Effect of Codon Optimization on Bacterial Translation Elongation Rates
Open Access
- Author:
- Swackhamer, Clay Dale
- Area of Honors:
- Agricultural and Biological Engineering
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Howard M Salis, Thesis Supervisor
Ali Demirci, Thesis Honors Advisor - Keywords:
- Clay Swackhamer Salis Synthetic Biology Biological Engineering C
- Abstract:
- Microorganisms are used in a variety of industries to produce bioproducts, including fuels, food products, specialty chemicals, and even lucrative biologic therapeutics. These products are created through manipulation of the natural ability of microorganisms to use genetic information to produce proteins. This is known as the central dogma of biology, and is mediated by the steps of DNA being transcribed to mRNA and then translated into protein. It is the task of engineers to use an understanding of these component biophysical processes to control protein synthesis through genetic level controls, and to create new systems for protein production using the principles of rational design. Using engineering knowledge to raise protein expression is a goal in numerous industries, as higher protein expression is a driver of overall profit. There are several genetic level points of control currently used by engineers to raise protein expression, one of which is codon optimization of the coding sequences used by microorganisms to produce proteins. Since there are 20 amino acids but 64 codons, there are instances were multiple codons are translated into the same amino acid. These are called degenerate codons. However, degenerate codons may not have the same translational efficiency. Traditional codon optimization methods in E. coli rely on the preference for certain codons across the entire genome, yet this is not the only possible approach. The principal challenge is that at high translation initiation rates, protein expression may plateau, and it is hypothesized that novel criteria for codon optimization of genes can be used to raise expression plateaus that occur when translation elongation becomes the rate limiting step in protein synthesis, and also allow for fine tuning of expression due to predicted differences in translational efficiency between degenerate codons. In this research, novel criteria for codon optimization were employed to design and create synthetic variants of a reporter gene that was then characterized in vivo using an expression construct. Fluorescence levels of cells expressing these constructs were measured and results suggest that protein expression plateaus may still be experienced, even by the sequences optimized for high efficiency. However, the new criteria for codon optimization, for example the statistical correlation between a degenerate codon and its presence in highly translated parts of the genome, are feasible for use in future projects. This may enable future researchers to optimize genes at the codon level with greater fidelity.