Difference between revisions of "Part:BBa T9002:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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This experience page is provided so that any user may enter their  
 +
 
 +
experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
  
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===Applications of BBa_T9002===
+
=Applications of BBa_T9002=
 +
 
 +
=User Reviews=
 +
 
 +
=== UNIPV-Pavia iGEM 2011 ===
  
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===User Reviews===
 
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<!-- DON'T DELETE --><partinfo>BBa_T9002 StartReviews</partinfo>
 
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<partinfo>BBa_T9002 AddReview number</partinfo>
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<partinfo>BBa_T9002 AddReview 5</partinfo>
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<I>Username</I>
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<I>UNIPV-Pavia iGEM 2011</I>
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Enter the review inofrmation here.
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''Antiquity. This review comes from the old result system and indicates that this part worked in some test.''
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<!-- End of the user review template -->
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'''2009 DNA Distribution quality control'''
 
'''2009 DNA Distribution quality control'''
  
−
The UNIPV-Pavia iGEM team sequenced T9002 part and found that it was completely confirmed, while iGEM QC results classified it as "inconsistent". DNA was resuspended from well 9A, kit plate 2, transformed in TOP10 E. coli and amplified inoculating a single colony from the grown LB agar plate in LB medium. Finally DNA has been miniprepped from the grown culture and sent to a BMR Genomics (Padova, Italy) for sequencing.
+
The UNIPV-Pavia iGEM team sequenced T9002 part and found that it was  
 +
 
 +
completely confirmed, while iGEM QC results classified it as  
 +
 
 +
"inconsistent". DNA was resuspended from well 9A, kit plate 2,  
 +
 
 +
transformed in TOP10 E. coli and amplified inoculating a single colony  
 +
 
 +
from the grown LB agar plate in LB medium. Finally DNA has been  
 +
 
 +
miniprepped from the grown culture and sent to a BMR Genomics (Padova,  
 +
 
 +
Italy) for sequencing.
  
 
'''Experimental measurements'''
 
'''Experimental measurements'''
  
−
The UNIPV-Pavia iGEM team tested T9002 BioBrick in several working conditions. Results are reported in <partinfo>BBa_F2620</partinfo> Experience page.
+
The UNIPV-Pavia iGEM team tested T9002 BioBrick in several working  
  
−
The Brown iGEM team conducted tests on this part in the summer of 2007. The results are depicted in the graphs below.
+
conditions. Results are reported in <partinfo>BBa_F2620</partinfo>
 +
 
 +
Experience page.
 +
 
 +
The Brown iGEM team conducted tests on this part in the summer of 2007.  
 +
 
 +
The results are depicted in the graphs below.
  
 
[[Image:AHL.JPG|700px]]   
 
[[Image:AHL.JPG|700px]]   
  
−
The first graph indicates that until a critical point is reached, increasing AHL concentration does increase GFP output. This is most easily noticeable between the concentrations of 20 nM, after which point the amount of GFP produced by cells begins to decrease. This may be due to one of two things: AHL quenches the signal from GFP, or too much AHL disrupts the cell's functions in a way that either kills it or prevents it from making as much GFP. This second hypothesis is partially confirmed by the second graph, which shows that adding more than 20 nM AHL causes a decline in cell density. On each graph, the different colored lines represent different time points after AHL was added to the cells. They are 4 hours, 5 hours, etc.
+
The first graph indicates that until a critical point is reached,  
  
 +
increasing AHL concentration does increase GFP output. This is most
  
−
{|width='80%' style='border:1px solid gray'
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easily noticeable between the concentrations of 20 nM, after which
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|-
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|width='10%'|
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<partinfo>BBa_T9002 AddReview 5</partinfo>
+
−
<I>Antiquity</I>
+
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|width='60%' valign='top'|
+
−
This review comes from the old result system and indicates that this part worked in some test.
+
−
|}
+
−
<!-- DON'T DELETE --><partinfo>BBa_T9002 EndReviews</partinfo>
+
  
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<!-- DON'T DELETE --><partinfo>BBa_T9002 StartReviews</partinfo>
+
point the amount of GFP produced by cells begins to decrease. This may
 +
 
 +
be due to one of two things: AHL quenches the signal from GFP, or too
 +
 
 +
much AHL disrupts the cell's functions in a way that either kills it or
 +
 
 +
prevents it from making as much GFP. This second hypothesis is
 +
 
 +
partially confirmed by the second graph, which shows that adding more
 +
 
 +
than 20 nM AHL causes a decline in cell density. On each graph, the
 +
 
 +
different colored lines represent different time points after AHL was
 +
 
 +
added to the cells. They are 4 hours, 5 hours, etc.
 +
 
 +
----
  
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<html></html>
 
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<!-- Template for a user review
 
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{|width='80%' style='border:1px solid gray'
 
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|-
 
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|width='10%'|
 
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<partinfo>BBa_T9002 AddReview 2</partinfo>
 
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<html><a name='t9002PV11'></a></html>
 
−
<I>Username</I>
 
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|width='60%' valign='top'|
 
−
Enter the review inofrmation here.
 
−
|};
 
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<!-- End of the user review template -->
 
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{|width='80%' style='border:1px solid gray'
 
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|width='10%'|
 
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<partinfo>BBa_T9002 AddReview 5</partinfo>
 
−
<I>UNIPV-Pavia iGEM 2011</I>
 
−
|width='60%' valign='top'|
 
 
<html>
 
<html>
  
−
BioBrick <a href="https://parts.igem.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non linear relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise. Referring to its activation formula, the calibration curve is shown below.<br></div>
+
BioBrick <a href="https://parts.igem.org/Part:BBa_T9002">BBa_T9002</a>  
 +
 
 +
is an HSL biosensor, which provides a non linear relationship between  
 +
 
 +
HSL input and S<sub>cell</sub> output. More precisely, the  
 +
 
 +
characteristic sigmoidal curve requires synthetic parameters for its  
 +
 
 +
accurate identification. These are the minimum and maximum values, the  
 +
 
 +
swtich point (i.e., the curve inflection point), and the upper and  
 +
 
 +
lower boundaries of linearity. This biosensor revealed greatly  
 +
 
 +
reliable, providing measurement repeatability and minimal experimental  
 +
 
 +
noise. Referring to its activation formula, the calibration curve is  
 +
 
 +
shown below.<br></div>
 
     <br>
 
     <br>
 
     <div style='text-align:center'>
 
     <div style='text-align:center'>
−
       <div class="thumbinner" style="width:100%;"> <a href="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage"  width="47%" height="50%"></a></div>
+
       <div class="thumbinner" style="width:100%;"> <a  
 +
 
 +
href="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg"><img  
 +
 
 +
alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg"  
 +
 
 +
class="thumbimage"  width="47%" height="50%"></a></div>
 
     </div>
 
     </div>
 
     <br>
 
     <br>
 
     <div style='text-align:center'>
 
     <div style='text-align:center'>
−
       <div class="thumbinner" style="width:100%;"> <a href="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage"  width="100%"></a></div>
+
       <div class="thumbinner" style="width:100%;"> <a  
 +
 
 +
href="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg"><img  
 +
 
 +
alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg"  
 +
 
 +
class="thumbimage"  width="100%"></a></div>
 
     </div>
 
     </div>
 
     <center>
 
     <center>
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     <br>
 
     <br>
 
     <br>
 
     <br>
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<div align="justify">In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.</div>
+
<div align="justify">In order to determine the threshold sensitivity of  
 +
 
 +
T9002 biosensor, experiments were performed with several HSL inductions  
 +
 
 +
minimally interspaced in the region of low detectability. Hypothesizing  
 +
 
 +
that the inducer is 1:20 diluted (as for all of our tests), the minimum  
 +
 
 +
detectable HSL concentration is 3 nM.</div>
 
<br><br>
 
<br><br>
 
<a name='t9002PV11'></a>
 
<a name='t9002PV11'></a>
−
This biosensor was used to measure HSL concentration for parts producing or degrading this signalling molecule. For each of these experiments, a calibration curve of the biosensor was built, inducing it with known HSL concentrations, evaluating for each of them the S<sub>cell</sub> signal and finally estimating the Hill curve parameters. Once identified the parameters, the unknown concentration of a sample can be evaluated from its S<sub>cell</sub> (provided that it has a value included in the linear zone of the biosensor), as shown below:
+
This biosensor was used to measure HSL concentration for parts  
  
−
<div class="center"><div class="thumbinner" style="width: 650px;"><a href="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div>
+
producing or degrading this signalling molecule. For each of these
 +
 
 +
experiments, a calibration curve of the biosensor was built, inducing
 +
 
 +
it with known HSL concentrations, evaluating for each of them the
 +
 
 +
S<sub>cell</sub> signal and finally estimating the Hill curve
 +
 
 +
parameters. Once identified the parameters, the unknown concentration
 +
 
 +
of a sample can be evaluated from its S<sub>cell</sub> (provided that
 +
 
 +
it has a value included in the linear zone of the biosensor), as shown
 +
 
 +
below:
 +
 
 +
<div class="center"><div class="thumbinner" style="width: 650px;"><a  
 +
 
 +
href="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"  
 +
 
 +
class="image"><img alt=""  
 +
 
 +
src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbim
 +
 
 +
age" width="40%"></a></div></div>
  
 
<br>
 
<br>
  
−
It is necessary, then, to multiply the measurement for the dilution factor used (in our experiments it was 20).
+
It is necessary, then, to multiply the measurement for the dilution  
 +
 
 +
factor used (in our experiments it was 20).
  
 
</html>
 
</html>
 
|}
 
|}
  
 +
=== Sevilla 2011 ===
 +
 +
{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='20%' valign='top'|
 
<partinfo>BBa_T9002 AddReview 2</partinfo>
 
<partinfo>BBa_T9002 AddReview 2</partinfo>
−
<html><a name='t9002PV11'></a></html>
 
 
<I>Sevilla 2011</I>
 
<I>Sevilla 2011</I>
 +
|width='50%' valign='top'|
 +
 +
<html><a name='t9002PV11'></a></html>
 +
 +
We used this construction as a model for a GFP-based characterization
 +
 +
method. We tried different concentrations, that turned to be too high,
 +
 +
for it seems that the media was quite saturated.
 +
 +
<nowiki>Fluorescence
  
−
We used this construction as a model for a GFP-based characterization method. We tried different concentrations, that turned to be too high, for it seems that the media was quite saturated.
+
---------------------------------------------------------- O.D.
  
−
<nowiki>Fluorescence ---------------------------------------------------------- O.D.(600nm)</nowiki>
+
(600nm)</nowiki>
  
 
[[Image:Fluabs.png]]
 
[[Image:Fluabs.png]]
Line 134: Line 225:
  
 
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 +
 +
|}

Revision as of 17:02, 26 October 2014

This experience page is provided so that any user may enter their

experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_T9002

User Reviews

UNIPV-Pavia iGEM 2011

•••••

UNIPV-Pavia iGEM 2011

Antiquity. This review comes from the old result system and indicates that this part worked in some test.


2009 DNA Distribution quality control

The UNIPV-Pavia iGEM team sequenced T9002 part and found that it was

completely confirmed, while iGEM QC results classified it as

"inconsistent". DNA was resuspended from well 9A, kit plate 2,

transformed in TOP10 E. coli and amplified inoculating a single colony

from the grown LB agar plate in LB medium. Finally DNA has been

miniprepped from the grown culture and sent to a BMR Genomics (Padova,

Italy) for sequencing.

Experimental measurements

The UNIPV-Pavia iGEM team tested T9002 BioBrick in several working

conditions. Results are reported in BBa_F2620

Experience page.

The Brown iGEM team conducted tests on this part in the summer of 2007.

The results are depicted in the graphs below.

AHL.JPG

The first graph indicates that until a critical point is reached,

increasing AHL concentration does increase GFP output. This is most

easily noticeable between the concentrations of 20 nM, after which

point the amount of GFP produced by cells begins to decrease. This may

be due to one of two things: AHL quenches the signal from GFP, or too

much AHL disrupts the cell's functions in a way that either kills it or

prevents it from making as much GFP. This second hypothesis is

partially confirmed by the second graph, which shows that adding more

than 20 nM AHL causes a decline in cell density. On each graph, the

different colored lines represent different time points after AHL was

added to the cells. They are 4 hours, 5 hours, etc.

BioBrick BBa_T9002 is an HSL biosensor, which provides a non linear relationship between HSL input and Scell output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise. Referring to its activation formula, the calibration curve is shown below.


Minimum [Scell] Maximum [Scell] Switch point [nM] Lower boundary of linearity [nM] Upper boundary of linearity [nM]
17.31 739.4 1.39 0.38 5.07


In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.


This biosensor was used to measure HSL concentration for parts producing or degrading this signalling molecule. For each of these experiments, a calibration curve of the biosensor was built, inducing it with known HSL concentrations, evaluating for each of them the Scell signal and finally estimating the Hill curve parameters. Once identified the parameters, the unknown concentration of a sample can be evaluated from its Scell (provided that it has a value included in the linear zone of the biosensor), as shown below:

It is necessary, then, to multiply the measurement for the dilution factor used (in our experiments it was 20).

Sevilla 2011

••

Sevilla 2011

We used this construction as a model for a GFP-based characterization

method. We tried different concentrations, that turned to be too high,

for it seems that the media was quite saturated.

Fluorescence ---------------------------------------------------------- O.D. (600nm)

Fluabs.png


Fluorescence/Absorbance ratio and average of these data

Fluoabsavrg.png


Variation of the fluo/Abs ratio in time

Variationfluoabs.png


GFPgraph.png


UNIQ48ceb0a8a506b81e-partinfo-00000006-QINU