Team:NTU-Singapore/Parts

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==Introduction==
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='''Characterization Introduction'''=
The group proposed to discuss and investigate the working range of the parts and devices of our system
The group proposed to discuss and investigate the working range of the parts and devices of our system
-
*The proposed variables to investigate are: <br> i) Concentration of activator <br> ii) Time <br> iii) Temperature
+
*The proposed variables to investigate are: <br>  
 +
 
 +
i) Concentration of activator<br>  
 +
 
 +
ii) Temperature <br>
 +
 +
iii) Time <br>
==Characterization of Parts==
==Characterization of Parts==
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We are interested in characterizing the parts  
+
We are interested in characterizing the following parts:
i) Lactose/IPTG sensitive Promoter pLac [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 BBa_R0010]
i) Lactose/IPTG sensitive Promoter pLac [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 BBa_R0010]
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ii) Iron Sensitive Promoter pFe [http://partsregistry.org/wiki/index.php?title=Part:BBa_I716014 BBa_I716014]
ii) Iron Sensitive Promoter pFe [http://partsregistry.org/wiki/index.php?title=Part:BBa_I716014 BBa_I716014]
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iii)  [https://2008.igem.org/Team:NTU-Singapore/Wetlab/Experimental_Results#Characterization_of_plsrA-YFP plsrA-YFP]<br>
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iii)  AI2 senstive Promoter pLsrA [http://partsregistry.org/wiki/index.php?title=Part:BBa_K117002 BBa_K117002] <br>
The characterization is carried out by ligating the desired promoter with the Green Fluorescence protein: [http://partsregistry.org/Part:BBa_E0240 BBa_E02040]
The characterization is carried out by ligating the desired promoter with the Green Fluorescence protein: [http://partsregistry.org/Part:BBa_E0240 BBa_E02040]
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*For the pLac BBa_R0010, it is already available in the form we desired in the registry as [http://partsregistry.org/wiki/index.php?title=Part:BBa_J04430 BBa_J04430]
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*For the pLac BBa_R0010, it is already available in the form that we desired in the registry as [http://partsregistry.org/wiki/index.php?title=Part:BBa_J04430 BBa_J04430]
 +
*For the pLsrA promoter, the Yellow Fluorescence protein was used instead to differentiate between our two different systems.
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==The FLx800™ Fluorescence Microplate Reader==
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-
[[image: Ntu@iGEM_FLx_800_pic.jpg]]
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=='''Experiments'''==
 +
==='''1) Characterization of existing LacI regulated promoter (pLacI) via GFP Activity'''===
 +
[[image: Ntu@iGEM_FLx_800_pic.jpg|400px|center|thumb|The FLx800™ Fluorescence Microplate Reader]]
The FLx800™ Fluorescence Microplate Reader was available for our use courtesy of Division of Bio-engineering lab. It was purchased from BioTek and for more information about the reader, you can refer to [http://www.biotek.com/products/product_detail.php?pid=117 The FLx800™ Fluorescence Microplate Reader]
The FLx800™ Fluorescence Microplate Reader was available for our use courtesy of Division of Bio-engineering lab. It was purchased from BioTek and for more information about the reader, you can refer to [http://www.biotek.com/products/product_detail.php?pid=117 The FLx800™ Fluorescence Microplate Reader]
-
The FLx800™ Fluorescence Microplate Reader has a computer software KCJunior that was installed in a computer adjacent to the reader for collection and processing of data from our samples.
+
The FLx800™ Fluorescence Microplate Reader has a computer software KCJunior that was installed in a computer adjacent to the reader for collection and processing of data from our samples. It was used for the characterisation of pLacI GFP.
 +
 
 +
 
 +
The objectives of the characterization experiment are to investigate the effects of concentration of inducer, temperature and time on the production of Green Fluorescence protein (GFP) by the Biobrick promoter part being experimented.
 +
A stronger fluorescence, as measured using FLx800™ Fluorescence Microplate Reader will indicate more GFP proteins being synthesised. This also directly corresponds to a higher promoter activity.
 +
 
 +
Top 10 competent cells are transformed with plasmid carrying the following gene sequence:
 +
[[Image:NTU@iGEM_PLacI_GFP_pic.JPG|center|thumb|400px|pLacI promoter with GFP reporting gene]]
 +
The cells are cultured and upon addition of the inducer, the reaction mixtures (with both cells and inducer) are fed into the Fluorescence Microplate Reader. This machine has a inbuilt software, KCJunior, which will translate the results into excellent data suitable for analysis. The samples’ fluorescence was measured for a period of 12 hours, at the temperatures of <br>
 +
i) 25°C <br>
 +
ii) 37°C <br>
 +
and iii) 42°C.<br>
 +
 
 +
The readings were than plotted to produce 3-dimentional graphs for detail analysis.
 +
 
 +
For more information please refer to [https://2008.igem.org/Team:NTU-Singapore/Parts/Characterization_of_LacI-GFP Characterization of pLacI-GFP]
 +
 
 +
==='''2) Characterization of our New promoter pLsrA-YFP'''===
 +
In order to simulate the AND gate detection system, characterization of pLsrA using the Yellow fluorescence protein (YFP) was done. The YFP production is analogous to the Lysis protein production in our AND gate detection system. When the pLsrA promoter is activated by the AI-2 molecules, transcription of the downstream gene, followed by the translation, results in YFP formed. The YFP production is than to be measured using the VICTOR 3 multilabel reader.
 +
 
 +
[[Image:NTU@iGEM_Victor3-plates-web4web.jpg|center|thumb|400px| VICTOR 3 multilabel reader]]<br>
 +
[[Image: NTU@iGEM_PLsrA_YFP_pic.JPG|center|thumb|400px| pLsrA promoter with YFP reporting gene]]<br>
 +
<br>
 +
 
 +
 
 +
For more information please refer to [https://2008.igem.org/Team:NTU-Singapore/Parts/Characterization_of_pLsrA-YFP Characterization of pLsrA-YFP]
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==The Standardistation==
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In the first edition of the newsletter, it elaborated a method similar to the approach our characterization procedure. We have decided to make use of the protocol given to us, however with some minor modifications.
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===Transformation of parts===
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document.clock.document.close();
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The parts transformed are:
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}
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#LacI-GFP [http://partsregistry.org/wiki/index.php?title=Part:BBa_I763004 BBa_I763004],
+
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#Standard promoter with GFP reporter [http://partsregistry.org/Part:BBa_I20260 BBa_I20260]
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#GFP reporter devices with Weak promoter[http://partsregistry.org/Part:BBa_J23150 BBa_J23150],
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#Medium promoter [http://partsregistry.org/Part:BBa_J23151 BBa_J23151] and
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#Strong promoter [http://partsregistry.org/Part:BBa_J23102 BBa_J23102]
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===Discussion===
+
-
With the parts transformed, we can proceed with the characterization based on the protocol we constructed. We make use of the FLx800™ Fluorescence Microplate Reader to take the readings of the fluorescence that is emitted by the GFP reporting devices by measuring the relative fluorescence units (RFU). The different strength of the promoters associated with the same GFP reporting device, E0240 will determine the rate of transcription of the GFP protein. The stronger the promoter, the higher is the rate of its transcription. This results in more GFP proteins formed which in turns relates to more fluorescence being emitted. This direct proportionality brings us closer to quantifying the strength of any promoter of interest.  Hence a standard promoter was used to compare the RFU with other promoters. Based on the newsletter, there are also the Low, Medium and High promoters.
+
-
===Results===
+
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We transformed all these parts and take their readings over 12 hours at 10 minutes intervals. The graphs below illustrates the results we obtained and the detailed data points and graphs plotting can be retrieved from the [[image: NTU@iGEM_Results_for_characterization_010708.xls]].
+
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[[image: NTU_Characterization_Graph_Standards_(12_hours).JPG |thumb|center|600px|Characterization Graph of parts over 12 hours]]
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[[image: NTU_Characterization_Graph_Standards_(3_hours).JPG|thumb|center|600px|Characterization Graph of parts after 3 hours]]
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[[image: NTU_Characterization_Graph_Standards_(3.5_hours).JPG|thumb|center|600px|Characterization Graph of parts after 3.5 hours]]
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Latest revision as of 10:15, 27 October 2008

Contents

Characterization Introduction

The group proposed to discuss and investigate the working range of the parts and devices of our system

  • The proposed variables to investigate are:

i) Concentration of activator

ii) Temperature

iii) Time

Characterization of Parts

We are interested in characterizing the following parts:

i) Lactose/IPTG sensitive Promoter pLac BBa_R0010

ii) Iron Sensitive Promoter pFe BBa_I716014

iii) AI2 senstive Promoter pLsrA BBa_K117002
The characterization is carried out by ligating the desired promoter with the Green Fluorescence protein: BBa_E02040

  • For the pLac BBa_R0010, it is already available in the form that we desired in the registry as BBa_J04430
  • For the pLsrA promoter, the Yellow Fluorescence protein was used instead to differentiate between our two different systems.


Experiments

1) Characterization of existing LacI regulated promoter (pLacI) via GFP Activity

The FLx800™ Fluorescence Microplate Reader

The FLx800™ Fluorescence Microplate Reader was available for our use courtesy of Division of Bio-engineering lab. It was purchased from BioTek and for more information about the reader, you can refer to The FLx800™ Fluorescence Microplate Reader

The FLx800™ Fluorescence Microplate Reader has a computer software KCJunior that was installed in a computer adjacent to the reader for collection and processing of data from our samples. It was used for the characterisation of pLacI GFP.


The objectives of the characterization experiment are to investigate the effects of concentration of inducer, temperature and time on the production of Green Fluorescence protein (GFP) by the Biobrick promoter part being experimented. A stronger fluorescence, as measured using FLx800™ Fluorescence Microplate Reader will indicate more GFP proteins being synthesised. This also directly corresponds to a higher promoter activity.

Top 10 competent cells are transformed with plasmid carrying the following gene sequence:

pLacI promoter with GFP reporting gene

The cells are cultured and upon addition of the inducer, the reaction mixtures (with both cells and inducer) are fed into the Fluorescence Microplate Reader. This machine has a inbuilt software, KCJunior, which will translate the results into excellent data suitable for analysis. The samples’ fluorescence was measured for a period of 12 hours, at the temperatures of
i) 25°C
ii) 37°C
and iii) 42°C.

The readings were than plotted to produce 3-dimentional graphs for detail analysis.

For more information please refer to Characterization of pLacI-GFP

2) Characterization of our New promoter pLsrA-YFP

In order to simulate the AND gate detection system, characterization of pLsrA using the Yellow fluorescence protein (YFP) was done. The YFP production is analogous to the Lysis protein production in our AND gate detection system. When the pLsrA promoter is activated by the AI-2 molecules, transcription of the downstream gene, followed by the translation, results in YFP formed. The YFP production is than to be measured using the VICTOR 3 multilabel reader.

VICTOR 3 multilabel reader

pLsrA promoter with YFP reporting gene



For more information please refer to Characterization of pLsrA-YFP

Retrieved from "http://2008.igem.org/Team:NTU-Singapore/Parts"