Team:Wageningen UR/Project/CompleteProject1Description

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Building a Synchronized Oscillatory System

Synchroscillator

Complete Project Description

1. Introduction

The aim of this project is to design and implement a system exhibiting sustained oscillatory protein expression which is synchronized across a population of spatially constrained E. coli cells. The principles that govern this type of behaviour have been studied both in theory and in practice, and as such there exists a solid foundation to apply these ideas in the context of the iGEM competition. In essence, this project consists of constructing a plasmid which contains genes encoding proteins which reciprocally affect each other’s expression in a reliable manner, and experimentally measuring the expression dynamics to test the predictive value of a mathematical model. Due to the specificity of the required system parameters, and resulting difficulty in experimentally verifying the phenomena we wished to observe, special considerations regarding the experimental set-up had to be made. We hope that this system might be employed as a pace-making device to drive more complex genetic circuits requiring time-dependent gene expression, or as a component in sophisticated metabolic engineering applications.

Fig.1. Artistic rendering of the Synchronized Oscillatory System.

2. Mechanism

There are a number of genetic circuit topologies that have the potential to exhibit oscillatory behaviour under the right conditions. However, the requirement that the oscillations be synchronized posed a constraint on the components that could be used. The starting point for our genetic circuitry was a design recently published in the article “A synchronized quorum of genetic clocks” by Danino et al. This design combines elements of the Vibrio fischeri quorum sensing system with a quorum quenching enzyme from Bacillus subtilis, resulting in coupled positive and negative feedback loops which regulate the expression of a reporter protein.

Fig.2. Genetic circuit of a synchronized oscillator (Danino et al. 2010)

Basic Components:

LuxR is a transcriptional regulator in the bioluminescent quorum-sensing system of the symbiotic deep sea bacterium Vibrio fischeri. It is induced by binding the autoinducer molecule N-(3-oxohexanoyl)-homoserine lactone (AHL). The AHL-LuxR complex controls expression of the lux regulon, which contains diverging pRight and pLeft promoter elements. The pRight element has low basal transcription, and is activated by AHL-LuxR; pLeft has higher basal expression, and is repressed by the AHL-LuxR complex. This dual activity makes LuxR a useful element for controlling interconnected genetic feedback loops. The unrestricted diffusion of AHL through the plasma membrane allows spatially proximate populations of cells to experience identical AHL conditions and synchronize AHL-dependent gene expression.

The enzyme LuxI is an acyl-homoserine-lactone synthase which produces the intercellular signalling molecule N-(3-oxohexanoyl)-homoserine lactone. Placing LuxI under control of the pRight promoter results in a positive feedback loop: when increases in cell density cause the intracellular AHL concentration to rise above the activation threshold of the pRight promoter, the transcription rate of the LuxI gene is increased which in turn results in the production of more AHL.

AiiA is an enzyme from B. subtilis which degrades AHL. Its biological function is to interfere with the quorum sensing signals of other bacteria. Placing it under control of the pRight promoter results in negative feedback as a response to increasing AHL concentrations. The reporter molecule Green Fluorescent Protein (GFP)is also regulated by the pRight promoter and provides a quantitative (albeit delayed) indication of the AHL concentration the cell is exposed to at a given point in time. LuxI, AiiA and GFP are all tagged for rapid degradation. Due to differences in the synthesis and degradation rates of LuxI and AiiA, there exists a space of conditions within which periodic oscillations in AHL concentration, and concomitant oscillatory protein expression can emerge. Under most conditions, the level of AHL within a population of cells will quickly reach a steady state. However, by simulating the system using a quantitative biochemical model, it is possible to predict conditions under which oscillations are likely to occur. See Modeling page for details [hyperlink]

Designs

“Hasty” system:

The first design we implemented was a BioBrick part based reconstruction of the plasmids used by Danino & Hasty. We intended to make as accurate a replica as possible in order to confirm the previously published results, and to test the viability of our experimental platform [hyperlink to Device Design]. However, there are a few differences in between the original Hasty system and our replica. While the Hasty system employs the natural lux promoter which contains divergent pLeft and pRight elements (Fig 4), the BioBrick parts we employed have both elements in the same orientation. Both the original and our system contain 3 copies of the luxR gene under control of the pLeft element. Furthermore, a different (high copy) backbone was used during the functional validation of the parts, as opposed to the low copy backbone employed by Hasty.

Fig.4. Synchronized Oscillator plasmids using natural quorum sensing system (Danino et al. 2010)

{

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@@ Line 30: / Line 30: @@
 ====1. Introduction====
-The aim of this project is to design and implement a system exhibiting sustained oscillatory protein expression which should be visible and synchronized on the scale of a physically constrained population of ''E. coli'' cells. The principles that govern this type of behaviour have been studied both in theory and in practice, and as such there exists a solid foundation to apply these ideas in the context of the iGEM competition. In essence, this project consists of constructing a plasmid containing genes, the products of which reciprocally affect each other’s expression in a reliable manner. Based on a previously established design, we intend to take advantage of the great variety of standardized, interchangeable and freely available Bio–Bricks to construct modified genetic circuits, aiming at an improved bacterial oscillator. Due to the difficulty in experimentally verifying the phenomena we wish to observe, special considerations regarding the experimental set-up had to be made.
+The aim of this project is to design and implement a system exhibiting sustained oscillatory protein expression which is synchronized across a population of spatially constrained E. coli cells. The principles that govern this type of behaviour have been studied both in theory and in practice, and as such there exists a solid foundation to apply these ideas in the context of the iGEM competition. In essence, this project consists of constructing a plasmid which contains genes encoding proteins which reciprocally affect each other’s expression in a reliable manner, and experimentally measuring the expression dynamics to test the predictive value of a mathematical model. Due to the specificity of the required system parameters, and resulting difficulty in experimentally verifying the phenomena we wished to observe, special considerations regarding the experimental set-up had to be made. We hope that this system might be employed as a pace-making device to drive more complex genetic circuits requiring time-dependent gene expression, or as a component in sophisticated metabolic engineering applications.
-The starting point for the genetic circuitry we intend to make is a design recently published in the article [http://www.nature.com/nature/journal/v463/n7279/abs/nature08753.html “A synchronized quorum of genetic clocks”] by Danino et al. This genetic circuit uses natural elements of bacterial quorum sensing systems to form coupled positive and negative feedback loops which control the expression of a reporter protein: the LuxI enzyme that catalyses the last step of the acyl-homoserine lactone (AHL) biosynthesis, the AHL-responsive transcriptional regulator LuxR, and the reporter Green Fluorescent Protein (GFP) (Figure 1). The AHL molecules can easily diffuse through cell membranes to the extracellular medium. This allows all the cells in a culture to influence each other’s activity in a uniform manner (quorum sensing). The result being that the oscillations arising from the genetic feedback loops are synchronized on the scale of a whole cell culture. All of the parts used in this design exist in the Registry of Standard Biological Parts and are freely available for our use.
 [[File:SOS_2.png]]
-'''Fig.1.''' ''What synchronized oscillation of Green Fluorescent Protein in E. coli cells might look like.''
+'''Fig.1.''' ''Artistic rendering of the Synchronized Oscillatory System.''
 ====2. Mechanism====
-There are a number of genetic circuit topologies that have the potential to exhibit oscillatory behaviour under certain conditions. One simple design is the Smolen Oscillator which consists of two genes connected through double feedback loops.  The mechanism we intend to construct consists of components of the lux quorum sensing system that are configured in such a way.
+There are a number of genetic circuit topologies that have the potential to exhibit oscillatory behaviour under the right conditions. However, the requirement that the oscillations be synchronized posed a constraint on the components that could be used. The starting point for our genetic circuitry was a design recently published in the article “A synchronized quorum of genetic clocks” by Danino et al. This design combines elements of the Vibrio fischeri quorum sensing system with a quorum quenching enzyme from Bacillus subtilis, resulting in coupled positive and negative feedback loops which regulate the expression of a reporter protein.
 [[File:mainproject01.png]]
-'''Fig.2.''' ''Basic oscillating genetic circuit as published by Danino & Hasty.''
+'''Fig.2.''' ''Genetic circuit of a synchronized oscillator (Danino et al. 2010)''
+'''Basic Components:'''
+'''LuxR''' is a transcriptional regulator in the bioluminescent quorum-sensing system of the symbiotic deep sea bacterium ''Vibrio fischeri''. It is induced by binding the autoinducer molecule N-(3-oxohexanoyl)-homoserine lactone (AHL). The AHL-LuxR complex controls expression of the lux regulon, which contains diverging pRight and pLeft promoter elements. The pRight element has low basal transcription, and is activated by AHL-LuxR; pLeft has higher basal expression, and is repressed by the AHL-LuxR complex. This dual activity makes LuxR a useful element for controlling interconnected genetic feedback loops. The unrestricted diffusion of AHL through the plasma membrane allows spatially proximate populations of cells to experience identical AHL conditions and synchronize AHL-dependent gene expression.
+The enzyme '''LuxI''' is an acyl-homoserine-lactone synthase which produces the intercellular signalling molecule N-(3-oxohexanoyl)-homoserine lactone. Placing LuxI under control of the pRight promoter results in a positive feedback loop: when increases in cell density cause the intracellular AHL concentration to rise above the activation threshold of the pRight promoter, the transcription rate of the LuxI gene is increased which in turn results in the production of more AHL.
+'''AiiA''' is an enzyme from B. subtilis which degrades AHL. Its biological function is to interfere with the quorum sensing signals of other bacteria. Placing it under control of the pRight promoter results in negative feedback as a response to increasing AHL concentrations.
+The reporter molecule Green Fluorescent Protein ('''GFP''')is also regulated by the pRight promoter and provides a quantitative (albeit delayed) indication of the AHL concentration the cell is exposed to at a given point in time.
+LuxI, AiiA and GFP are all tagged for rapid degradation. Due to differences in the synthesis and degradation rates of LuxI and AiiA, there exists a space of conditions within which periodic oscillations in AHL concentration, and concomitant  oscillatory protein expression can emerge. Under most conditions, the level of AHL within a population of cells will quickly reach a steady state. However, by simulating the system using a quantitative biochemical model, it is possible to predict conditions under which oscillations are likely to occur. See Modeling page for details [hyperlink]
+=====Designs=====
+'''“Hasty” system:'''
+The first design we implemented was a BioBrick part based reconstruction of the plasmids used by Danino & Hasty. We intended to make as accurate a replica as possible in order to confirm the previously published results, and to test the viability of our experimental platform [hyperlink to Device Design]. However, there are a few differences in between the original Hasty system and our replica. While the Hasty system employs the natural lux promoter which contains divergent pLeft and pRight elements (Fig 4), the BioBrick parts we employed have both elements in the same orientation. Both the original and our system contain 3 copies of the luxR gene under control of the pLeft element. Furthermore, a different (high copy) backbone was used during the functional validation of the parts, as opposed to the low copy backbone employed by Hasty.
+[[File:Mainproject02.png]]
+'''Fig.4.''' ''Synchronized Oscillator plasmids using natural quorum sensing system (Danino et al. 2010)''
+{|
+|'''A'''||:||AiiA
+|-
+|'''I'''||:||LuxI
+|-
+|'''Hi'''||:||internal AHL
+|-
+|'''He'''||:||external AHL
+|}
-The main elements that comprise this circuit are the genes LuxI, LuxR, AiiA, GFP, and the promoters that regulate their activity. LuxI encodes the enzyme LuxI which enzymatically produces the molecule acyl-homoserine lactone (AHL). When AHL is bound to the transcription factor LuxR, it becomes active and induces transcription from the LuxI promoter (which normally has very low basal expression). Subsequently, AiiA is expressed and negatively regulates the pRight promoter by catalysing the degradation of AHL. The feedback loop emerging from this configuration results in periodic oscillatory protein expression. AHL is soluble and can freely diffuse through the cell membrane into extracellular medium and into other cells, effectively normalizing the AHL concentration across an entire localized cell culture, which is how synchronization can occur. Placing the gene for a reporter molecule, such as Green Fluorescent Protein or Luciferase, under a copy the LuxI promoter allows the dynamics of protein expression to be monitored.
-====3. The designs====
-In the original design, Danino & Hasty placed the genes LuxI (from ''V. fischeri''), AiiA (from ''B. Thurigensis'') and yemGFP genes under the control of three identical copies of the natural Lux promoters of ''V. fischerii''. This promoter region actually consists of two promoters regulating transcription in opposite directions. To avoid complications resulting from this, the authors placed a copy of the LuxR gene behind each of the “pLeft” parts of the promoter. Though this in itself should cause (unwanted) oscillations of LuxR levels, the fact that three copies are expressed, and pLeft has intermediate basal expression, results in de facto constitutive expression, and is treated as such in the modelling work.
-[[File:mainproject02.png]]
-'''Fig.3.''' ''Synchronized Oscillator plasmid designs by Danino & Hasty. LuxRp is the “left” promoter (repressed by AHL-LuxR). LuxIp is the “right” promoter, which is activated by AHL-LuxR. Note that all of the proteins (except for LuxR) have C-terminal degradation tags, as this increases the frequency of the oscillation and reduces noise.''
-=====Replica of the construct designed by Danino & Hasty.=====
-The first construct we intend to make is an accurate replica of this original design using BioBrick parts from the Registry of Standard Biological Parts.  Given that it has been proven to work, it will be able to serve as a basic starting point for our attempt to observe oscillatory protein expression in ''E. coli''. However, we will also construct modified versions using existing parts which will be more streamlined, and of which, in some cases, might even show enhanced performance.
-[[File:mainproject03.png]]
-'''Fig.4.''' ''Circuit view of the Danino & Hasty design using BioBrick Parts.''