<p> The genetic code is highly conserved across all domains of life, enabling horizontal gene transfer (HGT) between organisms and across ecosystems via horizontally-transferred genetic elements such as viruses and plasmids. While HGT increases genetic diversity, it poses a risk to engineered biological systems by introducing new genes that destabilize engineered functions or allowing the expression of engineered genes in wild organisms with unknown effects. A model organism engineered with an alternative genetic code may provide new insight into the origins of the genetic code while also providing a stable chassis for engineered biological systems.</p><p> The Isaacs Lab recently developed an <i>Escherichia coli</i> strain lacking both UAG stop codons and Release Factor 1, resulting in the first genomically recoded organism (GRO) with an unassigned codon in its genetic code. Here, we demonstrate that this alternative genetic code lacking UAG codon assignment confers resistance to multiple viruses (λ, M13, PI, MS2) at titers up to 10<sup>11</sup> PFU/mL and impairs conjugative plasmid function (F and RK2) up to 10<sup>5</sup>-fold. Propagating viruses on a mixed microbial community containing standard and alternative genetic codes also reduced viral population fitness and prompted viral adaptation to the alternative genetic code. In investigating the molecular mechanism underlying the resistance to viruses and conjugative plasmids, we found that UAG-ending genes elicit ribosomal stalling and the tmRNAmediated ribosomal rescue response, resulting in degradation of UAG-ending proteins and suggesting that genomic recoding may be a broadly applicable strategy to impair horizontal gene transfer into other organisms.</p><p> To prevent the expression of engineered genes in wild organisms, we reassigned the UAG codon in the GRO to a sense codon incorporating the non-standard amino acid <i>4</i>-acetylphenylalanine (pAcF) through the introduction of an orthogonal translation system (OTS). We then created a library of UAG-containing variants and assessed escape of UAG-containing genes from the GRO into wild-type organisms for both a non-selective green fluorescent protein (GFP) and selective chloramphenicol acetyltransferase (CAT) gene. While 1 UAG codon impaired the expression of GFP in wild-type organisms, at least 2 UAG codons were required in CAT to consistently prevent escaped expression in wild-type organisms with a standard genetic code. Additionally, sequencing revealed that wild-type organisms enabled expression of CAT by mutating UAG codons to UGG coding for tryptophan or CAG coding for glutamine. By placing UAG at sites in proteins that cannot tolerate a tryptophan or glutamine substitution, we can create UAG-containing genes further isolated from expression in wild organisms.</p><p> As biotechnology increasingly targets open-environment applications such as bioremediation or disease treatment in humans, we require methods to stabilize and control the genetic information that we encode in engineered biological systems. Because alternative genetic codes can both confer resistance to horizontal gene transfer into an engineered system and restrict expression of engineered genes in wild-type organisms, genomic recoding of organisms to contain alternative genetic codes is a promising path towards increasing the stability and safety of engineered biological systems. However, open-environment applications will expose engineered biological systems to new stresses not represented in the laboratory environment, and further work is required to validate these methods will be robust in conditions of limiting nutrients or other cellular stresses. Additionally, while we have demonstrated genetic isolation of the GRO with respect to genes both entering and leaving the cell, we cannot currently have both properties simultaneously because UAG is the sole open codon. We envision that current research into further codon reassignments, including the reassignment of sense codons, will pave the way for alternate genetic codes with multiple codon reassignments. By expanding recoding efforts to multiple species, we envision the development of synthetic microbial communities with alternate genetic codes that are genetically isolated and robust to perturbation by HGT.</p>
Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:10584955 |
Date | 27 July 2017 |
Creators | Ma, Natalie Jing |
Publisher | Yale University |
Source Sets | ProQuest.com |
Language | English |
Detected Language | English |
Type | thesis |
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