Team:NTU-Singapore/Project/Wetlab

From 2009.igem.org

(Difference between revisions)
 
(31 intermediate revisions not shown)
Line 28: Line 28:
-
== Successful Constructs ==
 
-
'''The following are the constructs that we have shown to be working.'''
+
<div id="tock"> 
 +
<div class="spiffyfg">
 +
__TOC__
 +
</div><html>
 +
<b class="spiffy">
 +
  <b class="spiffy5"></b>
 +
  <b class="spiffy4"></b>
 +
  <b class="spiffy3"></b>
 +
  <b class="spiffy2"><b></b></b>
 +
  <b class="spiffy1"><b></b></b></b>
 +
</html></div>
-
# J23119-B0034-NorR-B0015 (''system construct'')
 
-
# J23119-B0034-GFP-B0015 (''characterization construct'')
 
-
# pLac(R0010)-B0034-CHE-B0015 (''test construct'')
 
-
# J23119-B0034-IFP-B0015-J23119-B0034-HO1(I15008)-B0015 (''test construct'')
 
 +
'''Our Working Constructs!!'''
-
The first construct is a system construct, in that it was intended for our system to feature this as a functional part.
 
-
The second construct is a characterization construct to characterize the modular function of the J23119 promoter in the transcription of NorR.
+
* '''J23119-B0034-NorR-B0015''', is a system construct, in that it was intended for our system to feature this as a functional part.
 +
* '''J23119-B0034-GFP-B0015''', is a characterization construct to characterize the modular function of the J23119 promoter in the transcription of NorR.
 +
* '''Lac(R0010)-B0034-CHE-B0015''', is also a test construct to only test and characterize the enzymatic activity of CHE.
 +
* '''J23119-B0034-IFP-B0015-J23119-B0034-HO1(I15008)-B0015''', is a test construct to only test the reporting ability of IFP.
-
The third construct is also a test construct to only test and characterize the enzymatic activity of CHE.
+
<br class="clear" />
-
The fourth construct is a test construct to only test the reporting ability of IFP.
 
-
=== J23119-B0034-NorR-B0015 (K256007) ===
 
-
NorR is an important NO sensor that triggers the transcription at pNO. Thus, we want an NorR producing construct to improve on the sensitivity of pNO.
 
 +
= <span class="title">NorR System Construct</span> =
 +
NorR is an important NO sensor that triggers the transcription at pNO. Thus, we want an NorR producing construct to improve on the sensitivity of pNO to optimise its efficiency at physiological levels.
-
'''Double Digest verification'''
 
 +
== Verification of construct ==
-
<div class="grid_6 alpha">[[Image:NTUnorrgel2.jpg|346px]]</div>
+
<div class="grid_5 alpha suffix_1">
 +
'''Double Digestion'''
-
''Successful construct verification of J23119-B0034-NorR-B0015 by Double Digest'''
 
-
<span class="grey">Lanes 2 and 3 are the subjects of interest. Double digestion of J23119-B0034-NorR-B0015 with Xba1 & PstI indicated that the ligation in lane 2 had failed while lane 3 contained the successful construct. Length of J23119-B0034-NorR-B0015 corresponds to the expected length of ~1750 bp.</span><br class="clear">
+
[[Image:NTUnorrgel2.jpg|385px]]
 +
<center>
 +
'''Successful construct verification''' of J23119-B0034-NorR-B0015
 +
''This indicates the presence of the construct.''
 +
</center>
-
'''SDS PAGE verification of NorR'''
+
</div>
 +
<div class="grid_5 omega">
 +
'''SDS PAGE'''
-
<div class="grid_6 alpha">[[Image:NTUnorrsds.jpg|346px]]</div>
+
[[Image:SDS page for NorR on 16 Sep 09-edited.jpg|285px]]
-
'''Successful expression of NorR'''
 
-
<span class="grey">55 kDa NorR has been successfully produced by the construct. NorR  band is highlighted with white arrow.</span>
+
<center>'''Successful production of NorR'''</center>
 +
</div>
 +
<br class="clear" />
-
'''SDS PAGE characterization of NorR'''
+
==== Characterization of NorR ====
-
Since the sole purpose of J23119-B0034-NorR-B0015 is to produce NorR, we shall dwell into the factors that might affect the production of the protein. The transformed cells were incubated at 3 different environmental temperatures (30 / 37 / 42 deg C).
 
 +
[[Image:NorR-temp.jpg|400px|center]]
-
<div class="grid_6 alpha">[[Image:NTUnorrsds2.jpg|346px]]</div>
+
<center>'''SDS PAGE results for NorR expressed at different inoculation temperatures'''</center>
-
'''SDS PAGE results for NorR expressed at different inoculation temperatures'''
+
 
 +
Since the sole purpose of J23119-B0034-NorR-B0015 (<partinfo>K256007</partinfo>) is to produce NorR, we shall dwell into the factors that might affect the production of the protein. The transformed cells were incubated at 3 different environmental temperatures (30 / 37 / 42°C).
 +
 
 +
'''SDS PAGE characterization was carried out''' (''top right'').
 +
 
 +
 
 +
It has been observed that at elevated temperature, i.e. 42°C, production of NorR had decreased as shown by the diminished intensity (see 2nd lane). The production of NorR at 30°C and 37°C carried no significant difference.
 +
 
 +
 
 +
 
 +
== Characterization of J23119 ==
 +
 
 +
'''Since J23119 (<partinfo>K256003</partinfo>) was used as the constitutive promoter for NorR producing construct, it is necessary for us to understand its modular function and how it would affect NorR production.'''
 +
 
 +
 
 +
[[Image:NTU119temp.png|651px]]
 +
 
 +
<center>'''Characterization of J23119 w.r.t Temp (deg C)'''</center>
 +
 
 +
 
 +
Preliminary characterisation of J23119 showed that the activity of the promoter to be the strongest at 25°C and weakest at 37°C with 30°C being something of an intermediate. Activity of the J23119 was independent of time. Noteworthy was that there was a drastic drop in activity of the J23119 promoter at 16min, 37°C.
 +
 
 +
 
 +
 
 +
[[Image:NTU119culture.png|651px]]
 +
 
 +
<center>'''Average GFP/OD of J23119-GFP in different medium'''</center>
 +
 
 +
 
 +
It shows that the supplemented M9 medium is the ideal choice for the characterization of the J23119 promoter. M9 is most likely to be the ideal medium for characterisation of other promoters in general.
 +
 
 +
 
 +
 
 +
[[Image:NTU119backbone.png|651px|]]
 +
 
 +
<center>'''Average GFP/OD of J23119-GFP in steady state (M9)'''</center>
 +
 
 +
 
 +
Vector backbone did not seem to have much of an effect on the strengths of J23119, although pSB1A2 has a slight positive effect on the activity of the J23119.
 +
 
 +
 
 +
 
 +
[[Image:NTU119diffprom.png|651px]]
 +
 
 +
<center>'''Comparison of J23119 with other promoters'''</center>
 +
 
 +
 
 +
Based on our preliminary characterization results, it appeared that J23101 produced a much stronger signal than J23119 and pTet. This was quite surprising because J23119 promoter is supposed to be stronger then the J23101.
 +
 
 +
 
 +
 
 +
[[Image:NTU119diffchassis.png|651px]]
 +
 
 +
<center>'''Characterization of J23119 in different chassis'''</center>
 +
 
 +
 
 +
J23119 performance appeared to be the best in Origami B.
 +
 
 +
 
 +
 
 +
Click here to see the detailed Wetlab regarding [[Team:NTU-Singapore/Prototype/Sense#Device_Characterization | Sensing Device Characterization]].
 +
 
 +
 
 +
 
 +
= <span class="title">Cholesteryl Esterase Expression & Characterization</span> =
 +
 
 +
To examine the enzymatic activity of cholesteryl esterase, a test construct, '''pLac (R0010)-B0034-CHE-B0015''' (<partinfo>K256028</partinfo>) was synthesized.
 +
 
 +
CHE production was induced by l0mM lactose. The cells were sonicated, and the cell lysate containing CHE was characterized using Amplex® Red Cholesterol Assay Kit. The characterization results is shown below.
 +
 
 +
 
 +
 
 +
==== Characterization of CHE ====
 +
 
 +
[[Image:NTUcheresult.png|651px]]
 +
 
 +
<center>'''Cholesterol Assay with CHE Construct'''</center>
 +
From the graph, the cell lysate containing CHE had achieved higher fluorescence readings than the negative control. The increase in fluorescence reading is due to enzyme-coupled reaction that detects free cholesterol concentration. Hence, we have proven that CHE had indeed been produced from this construct and had been working to our expectation.
 +
 
 +
 
 +
Click here to see the detailed Wetlab regarding [[Team:NTU-Singapore/Prototype/Degrade#Device_Characterization | Degradation Device Characterization]].
 +
 
 +
 
 +
 
 +
 
 +
= <span class="title">Infrared Signal from Imaging Device</span> =
 +
 
 +
The purpose of creating a constitutively expressed Heme oxygenase and Infrared protein (IFP) is to characterize the reporting ability of IFP. The exact construct used was '''J23119-RBS-IFP1.4-Term-J23119-RBS-HO1-Term'''.
 +
 
 +
 
 +
==== Verification of construct & Signal ====
 +
 
 +
<div class="grid_5 alpha suffix_1">
 +
[[Image:NTUifppcr.jpg|285px]]
 +
 
 +
'''PCR verification of IFP construct'''
 +
</div>
 +
 
 +
<div class="grid_5 omega">
 +
[[Image:NTUifpsds.jpg|285px]]
 +
 
 +
'''SDS PAGE verifying expression of IFP'''
 +
 
 +
</div><br class="clear">
 +
 
 +
 
 +
<div class="grid_7 alpha">[[Image:NTUifpsignal.png|407px]]</div>
 +
 
 +
 
 +
'''Spectrometer reading of IFP'''
 +
 
 +
<span class="grey">The cell culture were irradiated with a 660nm laser diode for excitation. A spectrometer was used to detect the infrared emission at circa 708nm.  Our emission peak (''purple'') closely follows the shape profile of our positive control, IFP1.4 from the Tsien Lab vector. On top of that, our signal is even slightly higher than the Tsien Lab signal (''green''). Future work is recommended on optimising the variables that affect the reproducibility of amplitude.</span>
<br class="clear" />
<br class="clear" />
-
It has been observed that at elevated temperature, i.e. 42 deg C, production of NorR had decreased as shown by the diminished intensity (see 2nd lane). The production of NorR at 30 deg C and 37 deg C carried no significant difference.
+
==== Characterisation of IFP ====
 +
 
 +
 
 +
'''Characterization of IFP with varying inoculation temperature'''
 +
 
 +
[[Image:NTUifpchar1.jpg|651px]]
 +
 
 +
[[Image:NTUifpchar2.jpg|651px]]
 +
 
 +
 
 +
'''Characterization of IFP with varying concentration'''
 +
 
 +
[[Image:NTUifpchar3.jpg|651px]]
 +
 
 +
 
 +
These are the results we have achieved with IFP characterization!
 +
Click here to see the detailed Wetlab regarding [[Team:NTU-Singapore/Prototype/Image#Device_Characterization | Imaging Device Characterization]].
</div></div>
</div></div>
{{Template:NTUfootalt}}
{{Template:NTUfootalt}}

Latest revision as of 03:44, 22 October 2009

Conclusive Results



Our Working Constructs!!


  • J23119-B0034-NorR-B0015, is a system construct, in that it was intended for our system to feature this as a functional part.
  • J23119-B0034-GFP-B0015, is a characterization construct to characterize the modular function of the J23119 promoter in the transcription of NorR.
  • Lac(R0010)-B0034-CHE-B0015, is also a test construct to only test and characterize the enzymatic activity of CHE.
  • J23119-B0034-IFP-B0015-J23119-B0034-HO1(I15008)-B0015, is a test construct to only test the reporting ability of IFP.




NorR System Construct

NorR is an important NO sensor that triggers the transcription at pNO. Thus, we want an NorR producing construct to improve on the sensitivity of pNO to optimise its efficiency at physiological levels.


Verification of construct

Double Digestion


NTUnorrgel2.jpg

Successful construct verification of J23119-B0034-NorR-B0015

This indicates the presence of the construct.

SDS PAGE

SDS page for NorR on 16 Sep 09-edited.jpg


Successful production of NorR



Characterization of NorR

NorR-temp.jpg
SDS PAGE results for NorR expressed at different inoculation temperatures


Since the sole purpose of J23119-B0034-NorR-B0015 () is to produce NorR, we shall dwell into the factors that might affect the production of the protein. The transformed cells were incubated at 3 different environmental temperatures (30 / 37 / 42°C).

SDS PAGE characterization was carried out (top right).


It has been observed that at elevated temperature, i.e. 42°C, production of NorR had decreased as shown by the diminished intensity (see 2nd lane). The production of NorR at 30°C and 37°C carried no significant difference.


Characterization of J23119

Since J23119 () was used as the constitutive promoter for NorR producing construct, it is necessary for us to understand its modular function and how it would affect NorR production.


NTU119temp.png

Characterization of J23119 w.r.t Temp (deg C)


Preliminary characterisation of J23119 showed that the activity of the promoter to be the strongest at 25°C and weakest at 37°C with 30°C being something of an intermediate. Activity of the J23119 was independent of time. Noteworthy was that there was a drastic drop in activity of the J23119 promoter at 16min, 37°C.


NTU119culture.png

Average GFP/OD of J23119-GFP in different medium


It shows that the supplemented M9 medium is the ideal choice for the characterization of the J23119 promoter. M9 is most likely to be the ideal medium for characterisation of other promoters in general.


NTU119backbone.png

Average GFP/OD of J23119-GFP in steady state (M9)


Vector backbone did not seem to have much of an effect on the strengths of J23119, although pSB1A2 has a slight positive effect on the activity of the J23119.


NTU119diffprom.png

Comparison of J23119 with other promoters


Based on our preliminary characterization results, it appeared that J23101 produced a much stronger signal than J23119 and pTet. This was quite surprising because J23119 promoter is supposed to be stronger then the J23101.


NTU119diffchassis.png

Characterization of J23119 in different chassis


J23119 performance appeared to be the best in Origami B.


Click here to see the detailed Wetlab regarding Sensing Device Characterization.


Cholesteryl Esterase Expression & Characterization

To examine the enzymatic activity of cholesteryl esterase, a test construct, pLac (R0010)-B0034-CHE-B0015 () was synthesized.

CHE production was induced by l0mM lactose. The cells were sonicated, and the cell lysate containing CHE was characterized using Amplex® Red Cholesterol Assay Kit. The characterization results is shown below.


Characterization of CHE

NTUcheresult.png

Cholesterol Assay with CHE Construct

From the graph, the cell lysate containing CHE had achieved higher fluorescence readings than the negative control. The increase in fluorescence reading is due to enzyme-coupled reaction that detects free cholesterol concentration. Hence, we have proven that CHE had indeed been produced from this construct and had been working to our expectation.


Click here to see the detailed Wetlab regarding Degradation Device Characterization.



Infrared Signal from Imaging Device

The purpose of creating a constitutively expressed Heme oxygenase and Infrared protein (IFP) is to characterize the reporting ability of IFP. The exact construct used was J23119-RBS-IFP1.4-Term-J23119-RBS-HO1-Term.


Verification of construct & Signal

NTUifppcr.jpg

PCR verification of IFP construct

NTUifpsds.jpg

SDS PAGE verifying expression of IFP



NTUifpsignal.png


Spectrometer reading of IFP

The cell culture were irradiated with a 660nm laser diode for excitation. A spectrometer was used to detect the infrared emission at circa 708nm. Our emission peak (purple) closely follows the shape profile of our positive control, IFP1.4 from the Tsien Lab vector. On top of that, our signal is even slightly higher than the Tsien Lab signal (green). Future work is recommended on optimising the variables that affect the reproducibility of amplitude.



Characterisation of IFP

Characterization of IFP with varying inoculation temperature

NTUifpchar1.jpg

NTUifpchar2.jpg


Characterization of IFP with varying concentration

NTUifpchar3.jpg


These are the results we have achieved with IFP characterization!


Click here to see the detailed Wetlab regarding Imaging Device Characterization.


TOP || iGEM '09

NTU@iGEMcc 2009. Some rights reserved.




Retrieved from "http://2009.igem.org/Team:NTU-Singapore/Project/Wetlab"