Pattern definition – taken a different viewpoint in terms of a interspecies pattern seen within an ecosystem as this principles can be used to recreate patterns within closed conditions and neighbouring colonies.Previous papers used QS elements – Danino – blue pulsation and chemostat Weiss and Arnold – sender and receiver – ring pattern formationReporter gene is constitutively expressed in each cell within the colony – more cell, the more fluorescent
A very early example of data collected from information of skins caught by trappers in Canada.Clearly shows peaks and troughs in population density, a peak in prey is followed by a peak in predatorNot entirely a closed system due to other external influences but a strong representation because only one main predator involved which influences population of the prey. (Although humans could also be included).Over a long period of time but can be recreated within E.coli over much shorter period. (might be 20 mins doubling time in optimal conditions and certain strains have increased ability and virulence).
Used both the Lax and Lux systems and therefore two variants of lactone AHL (Arrows)Red symbol represents the destructive killer geneGreen symbol represents the antidote gene
Reporter genes give a visual representation to the fluctuations in population
Main summary – Two QS systems – one in left and one in right orientation, Las = AHLC12 Lux = AHLC6 Three separate colonies that will not come into contact with each other Each colony gives a distinct coloured output Killer gene constitutively in predator and QS linked expression in prey Whole system inducible by Dox/aTC (tetracycline variants)
Smaller population increase would be required to reach sufficient thresholds for co related expression in the opposite population, but because the speed of the cycle is limited by cell growth, amplification would allow the oscillations to occur at a higher frequency, although would not reach same ranges of population increase and decrease.Right hand orientation – LasI expression leads to AHL variation 30C12HSL being synthesized in the predator.Left Hand orientation – involves the LuxI/LuxR QS system that is initially signalled in the Prey, amplified in the intermediate and received by the Predator.
The strain of ecoli within the intermediate could be different to the predator and prey to help amplificationReplication, doubling and growth time in predator and prey should remain the same i.e. same strain and plasmid used
Constitutive promoter such as lambda because very strong promoter that would allow expression of the protein from each cellAccumulation of fluorescence as population increases gives brighter outputPrevent residual protein still remaining from the previous cycle.Ideally want the coloured output to be removed from the cells that are still included within the population from last time roundCells that are destroyed by killer gene may still show fluorescence so requires degradation
Microchemostat – Balagadde is well known for his work in microchemostat development Has described there use to maintain a constant environment in controlled studies Prevent biofilm formation Miniaturization and chip based systems have been developed to reduce the need for large quantities of growth media and reagents Long term culture and monitoring of very small ecoli populationsOptical micrograph showing six microchemostats that operate in parallel on a single chip. Various inputs have been loaded with food dyes to visualize channels and sub-elements of the microchemostats. The coin is 18 mm in diameter. (B) Optical micrograph showing a single microchemostat and its main components. Scale bar, 2 mm.
This diagram shows the flow of both nutrient and intercellular signalling by AHL with the device. Both are separate from each otherWould require something similar to the Danino paper that has a continual supply of nutrientsAllows cells to continue to grow to a maximum level. Population density over maximum that can be contained in trapping chamber would means cells are lost as transferred to waste.Maintainsteady state conditions and allows the continued growth of the population whilst containing each colony within its own trapping chamber. The larger arrows indicate an amplification of the signal at the intermediate colony.
Addition of inducing chemical should only have to be added in the left orientation of the systemCould be added to direct flow of nutrients in the fluidity device.Increasing sensitivity would mean the fine tuning and slight changes could be made to give optimal performance of system.
Oscillatory cycle should continue over a period of daysThe same pattern should be maintained throughout
Graph shows the peaks and troughs of population densityInitially the population density of the three colonies should follow similar growth patternSystem is induced by addition of Dox/aTcA period of exponential growth will continue for the preyTo begin with the predator density will decrease as Dox induced signalling of destructive gene can now take placeEventually population density of the prey allows for critical threshold of AHL signalling of the antidote gene in the predatorThe predator population begins to increase until AHL level reaches threshold for expression of killer gene in preyIntermediate population will increase, and continue to grow, although population density is limited by space of trapping chamber but flourscent production is maintain because the cells are still growing/replication etc.
Also possible use in HIV, might contain and prevent the onset of full blown AIDS
A brief summary of the parts available in the registry of standard biological partsExtremely useful in constructing this systemFurther info on there functionality and usability as well as actual examples of where they have been appliedComplete sequence and available for orderOther advice on species, strains and vector use.
Circuit Design Presentation- Predator Prey
Predator – prey Ecosystem<br />Pattern Forming Circuit Design<br /> By Ben Mackrow<br />
Introduction<br />What defines a pattern?<br />Recreation of a oscillatory pattern seen within a larger interspecies ecosystem<br />This will be represented by the creation of individual colonies that represent predator and prey species.<br />Population density of one colony will directly influence the population of the other via intercellular communication.<br />Build upon the designs previously discussed in lectures, which focused on QS elements and transcriptional regulation.<br />Using a fluorescence reporter gene as direct representation of population density within the colony.<br />Functional applications of such a system?<br />
Predator Prey Oscillatory Patterns<br />The most well known example of predator prey interaction can be seen from the Snowshoe Hare and Canadian Lynx associations (MacLulich 1957)<br />
Balagadde paper (Balagadde 2008)<br />First representation of the predator prey interactions seen within an ecosystem using a synthetic circuit design. <br />Relies on bi directional communication using quorum sensing elements between two different populations of E.coli.<br />The density of both populations has an affect on the other colonies survival, due to intercellular signalling , which can lead to the expression of a destructive gene in the other colony<br />The simple diagram below shows the interaction between both populations:<br />
The basic principles<br /><ul><li>QS signal from the predator leads to the expression of a killer gene in the prey.
Killer gene is continually expressed within the predator itself, and requires the QS signal from the prey to express the antidote gene.
Therefore an increase in predator population will cause a decrease in prey population
This means that there will not be sufficient signalling back to the predator for expression of the antidote gene.
Eventually the predator population will decrease once again and the prey population will recover.
From these events an oscillatory cycle is created</li></li></ul><li>Controllable and predictable fluctuation in population density of both colonies produces a patterned outcome, which could be easily visualized by linking expression of the genes with fluorescent reporter genes.<br />Requires use of amicrochemostatsystem, ensures steady state conditions. (This would also be important in the design of my own system which I will explain later.)<br />There are two important differences between this system and natural predator prey interactions within an ecosystem.<br />The prey provides an antidote gene to the programmed cell death of the predator instead of acting as an actual food source.<br />The competition of space and nutrients that would be seen when culturing both populations would not be a normal interaction between predator and prey. Both would likely have different food sources within a natural environment.<br />The paper concludes by stating that additional control over population mixing or segregation would make it possible to program bacterial populations to mimic development and differentiation seen in multicellular organisms.<br />
My Design<br />A summary of the changes that were made<br /><ul><li>Addition of an intermediate colony that would be used to amplify both QS signals so that the oscillatory effects would be seen over a shorter period.
Expression of a fluorescence reporter gene within each population, that had been highly engineered to give a strong fluorescent colour. This would be used to give a clear indication of population density and create a visual pattern.
Each reporter gene will be tagged for degradation so that the output is only transient and prevent residual protein remaining during the next oscillatory cycle.
Use of strong promoters that are constitutively expressed within system in order to express genes required in both left and right orientated QS networks.
Intermediate Colony<br /><ul><li>Critical threshold of AHL amplification can be reached far more quickly
Simple positive feedback system that is enclosed from other external influences
Relies on equal amplification in both orientations and the ability of LuxR and LasR to induced gene expression downstream of the pLuxI promoter (Gray et al 1994).
Positive feedback loop within the intermediate in which active complex formation causes further signalling
The population density of the intermediate colony should not fluctuate because it is not influenced by the destructive gene and should have sufficient nutrients and optimal space within the steady state condition of the microchemostat.
Able to constitutively express a modified yellow fluorescence protein.</li></li></ul><li><ul><li>The intermediate colony could theoretically be developed using a positive feedback loop using the elements of the QS system
My research has shown that a positive feedback gene circuit which uses a LuxR variant and the creation of the LuxR/LuxI fusion protein can result in the amplification and significant increase in levels of membrane protein (Bansal 2010).
Alternative construct – use a plasmid vector with a high copy number, allowing for increased expression of LuxR and LasR in the intermediate
Therefore, very sensitive response to AHL signalling would be possible as well increasing the amount of AHL synthesised and exported out of the cell.
Plasmid vectors with higher copy numbers, suitable for use within E.Coli are readily available from the registry of standard biological parts.
Again this would rely on equal amplification of both QS systems, otherwise there will be bias in one orientation and prevent predictable oscillatory pattern that I am trying to create.</li></li></ul><li>Figure 1<br />Predator<br />Intermediate<br />Prey<br />λpl<br />GFP<br />YFP<br />LasI<br />LuxR<br />LasR<br />RFP<br />AHL (c12)<br />LasI<br />ccdB<br />pluxI<br />LuxR<br />LuxI<br />Dox / aTC<br />ccdA<br />AHL (c6)<br />Tet on<br />
Fluorescence Proteins<br /><ul><li>Each colony labelled with its own individual fluorescent protein, under the control of a constitutive promoter.
Changing intensity will be a direct reflection of the increase or decrease in population density of the colony.
The protein should each contain a degradation tag to remove residual protein come the next round of oscillation
Many fluorescent proteins are available as parts within the registry and have been modified to give the brightest possible colour as well as being labelled with degradation tags such as LVA tag.
This would be key to visualizing the pattern outcome of my system.</li></li></ul><li>Microchemostat (Balagadde 2005)<br />
Inducible system<br />Tet On promoter into my system replaces the Lac inducible promoter.<br />System could be induced by the introduction of Dox/aTc into the nutrient supply of the microchemostat.<br />Variations in Tet promoters have been developed that are particularly sensitive to Dox, called Tet On advanced (10 times more sensitive) and Tet On 3G (100 times more sensitive) (Lead 2007).<br />This could allow very sensitive and finely tuned induction of the system which is relevant when we consider that for the system to oscillate correctly the promoters inducible by Dox must remain active throughout<br />Level of Dox within the system will related to cycle duration and the degradation time of Dox itself.<br />
Firstly the populations are allowed to increase because the destructive gene is not expressed in any of the colonies.<br />The population densities increase to a sufficient level which meet the critical threshold of AHL levels. <br />The system is induced by Dox /aTc which leads to the activation of the QS system in the prey (LuxI) and the expression of the destructive gene in the predator (ccdB). The population density of the prey is able to increase and give brighter fluorescence whereas the population density is much lower in the predator due to activity of the destructive gene. <br />After a period of time the signalling from the prey will lead to production of the antidote in the predator and this process should be made quicker by the amplification of the intermediate. <br />Visualization of pattern<br />
5. Eventually the population density of the predator has increased enough so that it too can express high level of fluorescent protein.<br />6. The increased population density of the predator then causes a critical threshold of AHL signalling to be reached, which will again be expressed in the intermediate before it causes the expression of the destructive killer gene in the prey. <br />7. As prey levels decrease then so will the population density of the predator as insufficient expression of the antidote gene will occur in order to sustain such a large population.<br />**The intermediate should remain at a constant population throughout and hence its fluorescence should be continual and constitutively expressed.<br />
Patterned Outcome<br /><ul><li>Relies on sustainable and regular oscillatory cycle
Continuing change in population density should mean that the fluorescent output changes in intensity throughout.
The patterned output will be seen as three varying intensities of colour seen within the different colonies that should show similar peaks and troughs in each cycle.
Essence of the pattern is that by increasing the intensity in one area, the intensity will decrease in another.
This level of cellular communication and the ability to respond to threshold levels is commonly seen within development.
It is the ‘pattern’ of the morphogen distribution that makes it possible for the formation of the ‘French Flag model’ (Wolpert 1969).
Morphogenconcentration gradient determines that one gene will be activated from a very low concentration and another might only be active at a higher concentration,
Limb development - determines length and direction of growth.</li></li></ul><li>Dox/aTc introduced into system<br />Population Density<br />Time<br />
Functional Applications<br />The ability of one cell to cause a negative effect on another cell, such as that seen by the predator acting on the prey can draw similarities with digit formation<br />This involves degradation and destruction of cells via Apoptosis to destroy cells between the webbing of each finger and toe.<br />Example - Biosensor that could target the production of a tumour destructive chemical within the host, when the population of tumour-like cells reaches levels we would consider to be dangerous.<br />Preservation of the chemical and reduce work load of the synthetic system as well as both external and internal control of the system.<br />Targeted destruction - when the tumour cell population begins to decrease the destructive chemical would be produced in lower amounts and this would also limit the chances of non-tumour cell destruction by miss-targeting. <br />
“the creation of this design would require extensive modelling, preliminary testing, alterations in plasmid construction and changes to external inducers”<br />I have opted to expand on a previous publication and draw upon the knowledge I have gained so far from lectures and primary sources in circuit design and construction. <br />Clearly it is just one example of pattern formation, something which I have looked at on an intercellular and interspecies level.<br />Summary<br />
References<br />Balagaddé, F.K et al, 2005. Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat, Science: 309 (5731), 137-140. <br />Balagaddé, F.K. et al., 2008. A synthetic Escherichia coli predator-prey ecosystem. Molecular systems biology, 4(187), p.187<br />Bansal, K. et al., 2010. A positive feedback-based gene circuit to increase the production of a membrane protein. Journal of biological engineering, 4, p.6.<br />Basu, S. et al., 2005. A synthetic multicellular system for programmed pattern formation. Nature, 434(7037), pp.1130-4.<br />Danino, T. et al., 2010. A synchronized quorum of genetic clocks. Nature, 463(7279), pp.326-30. <br />Gray, K.M. et al., 1994. Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of bacteriology, 176(10), pp.3076-80. <br />Lead, F.E. & Models, T.M., 2007. The Tet System : Powerful , Inducible Gene Expression. Nature Genetics, (October), pp.1-2.<br />MacLulich D.A 1957. The Place of Change in Population Processes, The Journal of Wildlife Management, Vol. 21, No. 3 pp. 293-299 <br />Turing, A.M., Aug, N. & Turing, B.Y.A.M., 2007. The Chemical Basis of Morphogenesis Society, 237(641), pp.37-72.<br />