The Escherichia coli ribosome is a 2.4 MDa molecular machine that consists of a large subunit and a small subunit, and is the key catalyst in gene expression, responsible for synthesizing proteins from amino acids in a sequence-defined fashion with impressive speed and accuracy. Expanding the repertoire of ribosome substrates and functions would be greatly beneficial for the advancement of systems and synthetic biology. However, as with any biological system, engineering objectives are often completely opposed to the growth and reproduction objectives of the organism. This problem can be solved using a specialized orthogonal ribosome that translates only a specific type of engineered messenger RNAs (mRNAs) and avoids translation of native cellular mRNAs. Before this work, efforts to construct such orthogonal ribosomes focused on modifying the small subunit alone, as orthogonality is endowed by modifying the Shine-Dalgarno sequence of an mRNA and the complementary sequence in the 16S ribosomal RNA (rRNA) of the small subunit. Unfortunately, free exchange between the subunits meant the large subunit, which is responsible for peptide bond formation and protein excretion, could not be extensively engineered. Here we develop an engineered ribosome with tethered subunits (termed Ribo-T), which contains a single core hybrid rRNA composed of small and large subunit rRNA sequences, and is capable of protein synthesis in vitro and in vivo. Considering that the ribosome is one of nature’s most evolved, fine-tuned and conserved structures, it is especially surprising that Ribo-T can even fully support cell growth in E. coli strains lacking wild-type untethered ribosomes. One of the exciting implications of Ribo-T with an orthogonal small subunit (oRibo-T) is the possibility of introducing mutations in large ribosomal subunits that would be deleterious if introduced in an untethered wild-type ribosome, all in living E. coli. We show the ability to evolve oRibo-T by selecting otherwise dominantly lethal rRNA mutations in the large ribosomal subunit that facilitate translation of challenging protein sequences. The Ribo-T and orthogonal Ribo-T system was then improved significantly by tether and orthogonal pair optimization, expanding the utility of this system. Importantly, towards the goal of enabling non-canonical amino acid (ncAA) incorporation, the Ribo-T system was shown to work with established orthogonal translation systems to incorporate the ncAA pAzF into sf-gfp. We then outline work creating engineering strains towards genomic integration of Ribo-T into ribosomal operons, to enable ribosome engineering towards development of novel drugs that kill bacteria resistant to common antibiotics, designer therapeutics, and new classes of sequence-defined polymers with tunable properties such as shape memory and self-healing

Last modified

• 10/09/2018

Creator

• Erik Daniel Carlson

DOI

• https://doi.org/10.21985/N21T84

Subject

• Chemical and Biological Engineering

Keyword

• Synthetic Biology
• Ribosome engineering
• Orthogonal translation

Date created

• 2017-01-01

Resource type

• Dissertation

Rights statement

• In Copyright