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Expanding The Genetic Code
Abstract
Protein design is usually limited to 20 amino acids. Since the chemical abilities of these amino acids are limited, a lot of interesting functions are not applicable in protein design. We aim to enable the translational incorporation of non-canonical amino acids (ncAAs) through an expanded genetic code. First, we repurposed the rarely used amber stop codon for translational incorporation. Therefore, we adjusted aminoacyl-tRNA synthetases (aaRS) which charge the ncAA to the tRNA. We designed a library of aaRS and a suitable selection system for the development of customized aaRS which specifically incorporate the ncAA of interest. As a proof of concept, we applied different ncAAs e.g. for labeling, photoswitching, and photolysis of proteins. To further improve the translational incorporation of ncAAs, we explored a new way to expand the genetic code by development of a semisynthetic organism. An additional unnatiral base pair (UBP) generates 61 additional codons. Therefore, we improved an UBP uptake system and identified and characterized a biosynthesis pathway for in vivo production of isoG in Escherichia coli. We engineered a retention system and and established detection methods for the UBP.
... raw read qualities around Q20 (99% accuracy). Various DNA or RNA modifications can be analysed based on ONT sequence reads (Karsten et al., 2017;Parker et al., 2019). ...
Third-generation long-read sequencing is transforming plant genomics. Oxford Nanopore Technologies and Pacific Biosciences are offering competing long-read sequencing technologies and enable plant scientists to investigate even large and complex plant genomes. Sequencing projects can be conducted by single research groups and sequences of smaller plant genomes can be completed within days. This also resulted in an increased investigation of genomes from multiple species in large scale to address fundamental questions associated with the origin and evolution of land plants. Increased accessibility of sequencing devices and user-friendly software allows more researchers to get involved in genomics. Current challenges are accurately resolving diploid or polyploid genome sequences and better accounting for the intra-specific diversity by switching from the use of single reference genome sequences to a pangenome graph.
... Team Bielefeld-CeBiTec 2017 worked on expanding the genetic code with the unnatural base pair formed between isoguanosine (isoG) and 5-methyl-isocytosine (isoC m ), and non-canonical amino acids [239]. The team used CRISPR/ Cas9 to retain the unnatural base pair in a specified sequence. ...
Background
Biosafety is a key aspect in the international Genetically Engineered Machine (iGEM) competition, which offers student teams an amazing opportunity to pursue their own research projects in the field of Synthetic Biology. iGEM projects often involve the creation of genetically engineered bacterial strains. To minimize the risks associated with bacterial release, a variety of biosafety systems were constructed, either to prevent survival of bacteria outside the lab or to hinder horizontal or vertical gene transfer.
Main body
Physical containment methods such as bioreactors or microencapsulation are considered the first safety level. Additionally, various systems involving auxotrophies for both natural and synthetic compounds have been utilized by iGEM teams in recent years. Combinatorial systems comprising multiple auxotrophies have been shown to reduced escape frequencies below the detection limit. Furthermore, a number of natural toxin-antitoxin systems can be deployed to kill cells under certain conditions. Additionally, parts of naturally occurring toxin-antitoxin systems can be used for the construction of ‘kill switches’ controlled by synthetic regulatory modules, allowing control of cell survival. Kill switches prevent cell survival but do not completely degrade nucleic acids. To avoid horizontal gene transfer, multiple mechanisms to cleave nucleic acids can be employed, resulting in ‘self-destruction’ of cells. Changes in light or temperature conditions are powerful regulators of gene expression and could serve as triggers for kill switches or self-destruction systems. Xenobiology-based containment uses applications of Xeno-DNA, recoded codons and non-canonical amino acids to nullify the genetic information of constructed cells for wild type organisms. A ‘minimal genome’ approach brings the opportunity to reduce the genome of a cell to only genes necessary for survival under lab conditions. Such cells are unlikely to survive in the natural environment and are thus considered safe hosts. If suitable for the desired application, a shift to cell-free systems based on Xeno-DNA may represent the ultimate biosafety system.
Conclusion
Here we describe different containment approaches in synthetic biology, ranging from auxotrophies to minimal genomes, which can be combined to significantly improve reliability. Since the iGEM competition greatly increases the number of people involved in synthetic biology, we will focus especially on biosafety systems developed and applied in the context of the iGEM competition.
... The transcriptome resource of C. tiglium revealed an additional isoform of the GMPS gene involved in GMP synthesis (Supplementary File 3). Heterologous expression and in vitro enzyme assays of the purified enzymes followed by HPLC/MS analysis revealed substantial differences in the reaction products of the two isoforms that differ from a GMP standard, supporting the postulated involvement of this additional isoform in the isoguanosine biosynthesis (Karsten et al., 2017). ...
In this work, we present a comprehensive de novo transcriptome assembly of C. tiglium based on a normalized library to cover a huge variety of transcripts. In addition, tissue-specific transcript libraries were generated to enable differential gene expression analysis between tissues. This will facilitate the identification of candidate genes involved in growth, development, and metabolism of this plant species.
Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations. Here we describe the construction of a series of genomically recoded organisms (GROs) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼10(11) cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.
One of the major challenges in contemporary synthetic biology is to find a route to engineer synthetic organisms with altered chemical constitution. In terms of core reaction types, nature uses an astonishingly limited repertoire of chemistries when compared with the exceptionally rich and diverse methods of organic chemistry. In this context, the most promising route to change and expand the fundamental chemistry of life is the inclusion of amino acid building blocks beyond the canonical 20 (i.e. expanding the genetic code). This strategy would allow the transfer of numerous chemical functionalities and reactions from the synthetic laboratory into the cellular environment. Due to limitations in terms of both efficiency and practical applicability, state-of-the-art nonsense- or frameshift suppression-based methods are less suitable for such engineering. Consequently, we set out to achieve this goal by sense codon emancipation i.e. liberation from its natural decoding function - a prerequisite for the reassignment of degenerate sense codons to a new 21st amino acid. This is achieved by redesigning of several features of the posttranscriptional modification machinery which are directly involved in the decoding process. In particular, we present first steps towards the reassignment of 5797 AUA isoleucine codons in Escherichia coli by using efficient tools for tRNA nucleotide modification pathway engineering. This article is protected by copyright. All rights reserved.
Significance
The genetic alphabet encodes all biological information, but it is limited to four letters that form two base pairs. To expand the alphabet, we developed synthetic nucleotides that pair to form an unnatural base pair (UBP), and used it as the basis of a semisynthetic organism (SSO) that stores increased information. However, the SSO grew poorly and lost the UBP under a variety of standard growth conditions. Here, using chemical and genetic approaches, we report the optimization of the SSO so that it is healthy, more autonomous, and able to store the increased information indefinitely. This SSO constitutes a stable form of semisynthetic life and lays the foundation for efforts to impart life with new forms and functions.
Recoding and repurposing genetic codons
By recoding bacterial genomes, it is possible to create organisms that can potentially synthesize products not commonly found in nature. By systematic replacement of seven codons with synonymous alternatives for all protein-coding genes, Ostrov et al. recoded the Escherichia coli genome. The number of codons in the E. coli genetic code was reduced from 64 to 57 by removing instances of the UAG stop codon and excising two arginine codons, two leucine codons, and two serine codons. Over 90% functionality was successfully retained. In 10 cases, reconstructed bacteria were not viable, but these few failures offered interesting insights into genome-design challenges and what is needed for a viable genome.
Science , this issue p. 819
The ability to incorporate unnatural amino acids into proteins directly in living cells will provide new tools to study protein and cellular function, and may generate proteins or even organisms with enhanced properties. Due to the limited promiscuity of some synthetases, natural amino acids can be substituted with close analogs at multiple sites using auxotrophic strains. Alternatively, this can be achieved by deactivating the editing function of some synthetases. The addition of new amino acids to the genetic code, however, requires additional components of the protein biosynthetic machinery including a novel tRNA-codon pair, an aminoacyl-tRNA synthetase, and an amino acid. This new set of components functions orthogonally to the counterparts of the common 20 amino acids, i.e., the orthogonal synthetase (and only this synthetase) aminoacylates the orthogonal tRNA (and only this tRNA) with the unnatural amino acid only, and the resulting acylated tRNA inserts the unnatural amino acid only in response to the unique codon. Using this strategy, the genetic code of Escherichia coli has been expanded to incorporate unnatural amino acids with a fidelity rivaling that of natural amino acids. This methodology is being applied to other cell types and unnatural analogs with a variety of functionalities.
Researchers disagree over whether making heritable changes to human genes crosses a line.
The addition of new and versatile chemical and biological properties to proteins pursued through incorporation of non-canonical amino acids is at present primarily achieved by stop codon suppression. However, it is critical to find new "blank" codons to increase the variety and efficiency of such insertions, thereby taking 'sense codon recoding' to center stage in the field of genetic code expansion. Current thought optimistically suggests the use of the pyrrolysine system coupled with re-synthesis of genomic information towards achieving sense codon reassignment. Upon review of the serious experimental challenges reported in recent studies, we propose that success in this area will depend on the re-synthesis of genomes, but also on 'rewiring' the mechanism of protein synthesis and of its quality control.