fig.1: Total Fluorescence of pTet-GFP and pT7-GFP in vivo over 6 hours
Fig.2: pTet in vivo over 6 hours
Fig.2: pTet in vivo over 6 hours
pTet-GFP and pT7-GFP were both compared to a negative and positive control, where pTet-GFP showed significant fluorescence, while pT7 did not show any dramatic increase (Fig.1). When measured over intervals, pTet showed a steady increase in fluorescence (Fig.2). Fig.2 also suggests that there may be some variation across the samples that have been measured.


Test: 21-08-2007

Fig.3 - Total Fluorescence of pT7-GFP in vivo over 4 hours
Fig.3 - Total Fluorescence of pT7-GFP in vivo over 4 hours
Fig.4 - pT7-GFP in vivo over 4 hours
Fig.4 - pT7-GFP in vivo over 4 hours
Fig.3 shows that the pT7-GFP construct had significant fluorescence compared to the negative control, which seems to suggest that GFP has been expressed. However, when fluorescence correlated over the time period, there was little or no increase in fluorescence (Fig.4), suggesting little or no expression of GFP over the time course.



Controls:

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

Complete set of results and raw data


Discussion

pTet-GFP

Fig.1 and Fig.2 both strongly indicate that there was a good amount of expression of GFP (~54000) with the pTet-GFP construct, leading to an increase in fluoresence over time. This shows that the construct is functioning well in vivo.

Fig.2 however suggests that there may be significant variation in the measurement of our samples. This could be due to experimental methodology, or the intrinsic nature of variability of expression in the in vivo chassis.


pT7-GFP

As our initial tests of the pT7-GFP construct did not yield expected results, it was postualated that the absence of fluorescence increase could be due to several reasons. Firstly, the cells might not be properly induced, to which IPTG of a suitable concentration(~1mM) needs to be added, and the culture grown overnight. It could also be that the construct was not working at all, or that the culture was in stationary phase when it was tested, leading to little or not expression of GFP.

In the re-test, the culture had been properly induced with IPTG at growth phase. While absolute fluorescence levels indicate the presence of GFP in the culture, it was not as significant as that of pTet-GFP. In addition, there was little or no increase of fluorescence over the time period at which it was measured. This could be due to the fact that the pT7-GFP construct either did not express as quickly or strongly as pTet-GFP in vivo, or that the construct was simply not working. Further investigation into the construct also revealed a weaker ribosome binding site compared to the pTet-GFP construct which could account for the weak fluorescent signal.


Conclusion

We investigated the possibility of using different constructs, and strength of different promoters for our projects. To conclude,

  • pTet-GFP construct gave a strong fluorescent signal, indicating good expression of GFP in vivo.
  • pT7-GFP construct gave little or no fluorescent signal, to which several reasons had been suggested, and further experiments are required to corrobate them.
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