High Quality de Novo Transcriptome Assembly of Croton tiglium

Haak M, Vinke S, Keller W, et al. High Quality de Novo Transcriptome Assembly of Croton tiglium. Frontiers in Molecular Biosciences. 2018;5: 62

Expanding The Genetic Code

Karsten L, Bergen D, Drake C, et al. Expanding The Genetic Code. Bielefeld University; 2017.We worked in many different scientific fields to find suitable ways for the translational incorporate of non-canonical amino acids into proteins. Repurposing existing codons or incorporating new bases are two possible ways. We realized both ways to expand the genetic code of Escherichia coli. The repurposing of a codon for the incorporation of a non-canonical amino acid (ncAA) is possible using the rarely used amber stop codon UAG or other rarely used codons like the leucine codon CUA. To incorporate a non-canonical amino acid using these codons, an orthogonal tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is necessary, which can charge the ncAA to the tRNA. We designed and synthetized the novel ncAA Nγ‑2‑cyanobenzothiazol‑6‑yl‑L‑asparagine (CBT-asparagine). This ncAA has the chemical ability of perform a highly specific covalent binding reaction, which we wanted to incorporate into our target protein. Therefore, we created a library of aaRS with random mutagenized amino acid binding sites and a selection system to select for the aaRS that specifically incorporates the ncAA. In parallel to the libary and selection based approach, we modeled the aaRS which could incorporate our new amino acid CBT-asparagine. We demonstrated that both ways are suitable for the evolution of aaRS. Although incorporation of ncAAs through the amber codon works, there are challenges associated with this approach. The repurposing of codons leads to the decrease of the growth rate of E. coli and it is only feasible to incorporate up to two different ncAAs. Therefore, we took a new way to incorporate ncAAs. The incorporation of an unnatural base pair into the DNA generates 64 new codons. Our first challenge was the uptake of the unnatural base from the media, because E.coli has no nucleoside triphosphate transporter and is not able to synthetize the bases itself. We cloned a nucleoside triphosphate transporter that enables the uptake of both bases from the media. Furthermore, we analyzed the transcriptome of the plant Croton tiglium, which produces the unnatural base isoG. The transcriptome revealed an enzyme for the biosynthesis, which was cloned and characterized for the biosynthesis of isoG in E. coli. To detect the unnatural base we developed two orthogonal systems. A restriction experiment based on the software tool M.A.X. and an adaption of the Oxford Nanopore sequencing, which were combined into one software suite. To demonstrate the possibilities offered by the incorporation of ncAAs, we developed a toolbox containing five different tools. We chose seven different ncAAs for these five tools and demonstrated interesting applications for them. These ncAAs can be used for various approaches in basic research, medicine and manufacturing. Furthermore, with our submitted parts, every iGEM team can incorporate these ncAAs into their target proteins. Regarding our project, two of the ncAAs that are part of our toolbox perform an autocatalytic reaction upon irradiation with ultraviolet light. Therefore, we decided to build our own LED panel that allows us to perform experiments with these non‑canonical amino acids under reproducible irradiation conditions

Xenobiology in synthetic biology: From non-standard amino acid incorporation to biosecurity implications

Vinke S. Xenobiology in synthetic biology: From non-standard amino acid incorporation to biosecurity implications. Bielefeld: Universität Bielefeld; 2023.In the era of sustainability, synthetic biology has emerged as a game changing field, holding immense potential to create innovative solutions to restore the ecological balance. The ability to produce peptides and proteins that can aid in achieving sustainability goals greatly depends on the development of effective protein design methods. However, conventional protein design approaches are limited by the 20 standard amino acids. This limitation calls for the exploration of alternative strategies for protein design that can expand the range of amino acids and allow the creation of novel proteins with enhanced functionalities. This challenge can be addressed by incorporating xenobiology in protein design strategies. Xenobiology seeks to expand the genetic code beyond the four naturally occurring nucleotides (A, C, T and G) and the 20 standard amino acids to create new synthetic DNA and protein blocks. To incorporate non-standard amino acids (nsAA) during translation, an orthogonal aminoacyl/tRNA synthetase (aaRS) needs to be introduced to endow the cell with the ability to charge a tRNA that is able to suppress a repurposed codon with the nsAA of choice. To make this system work a perfect aaRS and a codon that can be repurposed without influencing endogenous translation, apart from the incorporation of the nsAA, are needed. Current evolution methods for aaRS are limited in library design and alternative continuous evolution systems are not straightforward and can be hard to evaluate. To address this first bottleneck, we established and validated a Nanopore sequencing guided phage-assisted evolution strategy. The evolution of a L-2-nitrophenylalanyl aaRS was used to demonstrate the power of this new evolution method. By designing a new software solution to filter sequencing errors, it was possible to deep sequence the complete CDS throughout the whole evolution. This resulted in the evaluation of the entire evolution by constructing evolutionary trees with data indicating the strength of every mutation enriched throughout the evolution. By performing the evolution in triplicates, it was shown that independent experiments produced proteins that show the same amino acid exchanges under the same selection pressure. This uncovered which mutation results in local and global maxima of the aaRS activity and showed that mutations outside the ligand binding pocket have a strong influence on enzyme activity and specificity. Next to the aaRS, the choice of the repurposed codon has a strong influence on protein production with the nsAA. While in the evolution the most common used codon, the amber stop codon, was used to incorporate the non-standard amino acid, genetically recoded organisms are an alternative to create codons that do not interfere with the translational machinery. Genetically recoded organism are strains with one or several codons exchanged to synonymous codons throughout all protein CDSs. In Syn61 Δ3, the amber stop codons and two of the serine codons are exchanged. In addition to production of non-standard amino acid containing proteins, strains also offer the ability to resist viral replication. When a phage enters Syn61 Δ3 cells, the 3 recoded codons cannot be read by the cell anymore, and the phage proteins are not produced, impairing viral replication. However, several phages bring their own tRNAs being able to replicate in the recoded strains. To block this replication, leucine-serine swapped tRNAs were developed in this work. Through library and selection, we identified viral leucine tRNAs that tolerate an anticodon swap to the forbidden serine codons. These tRNAs can out-compete the viral tRNAs and lead to leucine incorporation at the serine codons, which leads to misfolding of the phage proteins and blocking phage replication. This swap can also be used to contain genetic information, making this system combined with genetically recoded organism a powerful tool for xenobiology. Both publications demonstrate the high pace at which this research discipline develops. However, to make this field grow in a responsible way, biosafety and biosecurity regulations need to grow at the same pace. Since biosecurity governance is mainly limited to pathogen-related research, the assessment and mitigation on the xenobiology field needs to be done by the researchers. This work shows that there is a dramatic lack of knowledge regarding dual-use risks and little effective efforts to change this situation. To make xenobiology research live up to its potential, responsibility, safety and security need to become a crucial aspect in project planning and execution, starting by educating young scientists about these topics over establishing suitable frameworks in governance

How to teach life sciences students about dual-use research-a view from the field

Gemunden M, Vinke S. How to teach life sciences students about dual-use research-a view from the field. Canadian Journal of Microbiology . 2022.To reduce biological risks, raising awareness for dual-use issues already at the level of university education is essential. Currently, most life sciences education programs do not incorporate biosecurity and dual-use in their regular curricula. Consequently, the responsibility rests with individual lecturers and depends on their initiative to incorporate dual-use topics into teaching activities. Students interested in biosecurity and dual-use topics often only have the option toeducate themselves in external or online courses. Here, we provide practical guidance on how to initiate and integrate a dual-use education program within the curriculum and provide a selection ofexisting teaching materials. In addition, we suggest key learning objectives to guide the planning of dual-use courses. Different course formats like lectures, seminars, or stand-alone events are discussed regarding their advantages, disadvantages, and suitability for conveying the learning objectives to different educational stages and audiences. As a minimum, we recommend the incorporation of dual-use issues into at least one mandatory course. Ideally, students should additionally participate in in-depth seminars, which can be voluntary and offered in cooperation with external organisations

The dual-use education gap: Awareness and education of life science researchers on nonpathogen-related dual-use research

Vinke S, Rais I, Millett P. The dual-use education gap: Awareness and education of life science researchers on nonpathogen-related dual-use research. Health Security . 2022;20(1):35-42.With the rise of synthetic biology, dual-use research risks are not confined to pathogen-related research. However, existing measures to mitigate the risks of dual-use research, such as export control, are still designed to hinder access to pathogens and do not address the risks of nonpathogen-related dual-use research. The current self-regulatory approach requires scientists to be aware of their responsibility and know how to assess risks and establish countermeasures. The purpose of this study was to examine the state of knowledge about dual-use research among life science students and to test an alternative teaching approach on the importance of considering biosecurity risks for teams participating in the International Genetically Engineered Machine (iGEM) competition. We conducted an international survey from July 18 to September 13, 2018, which was completed by 192 respondents from 29 countries and 74 universities. Based on the results of the survey, we designed and tested a learning workshop on dual-use research within the iGEM community. Results from the workshop and the survey show that educational machinery so far have failed to integrate teaching about dual-use research issues

Application of Next Generation Sequencing (NGS) in Phage Displayed Peptide Selection to Support the Identification of Arsenic-Binding Motifs

Braun R, Schönberger N, Vinke S, Lederer F, Kalinowski J, Pollmann K. Application of Next Generation Sequencing (NGS) in Phage Displayed Peptide Selection to Support the Identification of Arsenic-Binding Motifs. Viruses. 2020;12(12): 1360.Next generation sequencing (NGS) in combination with phage surface display (PSD) are powerful tools in the newly equipped molecular biology toolbox for the identification of specific target binding biomolecules. Application of PSD led to the discovery of manifold ligands in clinical and material research. However, limitations of traditional phage display hinder the identification process. Growth-based library biases and target-unrelated peptides often result in the dominance of parasitic sequences and the collapse of library diversity. This study describes the effective enrichment of specific peptide motifs potentially binding to arsenic as proof-of-concept using the combination of PSD and NGS. Arsenic is an environmental toxin, which is applied in various semiconductors as gallium arsenide and selective recovery of this element is crucial for recycling and remediation. The development of biomolecules as specific arsenic-binding sorbents is a new approach for its recovery. Usage of NGS for all biopanning fractions allowed for evaluation of motif enrichment, in-depth insight into the selection process and the discrimination of biopanning artefacts, e.g., the amplification-induced library-wide reduction in hydrophobic amino acid proportion. Application of bioinformatics tools led to the identification of an SxHS and a carboxy-terminal QxQ motif, which are potentially involved in the binding of arsenic. To the best of our knowledge, this is the first report of PSD combined with NGS of all relevant biopanning fractions

Proceedings of the dual use research of concern panel discussion: Challenges and perspectives

Rohden F, Nelson CJ, Yost CK, et al. Proceedings of the dual use research of concern panel discussion: Challenges and perspectives. Canadian Journal of Microbiology . 2022.To address real and perceived emerging risks originating from the ever-accelerating breakthroughs in life science research, the Dual Use Research of Concern (DURC) Panel Discussion, organized by Synbio Canada and the Alberta RNA Research and Training Institute (ARRTI), took place on June 23rd, 2021. It brought together six stakeholders from different levels of academic research, administration, governance, and science publishing to explore the current and future challenges of addressing DURC. The technological advancements within the life sciences, especially within the field of omics technology, make it difficult to apply simple checklist for Dual Use assessment and require a continuous and integrated effort. Bottom-up approaches from within the scientific community are suggested by all stakeholders to enable efficient governance and address the true risks resulting from DURC, not just the alleged risks. To address such alleged risks, open and broadscale communication of DURC and its oversight policies may be required. At the same time, any form of open communication also contains the risk of information hazard, defined as potentially creating public fear or informing malicious actors. Here, an overview of the DURC panel and its outcomes is given

Data_Sheet_3_High Quality de Novo Transcriptome Assembly of Croton tiglium.fasta

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Table_1_High Quality de Novo Transcriptome Assembly of Croton tiglium.CSV

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