Developing a tunable, adaptive plasmid selection system to screen Aminoacyl-tRNA synthetase variants

Abstract

The genetic code refers to a set of rules used by living organisms to translate information encoded within the genetic material (DNA/mRNA sequences) into proteins. All organisms generally encode 22 proteinogenic or canonical amino acids using the ribosomal machinery. By expanding the genetic code, it is possible to incorporate an unnatural amino acid (UAA) into proteins using the cellular translational apparatus. The incorporation of non-canonical amino acids having various functional properties into proteins has garnered significant interest in recent years. Over 200 non-canonical amino acids have been site-specifically installed into recombinant proteins, thus enabling the expansion of genetic code. Evolution of Aminoacyl tRNA-synthetases typically involves building a library with randomized residues in the amino acid binding pocket of these enzymes and subjecting them to rounds of positive and negative selection. The long-term idea here is to develop and use an ‘adaptable’ selection system for a large library of aminoacyl tRNA/synthetase pair mutants and thus get orthogonal variants specific to a particular unnatural amino acid, and aminoacylate that suppressor tRNA. Traditionally, the selection technique used contained a two-step procedure and was used to evolve the residues in the binding pocket of tyrosyl tRNA synthetase from Methanococcus jannaschii to charge a variety of amino acid substrates (O-methyl tyrosine) other than phenol side chain from tyrosine.

In the initial part of this thesis, we describe the usage of the bacteriophage λ Red recombineering system encoded in pKD46 helper plasmid to knockout the upp gene sequence from DH10B strain. The knockout strain DH10B∆upp was generated as a prerequisite for conducting the UPRT negative selection with 5-Fluorouracil on E. coli, since its essential that cellular toxicity in response to 5-FU is due to the expression of upp from the selection vector and not due to the expression of the genomic copy. Before using the UPRT negative selection system with 5-FU on E. coli, it is essential that the strain being used in the selection is itself devoid of the upp gene, in order to ensure that whatever cellular toxicity (in response to 5-FU) arises is due to the external conditions only. (From the plasmid transformed into the cells) In this case the parent strains were DH10B and KL16 and from these DH10BΔupp strain was produced (discussed in detail in this study). This study aims to describe the knockout procedure adopted in order to produce these strains. Here in E. coli, the reporter genes are CAT and UPRT- both selectable markers being expressed and regulated constitutively under T7 promoter being carried by the vector. The bacteriophage λ Red recombineering system was used (encoded in pKD46 helper plasmid) to knockout the upp gene sequence from DH10B and KL-16 strains. The upp sequence was replaced with an antibiotic cassette flanked by FRT sites in the first step, after amplifying the latter via PCR. Subsequently, with the help of another helper plasmid pCP20 that induces Flippase enzyme action, was introduced to target the FRT sites and knock out the KanR cassette, leaving a FRT scar behind. The knockout strain DH10B Δupp was thus obtained.

The second part of this report is based on the development of the selection system and optimization of the conditions for carrying the selection using CAT and UPRT as the selectable markers. Through solid media LB-Agar spot growth assays and O.D.600 monitoring experiments, we validated both positive and negative selection steps. Using this validated selection procedure, we successfully demonstrated this technique on two engineered Methanococcus jannaschii aminoacyl-3-iodo-tyrosyl tRNA-synthetase (Mj3IYRS) and aminoacyl-4-borono-phenylalanyl tRNA-synthetase (pBoFRS). We also made an improvement over the previous fused cat-uprt selection construct used to carry out enrichment and selection of PylRS variants in other reports, by designing a degradation experiment for selectively chewing away the selection plasmid, thus doing away with the conventional time-taking and laborious sequential co-transformation and harvesting the synthetase genes between two- different plasmid systems after each round of selection.