Notebook:Federico Castro M/Projects/Diffusible Signal oscillator

Details

Last updated February/02/2008

Project status: Active

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.

Cl lam	luxl		________ luxr_________luxPL	________ tetR_________tetR	_______ Cl lam	_______ aiiA	EYFP
<bbpart>R0051</bbpart>	<bbpart>F1610</bbpart>	<bbpart>J23119</bbpart>	<bbpart>I1762001</bbpart>	<bbpart>Q04400</bbpart>	<bbpart>S0103</bbpart>	<bbpart>C0260</bbpart>	<bbpart>E0430</bbpart>

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

• The group of Prakash

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."[[1]] 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."[[2]] so it seems that lactones will remain there for large amounts of time even with the use of aiiA.

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.

• <bbpart>BBa_I4204</bbpart> (Being transformed)
• <bbpart>BBa_I4203</bbpart>
• <bbpart>BBa_I4202</bbpart>
• <bbpart>BBa_I4201</bbpart>
• <bbpart>BBa_I4200</bbpart>

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.

• The Group of McGill

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.

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

Further Work

The half life of lactones still bothers me, with aiiA, I could inhibit their effect but I'm afraid that lactones will remain in the medium for a long time. One solution could be the use of peptide signals, last year the Cambridge team constructed and sent <bbpart>BBa_I746200</bbpart> I hope that it will be avaliable for the 2008 kit plates. I don't know what is the half life of those peptides, I'll have to study them. The other solution would be to translate the whole construction to an eukaryotic organism and add an excretion signal to aiiA... I have never worked with any eukaryotic organism, but other organisms are harder to work with than bacteria.

For now I will just develop my skills at the lab, I will need aproval for a procedure to transform the constructions made by Pakrash and see how they work. Also I would like to assemble the Represilator just to practice my assembling skills but for now we still don't have SpeI, so I guess I will have to wait, that bothers me a lot.

Note: We should build a lux protein generator with R0063. We could score some points by making that part and it would be extremely useful. A procedure for recovering the biobricks is posted here and awaits for approval.

First Mecican part!