Notebook:Federico Castro M/Projects/Diffusible Signal oscillator

Details

Last updated February/02/2008

Project status: Active

The Design

I intended to create an artificial oscillator that would be able to synchronize large population ofbacteria.

I've designed four different constructions:

• 1 The first one was presented as part of the project of the UNAM-IPN iGEM team it was Simulated by Luis de Jesus Martínez, that construction now seems outdated having many design flaws, still it was very robust and I liked it for that.

• 2 The second one seems to be able to work but there is something on it that I dislike although I can't say exactly what... perhaps I dislike it because it seems bulky. Surely it needs refinement.

• 3 The third is a modification of the devise that McGill presented last iGEM. It is the simplest devise that is able to oscillate but it seems it's very dependent on the rates of the components and is very unstable.

• 4 This one is my actual favorite. It's very simple, I still have to simulate it but I bet this is a robust one.

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!