Team:Exeter/Project

From 2013.igem.org

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==Introduction==
+
== Introduction ==
-
Our project aims to produce a Red, Green and Blue ([http://en.wikipedia.org/wiki/Additive_color RGB]) light control brick for E. coli. To demonstrate the system we will control the production of Cyan, Magenta and Yellow ([http://en.wikipedia.org/wiki/Subtractive_color CMY]) pigments to produce the world's first colour bio-photograph or [http://parts.igem.org/Coliroid coliroid]. Throughout the project extensive modelling and characterisation of the system will quantify its behaviour for future users.
 
-
We will be building upon the work done by previous teams and institutions, most notably UT Austin 2004, Edinburgh 2010 and Uppsala 2011 iGEM teams.
 
-
In order to produce a full colour spectrum our E. coli will be modified to detect the primary colours; red, green and blue and produce the corresponding mix of cyan, magenta and yellow pigments. Ultimately, this system will be affixed to film and utilised in a camera system to take a full colour image. High spatial and medium temporal control over each output should also be possible by the use of multi-wavelength lasers. This has many potential applications from the manufacturing of composite materials to control of engineered organisms in an environment.
+
Our project aims to produce a Red, Green and Blue ([http://en.wikipedia.org/wiki/Additive_color RGB]) light control brick for E. coli. To demonstrate the system we will control the production of Cyan, Magenta and Yellow
 +
([http://en.wikipedia.org/wiki/Subtractive_color CMY]) pigments to produce the world's first colour bio-photograph or
 +
[http://parts.igem.org/Coliroid coliroid].
-
As a sideline, we’re also going to test a new way of preserving and presenting bacterial culture using pouring plastics. Unfortunately, this would kill the bacteria, but should give academics a new way to physically present colonies and cultures they have been working on. For us, it will allow a way of easily transporting our “photographs” without unwieldy plates and gels.
+
We will be building upon the work done by previous iGEM teams, most notably Texas/Austin 2004, Edinburgh 2010 and Uppsala 2011.
-
As mentioned we will be using additive and subtractive colour combinations to allow synthesis of the correct pigment output, corresponding the colours of light the bacteria are exposed to. For example the NOT gated input of red light represses synthesis of cyan pigment enabling an output of yellow and magenta pigment. We hope to analyse the absorption spectrum of each pigment, both alone and in combination. Visualisation of light sensitivity throughout the system will enable calibration of the light input ensuring reliable output. Further to this other teams reported their light sensing systems working better at temperatures less than 37 degrees, which we will also investigate.
+
== The Camera ==
-
<!--  The following paragraph needs diagrams otherwise it's a heavy read-->
+
Bio-Photography is the application of genetically engineered bacteria as the light sensor of a camera, replacing digital sensors ([https://en.wikipedia.org/wiki/Image_sensor#Sensors_used_in_digital_cameras CCD/CMOS]) or photographic film. The surface area of bacteria is on the order of microns, much smaller than a digital sensor. This gives bio-cameras the potential to produce images with far greater resolution.
-
For the blue and red light sensors, the presence of their corresponding wavelength of light causes autophosphorylation of an intermediate protein. Phosphorylated intermediates then freely bind as repressors to the corresponding output gene (cyan for red light, yellow for blue light). With the green light sensor, a signal inverter will be introduced, as the FixJ system acts as an activator not a repressor (the exact opposite of what we want). Instead of acting on the gene coding for the output (magenta) the phosphorylated intermediate binds instead to a gene coding for the cI protein used in the lambda phage (a bacteriophage which infects E. coli). Once synthesised the cl protein acts as the output (magenta) repressor.
+
-
You can follow the progress of our project on our [http://exeterigem.tumblr.com/ blog] and [https://twitter.com/Exeter_iGEM2013 twitter] pages.
+
We aim to produce the world's first colour bio-photograph by producing Cyan, Magenta and Yellow pigments in response to Red, Green and Blue light. We plan to combine this system with a lens to focus the image onto plated bacteria to form the world's first colour bio-camera.
 +
 
 +
=== Colour system ===
 +
 
 +
To produce the full colour spectrum requires all three [http://en.wikipedia.org/wiki/Primary_color primary colours]. This is because any colour can be produced by mixing the primary colours but primary colours cannot be produced by mixing any colours.
 +
 
 +
In our system we are using two sets of primary colours. Red, Green and Blue for light and Cyan, Magenta and Yellow for pigments. This is because light and pigment mix differently. Light is additive and pigment is subtractive:
 +
 
 +
<!-- Diagram needed -->
 +
 
 +
This is because light and pigment produce colour differently. While coloured light has a peak wavelength corrosponding to its colour, coloured pigment absorbs all wavelengths that do not corrospond to its colour and so reflect the corrosponding coloured light:
 +
 
 +
<!-- Diagram needed -->
 +
 
 +
In this system Red is the opposite of cyan, green opposite magenta and blue opposite yellow. Hence our choice of sensors and pigments.
 +
 
 +
It must be noted that while [http://en.wikipedia.org/wiki/RGB_color_model RGB] light give a full spectrum of colour and tone. CMY is a simplified version of the CYMK colour system used in printing. This was done because to engineer the pathways for[http://en.wikipedia.org/wiki/CMYK_color_model CYMK] colour system outputs is too complex. however as CMY is subtractive there will be tone as Cyan + Magenta and Yellow will approximate black.
 +
 
 +
 
 +
 
 +
== The Biology ==
 +
 
 +
The focus of our wet work is the creation of three independant pathways in E. coli. Each will control the transcription of selected genes using Red, Green or Blue light. Incident light will prevent the transcription of the selected gene.
 +
 
 +
In our bio-camera the controlled genes will be those that code for the production of cyan, magenta and yellow pigments:
 +
 
 +
*Red light prevents the production of cyan pigment
 +
*Green light prevents the production of magenta pigment
 +
*Blue light prevents the production of Yellow pigment
 +
 
 +
Each sensor prevents the production of it opposite pigment reproducing the colour of light that the sensor detects. The mix of pigments will reproduce the colour of light incident on the bacteria.
 +
 
 +
=== Red light pathway ===
 +
 
 +
This pathway uses Cph8 red light sensor (composing of Cph1 and Envz) which acts to phosphorylate OmpR to OmpRp in the absence of red light. OmpRp then binds to the OmpF promoter upstream of the HO-pcyA gene which codes for cyan pigment, and stop codon. This
 +
 
 +
is then transcribed and cyan pigment protein produced.
 +
 
 +
=== Green light pathway ===
 +
 
 +
The CcaS green light sensor phosphorylates CcaR to CcaRp in the presence of green light. CcaRp binds to a promoter upstream of a gene that codes for cl repressor protein. The cl protein binds to the promoter upstream of the magenta pigment gene and stop codon. This prevents transcription and stops production of the magenta pigment pigment.
 +
 
 +
=== Blue light pathway ===
 +
 
 +
The YtvA/FixL blue light sensor phosphorylates FixJ to Fixjp in the absence of blue light. FixJp then binds to the promoter for the Yellow pigment gene and stop codon. So in the absence of blue light the Yellow pigment is produced.
 +
 
 +
<!-- Diagrams needed -->
 +
 
 +
== The future ==
 +
 
 +
Our project aims to provide a foundation for the control of organisms using multiple wavelengths of light for future development. Using lasers to control bacteria will provide high spatial control while a well characterised system will provide good temporal control. This technology has many implications for the future.
 +
 
 +
 
 +
<!-- High spatial and medium temporal control over each output should also be possible
 +
 
 +
by the use of multi-wavelength lasers. This has many potential applications from the manufacturing of composite materials to
 +
 
 +
control of engineered organisms in an environment.
 +
 +
As a sideline, we’re also going to test a new way of preserving and presenting bacterial culture using pouring plastics.
 +
 
 +
Unfortunately, this would kill the bacteria, but should give academics a new way to physically present colonies and cultures they
 +
 
 +
have been working on. For us, it will allow a way of easily transporting our “photographs” without unwieldy plates and gels.
 +
 +
As mentioned we will be using additive and subtractive colour combinations to allow synthesis of the correct pigment output,
 +
 
 +
corresponding the colours of light the bacteria are exposed to. For example the NOT gated input of red light represses synthesis
 +
 
 +
of cyan pigment enabling an output of yellow and magenta pigment. We hope to analyse the absorption spectrum of each pigment, both
 +
 
 +
alone and in combination. Visualisation of light sensitivity throughout the system will enable calibration of the light input
 +
 
 +
ensuring reliable output. Further to this other teams reported their light sensing systems working better at temperatures less
 +
 
 +
than 37 degrees, which we will also investigate.
 +
 +
For the blue and red light sensors, the presence of their corresponding wavelength of light causes autophosphorylation of an
 +
 
 +
intermediate protein. Phosphorylated intermediates then freely bind as repressors to the corresponding output gene (cyan for red
 +
 
 +
light, yellow for blue light). With the green light sensor, a signal inverter will be introduced, as the FixJ system acts as an
 +
 
 +
activator not a repressor (the exact opposite of what we want). Instead of acting on the gene coding for the output (magenta) the
 +
 
 +
phosphorylated intermediate binds instead to a gene coding for the cI protein used in the lambda phage (a bacteriophage which
 +
 
 +
infects E. coli). Once synthesised the cl protein acts as the output (magenta) repressor. -->

Revision as of 16:55, 16 July 2013

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Contents

Introduction

Our project aims to produce a Red, Green and Blue ([http://en.wikipedia.org/wiki/Additive_color RGB]) light control brick for E. coli. To demonstrate the system we will control the production of Cyan, Magenta and Yellow ([http://en.wikipedia.org/wiki/Subtractive_color CMY]) pigments to produce the world's first colour bio-photograph or [http://parts.igem.org/Coliroid coliroid].

We will be building upon the work done by previous iGEM teams, most notably Texas/Austin 2004, Edinburgh 2010 and Uppsala 2011.

The Camera

Bio-Photography is the application of genetically engineered bacteria as the light sensor of a camera, replacing digital sensors (CCD/CMOS) or photographic film. The surface area of bacteria is on the order of microns, much smaller than a digital sensor. This gives bio-cameras the potential to produce images with far greater resolution.

We aim to produce the world's first colour bio-photograph by producing Cyan, Magenta and Yellow pigments in response to Red, Green and Blue light. We plan to combine this system with a lens to focus the image onto plated bacteria to form the world's first colour bio-camera.

Colour system

To produce the full colour spectrum requires all three [http://en.wikipedia.org/wiki/Primary_color primary colours]. This is because any colour can be produced by mixing the primary colours but primary colours cannot be produced by mixing any colours.

In our system we are using two sets of primary colours. Red, Green and Blue for light and Cyan, Magenta and Yellow for pigments. This is because light and pigment mix differently. Light is additive and pigment is subtractive:


This is because light and pigment produce colour differently. While coloured light has a peak wavelength corrosponding to its colour, coloured pigment absorbs all wavelengths that do not corrospond to its colour and so reflect the corrosponding coloured light:


In this system Red is the opposite of cyan, green opposite magenta and blue opposite yellow. Hence our choice of sensors and pigments.

It must be noted that while [http://en.wikipedia.org/wiki/RGB_color_model RGB] light give a full spectrum of colour and tone. CMY is a simplified version of the CYMK colour system used in printing. This was done because to engineer the pathways for[http://en.wikipedia.org/wiki/CMYK_color_model CYMK] colour system outputs is too complex. however as CMY is subtractive there will be tone as Cyan + Magenta and Yellow will approximate black.


The Biology

The focus of our wet work is the creation of three independant pathways in E. coli. Each will control the transcription of selected genes using Red, Green or Blue light. Incident light will prevent the transcription of the selected gene.

In our bio-camera the controlled genes will be those that code for the production of cyan, magenta and yellow pigments:

  • Red light prevents the production of cyan pigment
  • Green light prevents the production of magenta pigment
  • Blue light prevents the production of Yellow pigment

Each sensor prevents the production of it opposite pigment reproducing the colour of light that the sensor detects. The mix of pigments will reproduce the colour of light incident on the bacteria.

Red light pathway

This pathway uses Cph8 red light sensor (composing of Cph1 and Envz) which acts to phosphorylate OmpR to OmpRp in the absence of red light. OmpRp then binds to the OmpF promoter upstream of the HO-pcyA gene which codes for cyan pigment, and stop codon. This

is then transcribed and cyan pigment protein produced.

Green light pathway

The CcaS green light sensor phosphorylates CcaR to CcaRp in the presence of green light. CcaRp binds to a promoter upstream of a gene that codes for cl repressor protein. The cl protein binds to the promoter upstream of the magenta pigment gene and stop codon. This prevents transcription and stops production of the magenta pigment pigment.

Blue light pathway

The YtvA/FixL blue light sensor phosphorylates FixJ to Fixjp in the absence of blue light. FixJp then binds to the promoter for the Yellow pigment gene and stop codon. So in the absence of blue light the Yellow pigment is produced.


The future

Our project aims to provide a foundation for the control of organisms using multiple wavelengths of light for future development. Using lasers to control bacteria will provide high spatial control while a well characterised system will provide good temporal control. This technology has many implications for the future.


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