Revision as of 11:57, 20 September 2011

Team IGEM Paris 2011

The tRNA amber diffusion

The amber codon and the tRNA amber suppressor

The amber codon is one of the less used stop codon in bacteria. The principle of the artificial amber suppresor tRNA is to provide a tRNA for the stop codon. We explain how it works in the following paragraphs

When the ribosome transcripts the RNA into protein, it look for the RBS sequence, fix on it. Then, it tryes to fit with the codon it is located on with the tree bases complement of the tRNA flying round. When it finds the tRNA with the anti-codon of the start codon, with a methionine loaded on it, the translation starts. Then, codon after codon, the ribosome try to fit many tRNA on the codon it is placed on, until it find the correct one, fix the corresponding amino-acid and then and then moves one codon farther. When the ribosome doesn't find the correct tRNA for the codon it is located on, the ribosome declare this codon is a stop, and release the peptide and the mRNA.

The idea behind the tRNA amber supressor is to create an artificial tRNA, based on an existing tRNA that is loaded with a specific amino-acid, and to change its anti-codon, replacing it by the amber anti-codon. By expressing this artificial tRNA in the cell, the ribosome can find a tRNA that match with the amber codon, skip the stop and keep polymerasing the protein.

Fig1: Transcription schematics animation
(modified from this animation)

By creating a protein that carries amber mutation in the middle of its coding sequence, in the place of the amino-acid that loaded on the tRNA amber supressor, we create a protein that can be properly transcribed only if the artificial tRNA amber suppressor is present in the cell. This approach is sometime used in synthetic biology to create artificial AND gates.

Surprisingly, the cells survives the expression of the amber tRNA although it is a really lethal object, because it prevents the cell from expressing properly almost 20% of her endogenic proteins.

Building a new tRNA amber supressor for B. subtilis

There were no tRNA amber supressor in the registry for B. Subtilis. Using biofinformatics analysis we found out that the tRNA sequence is quite different from E. coli to B. subtilis. So we decided to build our new one. The problem was the choice of the amino-acid we wanted to hijack. We found that some of le loading proteins recognize the anti-codon. As we are going to modify it, we need to choose an tRNA in which the anti-codon is not strongly recognized by the loading protein.

We found out in this paper[1] that some people managed to create a tyrosine amber tRNA in B. Subtilis (BBa_K606034), so we decided to work on this amino-acid. Many question around the maturation of the mRNA into the tRNA remained unsolved so we decide to build it with and without a translation terminator.

We also had to build two kind of amber mutated proteins. A T7 amber (K606032) and a GFP amber (BBa_K606043) to characterize the tRNA. As we wanted a very clean system, we made two mutation amber in the raw for the T7 polymerase, because we wanted non leaky system, and the stop codon could be skipped by base-pair woobeling with a tyrosine.

Principle of the design

As in the other designs, we want an emittor cell to produce a message, that can be unambiguously interpreted by the receiver cell.

The emitter cell will produce this mutated taRNA and make it diffuse through the nanotubes.

The receiver cell will then be able to translate a protein (the T7 polymerase) which gene contain an amber mutation. This protein will trigger of a reporter system.

We summed up this principle in the scheme below:

Fig2: Complete schematic of the system

Here is the explanation step by step:

I. The tRNA is over expressed and matured in the emittor cell. The loading proteins add the amino-acid tyrosine on the tRNA. In the receiver cell, the transcription of the T7 RNA polymerase is blocked by the amber codons that are in the sequence. It cannot be matureated and so the reporter system is silent.

Fig3: tRNA amber is produced in the emittor cell. In the absence of the tRNA in the receiver cell, the T7 polymerase cannot be transcribed.

II. The connection with the nanotube is established. Some 200 tRNA diffuse to the receiver cell, and this is sufficient to for the ribosome to skip the two amber codons and polymerasing a few T7 RNA polymerase.

Fig4: When the tRNA pass through the nanotube, it helps the receiver cell to translate the T7 polymerase

III. Then, the T7 RNA polymerase is maturated, and become functionnal. It is looking for the pT7 promoter of the construct.

Fig5: The T7 polymerase trigger the self amplifying reporter switch

IV. The T7 polymerase triggers the self amplifying switch by producing a few normal T7 polymerases, that will keep self producing. In the mean time, because it is on the same mRNA, a proportionnal quantity of GFP will be produced.

Fig6: The positive feed back loop helps the signal to be amplified and be very strong fast

The reporter system is then active. We can look under the microscope. When a red cell (aka emitor cell) meet a dark cell (aka receiver cell) and that, a few minutes latter, the receiver cell becomes green, we are sure that the message have passed through the nanotubes.

Models and experiments

We have managed to build all these constructs, and to modelize this system. We kindly invites you to visit the corresponding pages:

Modeling

Experiments

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Team:Paris Bettencourt/tRNA diffusion

From 2011.igem.org