Team:Uppsala-SwedenProject

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= Levande Klocka(Live Clock): =
 
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Our project for [https://2010.igem.org iGEM 2010] involves building a biological concentration band detection sensor and demonstrating its usefulness in building a bio-clock and color display unit.
 
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Many previous iGEM teams have come up with sensors for various applications ranging from toxic metals to life saving enzymes. Our attempt is to create a concentration band detect component which can quantify the signal. This concentration band detection sensor thus serves as a quantitative sensor which can work in combination with any of these chemical sensors. Apart from this utility, we believe the concentration band detect sensor in different configurations can allow the creation of complex circuits ranging from simple oscillators to complex data processing machines which can exist on the same system without affecting other components of the system owing to its concentration band specificity.
 
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Using the concentration band detect sensors in combination with regulated transcription factors we could create a color display unit. Each pixel of this Color Display Cell (CDC) consists of the three colors black, green and black each of which can be controlled individually. These CDC's in combinations can produce complex color pictures and even movies when combined with an efficient color degrading system which could be controlled by electric current.
 
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The bio-clock is an attempt to see how robust the concentration band detection mechanism really is and if it could be used to build more complex devices. Although many different approaches have been made to build oscillators, an oscillator which displays time like a wrist watch or wall clock is a more unique proposition. We are using the concentration band detection sensors in combination to build such an oscillator which actually displays time as it oscillates.
 
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= Commercial value of the project: =
 
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This project has many specific as well as diverse applications which give it a huge commercial value. In its simplest form the concentration band detection sensor can be used in combination with other chemical sensors to quantify toxic chemicals or specific enzymes within the body. The sensor can be combined with drug delivery systems which delivers drugs at a particular time and place.
 
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Other application such as the Color Display Unit will help in further development of the field of bio-electronics. Bio-clock gives yet another approach to building an oscillator which has many applications of its own. Moreover, the concentration band detection sensor can be used in building complex devices that are not affected by noise due to their concentration band specific action.
 
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= Project concept: =
 
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The concentration band detect sensor is based on the concept of the ‘band detect cells’ designed by Basu et al 2005, at Princeton University. These band detect cells use two plasmids (pHD{x} and pLD) in combination to detect the AHL concentration band.
 
== Project Description: ==
== Project Description: ==
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The concentration band detection mechanism makes use of a pair of of promoters with different efficiencies to detect the quantitative value of the input signal.  
The concentration band detection mechanism makes use of a pair of of promoters with different efficiencies to detect the quantitative value of the input signal.  
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[[Image:Schematic_band_detect.png|border|]]
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[[Image:Live clock.png|border|]]
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Figure 1:Schematic showing quorum sensing behaviour and responses which relate to band detect. Adapted from Basu S et.al, 2005.
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[[Image:figure.jpg|600px|thumb|left|The interplay between the sender, the repressor and the receiver.]]
== Band Detect: ==
== Band Detect: ==
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== Previous Work ==
== Previous Work ==
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The below diagram explains how we plan to use two sensitivity tuners in combination to get the band detect behaviour.The sensitivity tuners which detect different levels of the input signal will repress the output at levels below or above the band detect.
The below diagram explains how we plan to use two sensitivity tuners in combination to get the band detect behaviour.The sensitivity tuners which detect different levels of the input signal will repress the output at levels below or above the band detect.
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[[Image:Sensor sketch.jpg|800px|border|]]
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[[Image:Modules1.png|800px|border|]]
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== Proof Of Concept and Characterisation: ==
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== Proof of Concept and Characterization: ==
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Under Construction
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Generally speaking, there are two type of constructs to characterize: the sender system and the receiver system. The sender cells secrete AHL, causing the receiver cells to emit fluorescence in the designated band-detection fashion. In order to obtain a fully functional band detection sensor, it is vital to characterize each component by itself before putting together the entire system.
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The sender system can be characterized either quantitatively, in term of the amount of AHL it produces, or in a qualitative fashion, by checking its effect on a reporter construct. This reporter construct could be as easy as two parts consisting of a regulatory promoter coupled to a GFP-gene. The quantitative measurement involves usage of equipment from the analytical chemistry discipline, e.g. HPLC and mass-spectrometry.
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The receiver constructs can be characterized using commercially available AHL. AHL can be applied to receiver cell cultures with concentration gradient. Their fluorescence intensity can be measured. Using concentration gradient is also crucial to our original band-detection concept, as different receiver constructs should only react to AHL in certain concentration intervals.
== References: ==
== References: ==
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2.Danino T, Mondragón-Palomino O, Tsimring L, Hasty J, A synchronized quorum of genetic clocks, 2010, Nature, Vol 463(7279): 301-2.
2.Danino T, Mondragón-Palomino O, Tsimring L, Hasty J, A synchronized quorum of genetic clocks, 2010, Nature, Vol 463(7279): 301-2.
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3. The iGEM Cambridge team 2009 Biobrick Characterisation
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https://2009.igem.org/Team:Cambridge/Project/Amplification/Characterisation

Latest revision as of 15:33, 27 October 2010


Project Description:

Our project for iGEM 2010 involves building a biological concentration band detection sensor and demonstrating its usefulness in building a bio-clock and color display unit.

The concentration band detection mechanism makes use of a pair of of promoters with different efficiencies to detect the quantitative value of the input signal.

Live clock.png


The interplay between the sender, the repressor and the receiver.

Band Detect:

Previous Work

Being an engineering competition iGEM promotes building on previous projects.The Cambridge 2007 and 2009 teams built upon each other to produce the Sensitivity Tuner using phage activators and promoters.These Sensitivity Tuner's can detect different levels of a input signal and generate a corresponding output. You can find details of how the teams built and characterised their parts in the last years website.

Detecting specific Bands or concentration ranges

The sensitivity tuners do a pretty good job of detecting a signal above certain specific values each. However, they will still get activated when the next sensitivity tuner detects the input signal. For example, the construct [http://partsregistry.org/wiki/index.php/Part:BBa_I746370 BBa_I746370] will start detecting at 0.85 uM while the construct [http://partsregistry.org/wiki/index.php/Part:BBa_I746371 BBa_I746371] will start detecting at 0.92 uM. Both the constructs will detect the input signal at say 0.95 uM. Although this is good enough for detecting chemical concentrations, it will have to be able to detect specific bands to be able to perform concentration band specific actions.

The below diagram explains how we plan to use two sensitivity tuners in combination to get the band detect behaviour.The sensitivity tuners which detect different levels of the input signal will repress the output at levels below or above the band detect.

Modules1.png

Proof of Concept and Characterization:

Generally speaking, there are two type of constructs to characterize: the sender system and the receiver system. The sender cells secrete AHL, causing the receiver cells to emit fluorescence in the designated band-detection fashion. In order to obtain a fully functional band detection sensor, it is vital to characterize each component by itself before putting together the entire system.

The sender system can be characterized either quantitatively, in term of the amount of AHL it produces, or in a qualitative fashion, by checking its effect on a reporter construct. This reporter construct could be as easy as two parts consisting of a regulatory promoter coupled to a GFP-gene. The quantitative measurement involves usage of equipment from the analytical chemistry discipline, e.g. HPLC and mass-spectrometry.

The receiver constructs can be characterized using commercially available AHL. AHL can be applied to receiver cell cultures with concentration gradient. Their fluorescence intensity can be measured. Using concentration gradient is also crucial to our original band-detection concept, as different receiver constructs should only react to AHL in certain concentration intervals.

References:

1.Basu S, Gerchman Y, Collins C.H, Arnold F.H, Weiss R, A synthetic multicellular system for programmed pattern formation, 2005, Nature, Vol 434, 1130-1134.

2.Danino T, Mondragón-Palomino O, Tsimring L, Hasty J, A synchronized quorum of genetic clocks, 2010, Nature, Vol 463(7279): 301-2.

3. The iGEM Cambridge team 2009 Biobrick Characterisation https://2009.igem.org/Team:Cambridge/Project/Amplification/Characterisation

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