Team:NTU-Singapore/Parts

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<div id="body" style="margin-top:200px;width:950px;padding:5px;">
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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>  
*The proposed variables to investigate are: <br>  
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i) Concentration of activator<br>  
i) Concentration of activator<br>  
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ii) Time <br>  
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ii) Temperature <br>
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iii) Temperature
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iii) Time <br>
==Characterization of Parts==
==Characterization of Parts==
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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 AI2 senstive Promoter pLsrA]<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]
*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 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.
*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.
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=Protocols=
 
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==Characterization of standard Biobrick via GFP Activity==
 
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.  
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 NTU FLx800™ Fluorescence Microplate Reader will indicate more GFP proteins being synthesised. This also corresponds to higher promoter activity.
+
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.
-
Top10 competent cells are transformed with plasmid carrying the following gene sequence:
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Top 10 competent cells are transformed with plasmid carrying the following gene sequence:
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[[Image:PLacI-GFP.JPG|center]]
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[[Image:NTU@iGEM_PLacI_GFP_pic.JPG|center|thumb|400px|pLacI promoter with GFP reporting gene]]
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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 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>
+
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>  
i) 25°C <br>  
ii) 37°C <br>
ii) 37°C <br>
and iii) 42°C.<br>
and iii) 42°C.<br>
-
The readings were than plotted to produce a 3-dimentional graph for detail analysis.
+
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]
For more information please refer to [https://2008.igem.org/Team:NTU-Singapore/Parts/Characterization_of_LacI-GFP Characterization of pLacI-GFP]
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==Characterization of pLsrA - YFP==
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==='''2) Characterization of our New promoter pLsrA-YFP'''===
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1) The successfully ligated plasmid with pLsrA-YFP gene was first transformed into chemically competent LuxS(-) cells. <br><br>
+
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.
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2) The next day, one colony of cell with pLsrA-YFP plasmid was inoculated in 5ml LBA for 16 hours at 37oC and shaked at 225 rpm.<br><br>
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3) Overnight cell culture was then centrifuged at 4000 rpm and 4oC for 10 minutes. <br><br>
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[[Image:NTU@iGEM_Victor3-plates-web4web.jpg|center|thumb|400px| VICTOR 3 multilabel reader]]<br>
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4) The supernatant was discarded and cell pellets were re-suspended in 5ml of Ampicilin-containing M9 medium. The amount of M9 medium was adjusted until cell suspension had an OD600 of 1. <br><br>
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[[Image: NTU@iGEM_PLsrA_YFP_pic.JPG|center|thumb|400px| pLsrA promoter with YFP reporting gene]]<br>
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5) The cell suspensions were then pipetted into 96-well microplate wells. This was followed by the adding of 50µl AI-2-containing supernatant into corresponding wells. <br><br>
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6) There were 6 different supernatant solutions used, which correspond to the time points when they were obtained: 2, 3, 4, 5, 6, 8 hours. <br><br>
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7) There were 3 different negative control samples being used for this study.<br>
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  * The First control sample contained cell suspension only. <br>
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  * The Second control sample was cell suspension with 50µl pure water added.<br>
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  * And as the supernatants also contain large amount of LB, cell suspension with 50µl <br>
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    LB added was also used as another control sample. <br>
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<br>
<br>
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8) YFP measurement was carried out by VICTOR 3 multilabel reader at excitation wavelength of 490 nm and emission wavelength of 535 nm. <br><br>
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9) Data were automatically gathered every 10 minutes and temperature was set at 37oC.
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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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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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 BBa_R0010]

ii) Iron Sensitive Promoter pFe [http://partsregistry.org/wiki/index.php?title=Part:BBa_I716014 BBa_I716014]

iii) AI2 senstive Promoter pLsrA [http://partsregistry.org/wiki/index.php?title=Part:BBa_K117002 BBa_K117002]
The characterization is carried out by ligating the desired promoter with the Green Fluorescence protein: [http://partsregistry.org/Part:BBa_E0240 BBa_E02040]

  • 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.


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 [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. 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"