Team:Queens Canada/Guide/mRNA

When constructing fusion proteins, it is necessary to make sure that stop codons only appear at the end of the DNA sequence so that translation of the resulting mRNA does not stop prematurely. If it is known that the first coding sequence terminates in a stop codon, it must be removed, such as designing primers to PCR amplify the sequence without the stop codon. after which the two coding sequences can be combined either by ligation or overlap extension PCR. Once the parts are assembled together, new stop codons may appear if the reading frame is shifted. It is useful to run the entire composite gene sequence through a software such as Synbiota’s GENtle, to check for stop codons.

The current Biobrick assembly standard RFC[10] does not support the construction of fusion proteins because ligating two parts together results in a 8-bp scar that shifts the reading frame of the second protein. Thus, another assembly standard must be chosen when building chimeric proteins, such as the BB-2 standard RFC[12], which allows for the in-frame fusion of parts with its 6-bp ligation scar.

The frequencies of codons present in an organism’s genome vary widely. A rare codon is defined as a codon that is used infrequently in a genome, and consequently is decoded by a rare tRNA that is present in low abundance [1]. This causes inefficient expression of proteins that contain these rare codons in their gene sequence. In E. coli, the rare codons are arginine (AGG, AGA, CGA), leucine (CTA), isoleucine (ATA), and proline (CCC). The Silver lab biofusion standard RFC[23] contains the rare codon AGA in the ligation scar, which is why we opted to use Tom Knight’s BB-2 standard.

[1] Chen, D. and Texada, D. E. (2006) Low usage codons and rare codons of Escherichia coli. Gene Ther Mol Biol Vol 10(1): 1-12.