A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction.

BACKGROUND: Chinese Hamster Ovary (CHO) cells are the current industry standard for production of therapeutic monoclonal antibodies at commercial scales. Production optimisation in CHO cells hinges on analytical technologies such as the use of the polymerase chain reaction (PCR) to quantify genetic factors within the CHO genome and to detect the presence of contaminant organisms. PCR-based assays, whilst sensitive and accurate, are limited by (i) requiring lengthy sample preparation and (ii) a lack of standardisation. RESULTS: In this study we directly assess for the first time the effect of CHO cellular material on quantitative PCR (qPCR) and end-point PCR (e-pPCR) when used to measure and detect copies of a CHO genomic locus and a mycoplasma sequence. We also perform the first head-to-head comparison of the performance of a conventional qPCR method to that of the novel linear regression of efficiency (LRE) method when used to perform absolute qPCR on CHO-derived material. LRE qPCR features the putatively universal 'CAL1' standard. CONCLUSIONS: We find that sample preparation is required for accurate quantitation of a genomic target locus, but mycoplasma DNA sequences can be detected in the presence of high concentrations of CHO cellular material. The LRE qPCR method matches performance of a conventional qPCR approach and as such we invite the synthetic biology community to adopt CAL1 as a synthetic biology calibration standard for qPCR

A Systematic Review of the Criminogenic Potential of Synthetic Biology and Routes to Future Crime Prevention

Synthetic biology has the potential to positively transform society in many application areas, including medicine. In common with all revolutionary new technologies, synthetic biology can also enable crime. Like cybercrime, that emerged following the advent of the internet, biocrime can have a significant effect on society, but may also impact on peoples' health. For example, the scale of harm caused by the SARS-CoV-2 pandemic illustrates the potential impact of future biocrime and highlights the need for prevention strategies. Systematic evidence quantifying the crime opportunities posed by synthetic biology has to date been very limited. Here, we systematically reviewed forms of crime that could be facilitated by synthetic biology with a view to informing their prevention. A total of 794 articles from four databases were extracted and a three-step screening phase resulted in 15 studies that met our threshold criterion for thematic synthesis. Within those studies, 13 exploits were identified. Of these, 46% were dependent on technologies characteristic of synthetic biology. Eight potential crime types emerged from the studies: bio-discrimination, cyber-biocrime, bio-malware, biohacking, at-home drug manufacturing, illegal gene editing, genetic blackmail, and neuro-hacking. 14 offender types were identified. For the most commonly identified offenders (>3 mentions) 40% were outsider threats. These observations suggest that synthetic biology presents substantial new offending opportunities. Moreover, that more effective engagement, such as ethical hacking, is needed now to prevent a crime harvest from developing in the future. A framework to address the synthetic biology crime landscape is proposed

A systematic review protocol for crime trends facilitated by synthetic biology

BACKGROUND: When new technologies are developed, it is common for their crime and security implications to be overlooked or given inadequate attention, which can lead to a 'crime harvest'. Potential methods for the criminal exploitation of biotechnology need to be understood to assess their impact, evaluate current policies and interventions and inform the allocation of limited resources efficiently. Recent studies have illustrated some of the security implications of biotechnology, with outcomes of misuse ranging from compromised computers using malware stored in synthesised DNA, infringement of intellectual property on biological matter, synthesis of new threatening viruses, 'genetic genocide,' and the exploitation of food markets with genetically modified crops. However, there exists no synthesis of this information, and no formal quality assessment of the current evidence. This review therefore aims to establish what current and/or predicted crimes have been reported as a result of biotechnology. METHODS: A systematic review will be conducted to identify relevant literature. ProQuest, Web of Science, MEDLINE and USENIX will be searched utilizing a predefined search string, and Backward and Forward searches. Grey literature will be identified by searching the official UK Government website (www.gov.uk) and the Global database of Dissertations and Theses. The review will be conducted by screening title/abstracts followed by full texts, utilising pre-defined inclusion and exclusion criteria. Papers will be managed using Eppi-center Reviewer 4 software, and data will be organised using a data extraction table using a descriptive coding tool. A predefined rating system (speculative, experimental or currently occurring) will be used to sort studies, and a thematic synthesis of the results will be presented. DISCUSSION: Despite the concerns raised about the misuse of biotechnology, no previous work has been conducted from a Crime Science perspective to collate and assess the literature. This systematic review aims to identify the types of offending activity facilitated by biotechnology, including synthetic biology and genetic engineering. The objective of the review is to examine whether this offending activity can be prevented by assessing the conditions necessary for the crime events to occur. It is anticipated that evidence generated from this review will guide future research in this area and aid relevant stakeholders to prioritise and allocate limited resources to biotechnology crime prevention. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42019131685

Genomic data mining reveals the transaminase repertoire of Komagataella phaffii (Pichia pastoris) strain GS115 and supports a systematic nomenclature

Transaminases are an industrially important class of enzyme, due to their ability to catalyse amination reactions for production of chiral amines, and are key building blocks of small molecule pharmaceuticals. We analysed the genome of strain GS115 of the methylotrophic yeast Komagataella phaffii, formerly known as Pichia pastoris, to identify the transaminase genes and propose a systematic nomenclature based on both phylogeny and structuro-functional features. K. phaffii is an increasingly attractive industrial host cell due to its ability to grow to high biomass, up to 60% wet cell weight by volume, using methanol as carbon source and inducer of transgene expression. Thirty-nine UniProt database hits were reduced to 19 on the basis of sequence similarity and hidden Markov model. Of the 19 genes, the open-reading frames of three (KpTam I-II.1b, KpTam I-II.7 and KpTam V.2) had strong homology with no characterized protein and four (KpTam III.1a, KpTam III.1b, KpTam III.2a and KpTam III.2b) had relatively high sequence similarity to ω-type transaminases, a subtype that typically accepts the broadest range of substrates. Comparison with Saccharomyces cerevisiae S288C suggested functions for KpTam I-II.1b and KpTam I-II.7. K. phaffii GS115 was originally generated by mutagenesis of K. phaffii CBS7435 and comparison revealed that one transaminase gene may have been deleted during this mutagenesis. These insights can advance fundamental understanding of yeast biology and can inform industrial screening and engineering of yeast transaminases

IMAC capture of recombinant protein from unclarified mammalian cell feed streams

Fusion-tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300-500 μm diameter agarose resin beads that allow free passage of cells but capture His-tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His-tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼8 U/mL and 2 ng/μL in column flow-through, respectively. Recovery of His-tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2015;9999: 1-11. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc

Towards an Aspect-Oriented Design and Modelling Framework for Synthetic Biology

Work on synthetic biology has largely used a component-based metaphor for system construction. While this paradigm has been successful for the construction of numerous systems, the incorporation of contextual design issues—either compositional, host or environmental—will be key to realising more complex applications. Here, we present a design framework that radically steps away from a purely parts-based paradigm by using aspect-oriented software engineering concepts. We believe that the notion of concerns is a powerful and biologically credible way of thinking about system synthesis. By adopting this approach, we can separate core concerns, which represent modular aims of the design, from cross-cutting concerns, which represent system-wide attributes. The explicit handling of cross-cutting concerns allows for contextual information to enter the design process in a modular way. As a proof-of-principle, we implemented the aspect-oriented approach in the Python tool, SynBioWeaver, which enables the combination, or weaving, of core and cross-cutting concerns. The power and flexibility of this framework is demonstrated through a number of examples covering the inclusion of part context, combining circuit designs in a context dependent manner, and the generation of rule, logic and reaction models from synthetic circuit designs

Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules

Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (β2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery

Complex and unexpected dynamics in simple genetic regulatory networks

Peer reviewedPublisher PD

An Efficient Vector System to Modify Cells Genetically

The transfer of foreign genes into mammalian cells has been essential for understanding the functions of genes and mechanisms of genetic diseases, for the production of coding proteins and for gene therapy applications. Currently, the identification and selection of cells that have received transferred genetic material can be accomplished by methods, including drug selection, reporter enzyme detection and GFP imaging. These methods may confer antibiotic resistance, or be disruptive, or require special equipment. In this study, we labeled genetically modified cells with a cell surface biotinylation tag by co-transfecting cells with BirA, a biotin ligase. The modified cells can be quickly isolated for downstream applications using a simple streptavidin bead method. This system can also be used to screen cells expressing two sets of genes from separate vectors