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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Aim)
Line 1: Line 1:
-
<br><span style="font-size: 180%;"> In Vivo Tetsing'''</span>
+
<br><span style="font-size: 180%;"> In Vivo Testing'''</span>
<hr>
<hr>
__NOTOC__
__NOTOC__
Line 6: Line 6:
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13522 '''pTet-GFP''']  
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13522 '''pTet-GFP''']  
*[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 '''pT7-GFP''']
*[http://parts.mit.edu/registry/index.php/Part:BBa_E7104 '''pT7-GFP''']
 +
The testing was comprised of several tests:
The testing was comprised of several tests:
*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]]
 +
 +
Controls:
 +
*Positive control - diluted GFP solution of equal volume
 +
*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"
-
| width="200px"|[[Image:IC2007 Experiments Phase1 Protocol1-1Res.JPG|thumb|300px|<font color=blue>17-08-2007</font>- Results of pTet and pT7]]
+
| width="200px"|[[Image:IC2007 Experiments Phase1 Protocol1-1Res.JPG|thumb|300px|Fig.1: Total Fluorescence of pTet and pT7 ''in vivo'' over 6 hours]]
-
| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1.jpg|thumb|300px|<font color=blue>17-08-200707</font>- Results of pTet in vivo over 1 hour]]
+
| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1.jpg|thumb|300px|Fig.2: pTet ''in vivo'' over 6 hours]]
| width="50px"|
| width="50px"|
-
|width="300px"| The results show that pTet works in vivo whereas the pT7 does not show any dramatic increase in fluorescence. Both the samples were compared to a negative and positive control to determine if there was any significant fluorescence. The fluorescence of pTet over 1 hour is shown, the results do show an increase. In addition it can be seen that there is a massive variation  
+
|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.
|}
|}
<br clear=all>
<br clear=all>
Line 22: Line 27:
====<font color=darkblue>''Test: 21-08-2007</font>''====
====<font color=darkblue>''Test: 21-08-2007</font>''====
{|align="left"
{|align="left"
-
| width="200px"| [[Image:IC2007 Experimental design protocol1-1res2 .PNG|thumb|280px|<font color=blue>21-08-2007</font>- Results of retesting pT7 in vivo repeat]]
+
| width="200px"| [[Image:IC2007 Experimental design protocol1-1res2 .PNG|thumb|280px| Fig.3 - Total Fluorescence of pT7 ''in vivo'' over 4 hours]]
-
| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1Res1.PNG|thumb|310px|<font color=blue>21-08-2007</font>- Results of restesting pT7 in vivo over 1 hour]]
+
| width="200px"| [[Image:IC2007 Experiments Phase1 Protocol1-1Res1.PNG|thumb|310px|Fig.4 - pT7 ''in vivo'' over 4 hours]]
| width="50px"|
| width="50px"|
-
|width="300px"| The fluorescence levels were recorded over a range of 4 hours for 3 repeats and compared, as in the case of pTet, to a positive control of diluted GFP. The fluorescence activity of pT7-GFP is minimal and remains at a constant basal level. 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.  
+
|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.  
 +
 
|}
|}
<br clear="all">
<br clear="all">

Revision as of 06:37, 14 October 2007


In Vivo Testing


Aim

To Determine if the following constructs work in vivo:

The testing was comprised of several tests:

Controls:

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

Results

Test: 17-08-2007

Fig.1: Total Fluorescence of pTet and pT7 in vivo over 6 hours
Fig.1: Total Fluorescence of pTet and pT7 in vivo over 6 hours
Fig.2: pTet in vivo over 6 hours
Fig.2: pTet in vivo over 6 hours
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.


Test: 21-08-2007

Fig.3 - Total Fluorescence of pT7 in vivo over 4 hours
Fig.3 - Total Fluorescence of pT7 in vivo over 4 hours
Fig.4 - pT7 in vivo over 4 hours
Fig.4 - pT7 in vivo over 4 hours
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.


Complete set of results and raw data


Discussion



Test: 17-08-2007

pTet-GFP
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.

pT7-GFP
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.



Test: 21-08-2007

pT7-GFP
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:

  1. The bacteria cells were in stationary phase
  2. The pT7 construct is not working.

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

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.

Personal tools