Notebook:Federico Castro M/Projects/Diffusible Signal oscillator

=Details=

Last updated March/09/2008

Project status: Frozen

On Pattern Formation
Information about the mechanisms underlying the process of morphogenesis traditionally has been obtained by the analysis of variation in naturally occurring patterns. A different approach to the problem; the construction of synthetic networks that produce patterns in organisms that previously didn’t have them, might to be an insightful and refreshing alternative..

While synthetic devises may not resemble natural ones and pale in comparison, their successful construction would allow us to establish whether or not the basic elements necessary for the process are complete and well understood and even reveal what would be needed for cells to differentiate.

Accordingly to the actual paradigm on morphogenesis, a population of cells relies on differential activation of genes triggered by one or more signals to differentiate. Signals might be inherent to the environment in which cells develop such as gravity, a maternally generated protein gradient or the cell’s point of attachment to the substrate or might come from neighboring cells as diffusible morphogens.

A problem arises if the media is homogeneous or almost so; signals responsible for the process of morphogenesis would be equally strong in the environment and might not produce differential activation of genes, thereby keeping the cells in a population altogether in the same stage.

Some mechanisms have been proposed, such as the Turing system exposed by Gierer and Meindhart, that could generate patterns from an apparently homogeneous media. It’s very controversial whether or not those patterns could account for patterns observed in natural organisms. To artificially reproduce the rupture of symmetry in organism would be a great feat and would demonstrate the feasibility of the phenomena independently of its natural occurrence.

The Design
Here I propose an artificial genetic network that, if implanted onto Escherichia coli, will confer the host the capability to synchronize by means of the expression of Luxl/R as well as to oscillate between various states and possibly generating complex patterns.

While oscillations are not necessary for the differentiation, the different states that are part of the oscillations are essential and the genetic network comprised could ensure a tight regulation of gene expression that otherwise would be difficult to adjust.

The genetic network is designed in such a way that under the threshold levels of the inducer, the cells are fixed in on stage in which all of them produce lactones that diffuse within the media and the population until the threshold level is reached, thereafter cells move onto the next stage and eventually cells complete the oscillation and return to the first stage.

While the design of the devise allows several bacteria to oscillate in a synchronized way, complex behaviors might be observed if the inducer concentrates in some area allowing some bacteria to reach the threshold levels of activation before others can. This heterogeneous concentration of inducer might arise due to some instabilities in the media and bacteria metabolism, a fast degradation of the inducer a large distance between bacteria colonies or a slow diffusion of the inducer.

Waves, like those formed by a drop of water falling into a pond, could arise in bacteria surrounding a lower concentration of the inducer, allowing bacteria in those areas to change of phase and oscillate while surrounding bacteria will be locked between two or more oscillating colonies by their constant signal that keeps them perpetually activated by the inducer. The complex patterns that could arise could range from intermixing waves to stripes or even small spots.

Whether or not the concentration of the diffusible signal will give rise to complex patterns remains to be tested, discontinuities in the concentration of the inducer will be present for sure yet they might be too small to produce an observable temporal or spatial patterns.

Parallel work
A couple weeks before the Jamboree I discovered a very interesting work made at the IAP 2003, a work from the group of Prakash. They basically designed a synchronized oscillator with many similarities with the one I designed. They also remarked a point that I had not noticed "...The half life of HSL is 24 hrs at pH of 7.5 and it is important that a degradation mechanism that is faster than the desired period of oscillations be introduced into the system. Cyclical degration of HSL would generate better synchronisation signals than a constant degradation mechanism."[] They sugested the use of aiiA, an enzime that degrades the lactones, but it seems that the enzyme does not difuse to the medium "The protein has no hydrophobic signal pepetide at the N-terminus and therefore it is believe that it is not secreted. This is supported by the observation that when aiiA is expressed in E.coli DH5alpha or Bacillus 240B1 cells no autoinducer inactivation is detected in the supernatants of these cultures."[] so it seems that lactones will remain there for large amounts of time even with the use of aiiA.
 * The group of Prakash

Apparently the work at the IAP 2003 was mainly theoretical but later on, someone actually assembled the constructions that the Pakrash group designed and they are available at the iGEM 2007 kit plates.


 * BBa_I4204 (Succesfully recovered)
 * BBa_I4203
 * BBa_I4202
 * BBa_I4201
 * BBa_I4200

I have analyzed some of them and their design seems incoherent to me... perhaps I just don't understand them well. Anyway, I can't wait to recover those constructions and test them.

I was very surprised to find out that another iGEM team was developing a two phase synchronized oscillator. Like us, they were unable to assemble the whole thing so they only had theoretical work, they say that their construction also produces oscillations... I have my doubts.
 * The Group of McGill

They also found the same problem with the degradation of lactones and they also used aiiA.

Further Work
Here I pressent some work that is needed in order to complete the project and that we can do with the materials that we have available at the lab.

Work to do
 * Recover BBa_I4203 (One of Prakash's oscillators)
 * Recover BBa_I4202 (One of Prakash's oscillators)
 * Recover BBa_I4201 (One of Prakash's oscillators)
 * Recover BBa_I4200 (One of Prakash's oscillators)


 * Recover BBa_E0430 (Component of the oscillator, necesary for EYFP expression)
 * Recover BBa_E0430 (Component of the oscillator, Tet inverter)
 * Recover BBa_BBa_T9002 (screening plasmid estandarized part, useful for HSL detection)
 * Recover BBa_S03151 (Component for the repressilator)


 * Test BBa_I4203 for oscillations (One of Prakash's oscillators)
 * Test BBa_I4202 for oscillations (One of Prakash's oscillators)
 * Test BBa_I4201 for oscillations (One of Prakash's oscillators)
 * Test BBa_I4200 for oscillations (One of Prakash's oscillators)


 * Sequence the new part made at the IBT.

Explanation

We shouldn’t build the whole devise until we are sure that aiiA can diffuse trough the media. In order to know whether or not the aiiA is able to diffuse, it would be very useful to observe the behavior of the Prakash’s oscillators. One of them (BBa_I4204) have already been successfully recovered and awaits analysis while the others are still in the iGEM kit plates. If any of the recovered devises are able to oscillate it would mean that aiiA action prevents lactones to accumulate in the media thereby allowing the devise to oscillate. The rate of the oscillations, the intensity of the fluorescence and the periods of fluorescence will be important factor to check for they will reveal information about the production of proteins and would be a kind of PoPS indicator (although a rather inaccurate one) and the data will enable us to finely tune our devise so that it would be able to produce complex patterns it will also be very helpful to know which devise works. There are some variables that we have to exclude in order to look for fluorescence: -We have never seen EYFP, although it should be similar that with GFP or RFP, it would be a good idea to extract a functional devise that would enable us to see fluorescence. I suggest recovering the Repressilator for it will enable us to see EYFP and see the behavior of a real oscillator. If we have SpeI we can make it from BBa_S03151 and BBa_E0430 thereby extracting a useful part that will also is used for the latter construction of the devise. -If the media don’t allow diffusion of lactones the devise will not work, even when this is a small possibility we should culture the bacteria in liquid media and then extract the aliquot and check for fluorescence. -If none of the devises seem to work we have to be sure that this is due to the accumulation of lactones. We could exclude this possibility with a CVO 25 assay like the one that Cambridge used for the presence of lactones in 2006 for their iGEM project but ‘m afraid that we don’t have the LacZ required strain of bacteria needed for that. I suggest recovering BBa_T9002 for it’s a well characterized part that will enable us to detect the presence of lactones in the media via fluorescence and will help us latter on to characterize our parts.

If none of the Prakash’s oscillators work due the accumulation of lactones we would be able to redesign the devise for the use of peptide signals but I guess that for now I would take a couple months to check if the Prakash’s oscillators work.

There are also some parts that we should recover regardless of the viability of Prakash’s oscillators. E0430- A reporter that will be useful in all our devises. Q04400- A Tet inverter, it can easily fit in a lot of devises and will be extremely useful. We should also check that the sequence of the recently assembled part is correct so we should extract the part and sequence it.