"
Team:Paris Bettencourt/tRNA diffusion
From 2011.igem.org
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 suppressor tRNA is to provide a tRNA corresponding to this stop codon. When the ribosome translates the RNA into a protein, it first looks for the RBS sequence and fixes on it. It tries to fit the codon it is located on with the three complementary bases of tRNA flying round. When it finds the correct tRNA with the anti-codon of the start codon, with a methionine loaded on it, the translation starts. Then, codon after codon, the ribosome tries to fit many tRNA on the codon it is placed on, until it find the correct one, fixes the corresponding amino-acid and then moves to the next codon. When the ribosome does not find the correct tRNA for the codon it is located on, the ribosome declares this codon is a stop, and releases 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 matches the amber codon, skip the stop and keep polymerasing the protein. |
Fig1: Transcription schematics animation |
By creating DNA that carries Surprisingly, the cells survive 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.
There was no tRNA amber supressor in the registry for B. Subtilis. Using biofinformatics analysis we found out that the tRNA sequence is quite different than the one of E. coli. We therefore decided to build our new tRNA amber suppressor. The problem was the choice of the amino-acid we wanted to hijack. We found that some of the loading proteins recognize the anti-codon. As we were going to modify it, we needed to choose a tRNA that the anti-codon is not strongly recognized by the loading protein. We found out in a paper by Grundy et al. [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 questions 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 implemented two amber mutations for the T7 polymerase. This should reduce leaking to a minimum. As in the other designs, we wanted an emitter cell producing a message, that can be unambiguously interpreted by the receiver cell.Building a new tRNA amber supressor for B. subtilis
Principles of the design
We summed up this principles in the scheme below:
Fig2: Complete schematic of the system
Explanation step by step:
I. The tRNA is over expressed and matured in the emitter cell. The loading proteins add the amino-acid tyrosine on the tRNA. In the receiver cell, the translation of the T7 RNA polymerase is blocked by the amber codons in the sequence. It cannot be maturated 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 translated.
II. The connection through the nanotube is established. Some tRNA diffuse to the receiver cell, and this is sufficient for the ribosome to skip the two amber codons and polymerasing a few T7 RNA polymerase.
Fig4: When tRNA passes through the nanotube, it allows the receiver cell to translate the T7 polymerase
III. Then, the T7 RNA polymerase is maturated and becomes functionnal. It can trigger the pT7 promoter of the construct.
Fig5: The T7 polymerase triggers the self amplifying reporter switch
IV. The T7 polymerase triggers the self amplifying switch by producing a few normal T7 RNA polymerases, which 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
The reporter system is active. We can look under the microscope. When a red cell (emitter cell) meets a dark cell (receiver cell) and that, a few minutes later, the receiver cell becomes green, we are sure that the message has passed through the nanotubes.
Models and experiments
We have managed to build all these constructs, and to model this system. We kindly invite you to visit the corresponding pages:
