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Team:Paris Bettencourt/tRNA diffusion
From 2011.igem.org
The tRNA amber diffusion
The amber codon and the tRNA amber suppressor
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The amber codon is one of the less used stop codon in bacteria. When the ribosome transcripts the RNA into protein, it look for the RBS sequence, fix on it, and try many tRNA, until it find the correct one, and go one amino-acid farther. When the ribosome doesn't find the correct tRNA for the codon it is looging for, the ribosome declare this is a stop, and release the peptine and the mRNA. The idea behind the tRNA amber is to create an artificial tRNA, using an existing tRNA that is loaded with a specific amino-acid, and to change its codon, replacing it by the amber 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. By creating a protein that carries amber mutation in the middle of the coding sequence, 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 and gates. The cells survives the expression of the amber tRNA although it is a really deadly object, because it prevents the cell from expressing properly almost 20% of her endogenic proteins. |
Fig1: Transcription schematics. |
What we call a tRNA amber is a transfer RNA which anticodon has been modified for it to be complementary with a stop codon and especially with an amber stop codon (UAG). Our idea was to use the nanotubes to allow a receiver cell to translate an mRNA containing an amber mutation. The emitter cell will produce this mutated transfer RNA (we mutated a YtRNA for it to recognise the stop codon) and the receiver cell will then be able to translate a protein (we chose the T7 polymerase) which gene contain an amber mutation. This protein will part of a reporter system.
We summed up this principle in the scheme below:
The tRNA amber system preparation
The first step of this preparation was to choose the tranfer RNA and the amino acids to modify in the T7 polymerase gene. We found the article of Grundy and Henkin in 1994 that led us to use the YtRNA. We synthetysed it by an elongation of primers. Now that we know which codon we have to modify within the T7 polymerase sequence. We looked for those codon in the T7 polymerase sequence and realised a quick change mutagenesis, on it to modify two codons. Indeed we decided that only one modified codon would be too leaky, therefore we changed two of them and in the beginning of the sequence for the ribosome to still be tightly attached to the mRNA.
