Team:Penn State/Multiple Start Codons

Background

mRNA is the molecule that carries information about the sequence of amino acids in a protein. However, much like the lines on a sheet of paper, the protein coding sequence of an mRNA molecule does not start right at the beginning, or top of the page. Instead, once the mRNA is bound by a ribosome, a start codon must first be read before the protein can be translated. This start codon is generally AUG, or Methionine.

The Problem

Once this start codon in read the ribosome will continue reading and building the polypeptide (protein) until a stop codon is reached. But what happens if you have two AUG codons close together? That is the question we are attempting to answer.

We are trying to understand what happens when there are two start codons very close together, but out of frame. Out of frame refers to how the ribosome reads the mRNA. Remember those codons and how they are groups of three bases on the mRNA? The reading frame refers to which group of three. If you start at one base and read the bases in groups of three from that point on, that is one frame of reference. If you then move your start point ahead one base, then you are reading in a new reading frame. If you advance you starting point one more base, that is the third reading frame. If you advance it again you are now back in your first reading frame, but you have skipped the first codon. We are looking into what happens when you have multiple start codons close together, but in different reading frames. Which frame will be preferred?

The Objective

This construct aims to test the possibility of multiple start codons on a single mRNA strand. A plasmid has been engineered with two start codons slightly out of frame from each other, but close enough to test start codon slippage. Testing the fluorescence of E. coli carrying the plasmid will determine the rate of codon slippage