The Future of Biotechnology Crime: A Parallel Delphi Study with Non-Traditional Experts

BACKGROUND: The way science is practiced is changing and forecasting biotechnology crime trends remains a challenge as future misuses become more sophisticated. METHODS: A parallel Delphi study was conducted to elicit future biotechnology scenarios from two groups of experts. Traditional experts, such as professionals in national security/intelligence, were interviewed. They were asked to forecast emerging crime trends facilitated by biotechnology and what should be done to safeguard against them. Non-traditional experts, such as “biohackers” who experiment with biotechnology in unexpected ways, were also interviewed. The study entailed three rounds to obtain consensus on (i) biotechnology misuse anticipated and (ii) potential prevention strategies expected. RESULTS: Traditional and non-traditional experts strongly agreed that misuse is anticipated within the cyber-infrastructure of, for example, medical devices and hospitals, through breaches and corporate espionage. Preventative steps that both groups strongly advocated involved increasing public biosecurity literacy, and funding towards addressing biotechnology security. Both groups agreed that the responsibility for mitigation includes government bodies. Non-traditional experts generated more scenarios and had a greater diversity of views. DISCUSSION: A systematic, anonymous and independent interaction with a diverse panel of experts provided meaningful insights for anticipating emerging trends in biotechnology crime. A multi-sector intervention strategy is proposed

Enhancing the productivity of supercoiled plasmid upstream bioprocessing through plasmid engineering

This study was set out to develop an approach for producing highly supercoiled plasmid DNA. Potentially, the level of supercoiling can have an impact on ease of downstream processing. A 7.2kb plasmid was developed by cloning of Bacteriophage-Mu Strong gyrase-binding sequence (Mu-SGS) into 6.8kb pSVβ-Gal. Four E. coli strains were transformed with both the modified pSVβ-Gal398 plasmid and pSVβ-Gal. Small scale fermentations and analysis were carried out in triplicate cultures to screen for best performing strains. Two of the four strains selected amplified the plasmids efficiently. There was over 20% increase in the total plasmid yield with pSVβ-Gal398 in both strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The two strains were investigated further in shake flasks. Increases in supercoiling and plasmid yield were also observed. The extent of supercoiling was examined by superhelical density quantification, with pSVβ-Gal398 maintaining a supercoil density of -0.022 and pSVβ-Gal -0.019 in both strains. The compactness of the plasmid DNA was also quantified by hydrodynamic diameter measurement using the Nanoparticle Tracking Analysis (NTA) and it was observed that pSVβ-Gal398 was more compact with a Dh of 40-59nm compared to pSVβ-Gal with Dh of 70-90nm for both strains examined. The report of this study has shown that plasmid engineered to contain the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing. References: Hassan, S., Keshavarz‐Moore, E., & Ward, J. (2016). A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy. Biotechnology and bioengineering, 113(9), 2064-2071. Yau, S. Y., Keshavarz‐Moore, E., & Ward, J. (2008). Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes. Biotechnology and bioengineering, 101(3), 529-544

One-pot synthesis of amino-alcohol using a de novo transketolase: Transaminase pathway in Pichia pastoris strain GS115

Pichia pastoris (P. pastoris) is an attractive industrial host cell due to its ability to grow up to 60% wet cell weight (WCW) by volume, a far higher level of biomass than the typical values reached by Escherichia coli (E. coli) and Saccharomyces cerevisiae. This thesis seeks to explore how the genetic tractability and high cell densities characteristic of P. pastoris can be exploited to intensify whole-cell biocatalysis. Chiral amino alcohols such as 2-amino-1,3,4-butanetriol (ABT) are key building blocks of small molecule pharmaceuticals and have previously been produced by whole-cell biocatalysis using cells engineered to overexpress a de novo enzyme pathway consisting of transketolase and transaminase. Within this work, native and foreign P. pastoris transaminases were characterized with respect to their biocatalytic potential. Genomic data mining was performed to explore the GS115 strain genome, allowing the selection of three putative Class III transaminase genes and the construction of overexpressor strains PpTAm107, PpTAm677 and PpTAm410. The well-studied ω-transaminase CV2025 from Chromobacterium violaceum was also successfully engineered to generate two strains; PpTAmCV708 for single expression of CV2025, and PpTAm-TK16 strain for CV2025 co-expression alongside a native transketolase previously characterized for L-erythrulose production. The rapid growth and high biomass characteristics of P. pastoris were successfully exploited for production of ABT by whole-cell biocatalysis. At high cell density, the best performance for the de novo pathway was obtained with the engineered PpTAm-TK16 strain, which tolerated high concentrations of substrate to achieve STY 0.57 g L-1 h-1 of ABT, 40-fold higher than levels previously achieved with E. coli for the same reaction

A biological OR(XNOR) logic gate couples carbon source and transgene expression switching in a Komagatella phaffii (Pichia pastoris) strain co-producing process-enhancing lipase and a virus-like particle (VLP) vaccine

We constructed a three-input biological logic gate: S OR (G XNOR M), where S is sorbitol, G is glycerol, and M is methanol, to optimize co-expression of two transgenes in Komagataella phaffii using batch-mode carbon source switching (CSS). K. phaffii was engineered to harbor transgenes encoding a Candida rugosa triacylglycerol lipase, which can enhance downstream processing by removing host cell lipids from homogenates, and the hepatitis B virus surface antigen (HBsAg), a protein that self-assembles into a virus-like particle (VLP) vaccine. Using the native alcohol oxidase 1 (PAOX1) and enolase 1 (PENO1) promoters to direct VLP vaccine and lipase expression, respectively, successfully provided an OR(XNOR) gate function with double-repression as the output. This logic gate functionality enabled use of CSS to ensure that approximately 80% of total VLP yield was accumulated before cells were burdened with lipase expression in 250 mL DasGip bioreactor cultivation

Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 105 TU/mL (±3.34 × 104 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions

Synthetic biology tools for engineering Goodwin oscillation in Trypanosoma brucei brucei.

Kinetoplastid protozoa possess properties that are highly divergent from the mammalian, yeast and bacterial cells more commonly used in synthetic biology and represent a tantalisingly untapped source of bioengineering potential. Trypanosoma brucei brucei (T. b. brucei), an established model organism for studying the Kinetoplastida, is non-pathogenic to humans and provides an interesting test case for establishing synthetic biology in this phylogenetic class. To demonstrate further the tractability of Kinetoplastida to synthetic biology, we sought to construct and demonstrate a Goodwin oscillator, the simplest oscillatory gene network, in T. b. brucei for the first time. We report one completed iteration of the archetypal synthetic biology Design-Build-Test-Learn (DBTL) cycle; firstly, using Ab initio mathematical modelling of the behaviour a theoretical, oscillatory, trypanosomal synthetic gene network (SGN) to inform the design of a plasmid encoding that network. Once assembled, the plasmid was then used to generate a stable transfectant T. b. brucei cell line. To test the performance of the oscillatory SGN, a novel experimental setup was established to capture images of the fluorescent signal from motion-restricted live cells. Data captured were consistent with oscillatory behaviour of the SGN, with cellular fluorescence observed to oscillate with a period of 50 min, with varying amplitude and linear growth trend. This first DBTL cycle establishes a foundation for future cycles in which the SGN design and experimental monitoring setup can be further refined

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

Salmonella Exhibit Altered Cellular Localization in the Presence of HLA-B27 and Codistribute with Endo-Reticular Membrane

Salmonella enteritica (S. enteritica) induce and require unfolded protein response (UPR) pathways for intracellular replication. Salmonella infections can lead to reactive arthritis (ReA), which can exhibit associations with Human Leucocyte Antigen (HLA)-B ∗ 27 : 05. S. enteritica normally reside in a juxtanuclear position to the Golgi apparatus, representing the formation and residence within the Salmonella-containing vacuole (SCV). Changes in cellular localization of infecting Salmonella can alter their ability to replicate. We therefore used isogenic epithelial cell lines expressing physiological levels of HLA-B ∗ 27 : 05 heavy chain (HC) and a control HLA-B allele, HLA-B ∗ 35 : 01.HC to determine any changes in Salmonella localization within epithelial cells. Expression of HLA-B ∗ 27 : 05 but not HLA-B ∗ 35 : 01 was associated with a quantifiable change in S. enteritica cellular distribution away from the Golgi apparatus. Furthermore, the Salmonella requirements for UPR induction and the consequences of the concomitant endoplasmic reticulum (ER) membrane expansion were determined. Using confocal imaging, S. enteritica bacteria exhibited a significant and quantifiable codistribution with endo-reticular membrane as determined by ER tracker staining. Isogenic S. enterica Typhimurium mutant strains, which can infect but exhibit impaired intracellular growth, demonstrated that the activation of the UPR was dependent on an integral intracellular niche. Therefore, these data identify cellular changes accompanying Salmonella induction of the UPR and in the presence of HLA-B27

Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication

A.N.A was funded by ARUK Fellowships Non-Clinical Career Development Fellowship Ref No: 18440. I.L. was funded by an ARUK PhD studentship Ref No: 17868. A.N.A and S.J.P were also in part funded by ARUK (grant 21261)Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. Methods Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. Conclusions HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.Publisher PDFPeer reviewe

Complex and unexpected dynamics in simple genetic regulatory networks

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