Effects of Synonymous, Consecutive, Repetitive Codons on Bacterial Translation Elongation Rates

Open Access
Author:
Heasley, Victoria Lynn
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Howard M Salis, Thesis Supervisor
  • William O Hancock, Honors Advisor
  • Peter J Butler, Faculty Reader
Keywords:
  • repetitive codon sequences elongation
  • affects on translation elongation rates
  • synthetic biology
  • genetic engineering
Abstract:
Microorganisms can be engineered to produce a wide range of chemical products including therapeutic drugs, consumer plastics, and biofuels. These advancements are made possible because there is a direct relationship between the genetic information of an organism and the composition of its functional protein machinery. The relationship is described by the central dogma, which states genes are first transcribed from DNA to RNA, and the RNA in turn is translated into proteins. By manipulating the DNA of an organism we can alter its protein machinery to modify its metabolic pathways so as to make products of our interest. In order for protein translation to occur, a ribosome reads the triplet codons on the messenger RNA and synthesizes a polypeptide adding a specific amino acid for each codon. There are 64 possible codons, of which 61 code for 20 amino acids. Consequently, some amino acids are coded for by a degenerate set of codons. Previous research suggests that there are differences in efficiency of amino acid incorporation for different codons within a degenerate set, as well as between amino acids. Here we design experiments to quantify these differences. We designed and constructed an expression construct in order to quantitatively estimate translation elongation rates when introducing synonymous rare codons into the protein’s coding sequence. We hypothesize that adding adjacent, repetitive, rare codons will affect the ribosome’s elongation rate in a codon-dependent manner, which can be measured by using our expression system. We designed a genetic construct containing an mCherry fusion protein with an N-terminus leader peptide sequence followed by our varying codon repeats. Fluorescence levels of cells containing these constructs, measured at steady-state conditions, will provide a quantitative estimate of the elongation rate of the protein. Comparing the elongation rates for our different variants will help establish the relative speeds of codons within and between degenerative sets. Overall, the project will measure how protein elongation rate is affected by introducing synonymous, codon repeat sequences, for the amino acids: threonine, alanine, and serine.