Team:UTK-Knoxville/Project

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Project Overview

Abstract

Project Details

The Experiments

Results

Synthetic biology has become a staple of chemical engineering, used to produce protein products, biofuels and many other important compounds. Bacteria such as Escherichia coli can be easily manipulated to produce a desired product and to do so efficiently. However it is often advantageous to use a eukaryotic host, such as Saccharomyces cerevisiae. While many techniques have been developed for the genetic manipulation of S. cerevisiae, there are still many challenges with this work, including the difficulty of creating multicistronic constructs so that multiple genes can be under the control of a single promoter.

Cap Dependent Translation Initiation

In eukaryotes, translation initiation is typically dependent on a 7 methyl guanisine cap, located at the 5’ end of the mRNA, upstream of an open reading frame. This cap interacts with the poly-adenine tail of the mRNA and with several eukaryotic initiation factors (eIFs) to recruit the ribosome to the mRNA, allowing translation to begin. This mechanism is known as cap-dependent translation initiation. At the end of the open reading frame, secondary structure in the mRNA causes the ribosome to disassociate, meaning that a second open reading frame in the same piece of mRNA will not be translated. As a result, every gene must have its own promoter. This presents many problems to synthetic biologists. Promoters are large and can greatly increase plasmid size. In addition, using the same promoter for several genes in a pathway can result in recombination events that can delete pieces of the plasmid. Moreover, using different promoters for each gene can cause flux balance issues, as every promoter is transcribed differently. Luckily, nature has come up with its own remedy for this problem, known as an internal ribosomal entry site (IRES). IRESs are highly structured regions of mRNA located in the untranslated region between two open reading frames. This secondary structure works with primary structure motifs to recruit eIFs to the mRNA, resulting in the binding of the ribosome. This allows for translation of the second gene in a multicistronic operon and is known as IRES mediated translation initiation.

IRES Mediated Translation Initiation

Although IRESs are an important part of eukaryotic cloning, they are still poorly understood and poorly characterized. The UTK-Knoxville iGEM team is attempting to remedy that problem. We have selected seven IRESs, including two commercial IRESs and five from S. cerevisiae genomic DNA. Based on the methodology put forth by Kelly et al in the Journal of Biological Engineering, we will characterize these IRESs to determine a the relative strengths (Kelly et al., 2001). This will be accomplished using a constitutive promoter, two fluorescent proteins, an IRES, and a terminator built into a single construct as shown below. By comparing the fluorescence produced under the control of each IRES, we can create a relative strength profile.

Internal ribosomal entry sites (IRES) are an important but poorly understood part of the eukaryotic translational machinery, allowing cap-independent translation initiation. Unfortunately, the Registry of Standard Biological Parts contains few IRESs and even those are poorly annotated. In this work, a group of IRESs were characterized for relative strength and submitted to the registry. To do this, we developed a methodology of determining relative IRES strength, as modeled by the methodology put forth by Kelly et al in the Journal of Biological Engineering (Kelly et al., 2001). Using flow cytometry, we determined the relative levels of fluorescent protein under the control of each IRES, as expressed in S. cerevisiae. Finally, we will show a practical example of how IRESs can be used in chemical engineering and synthetic biology. This work will not only be useful for other projects within our sponsor lab, but will also serve the greater synthetic biology community by initiating the growth of a library of IRESs and a protocol to allow contribution by all members of the community.