Low-cost and user-friendly biosensor to test the integrity of mRNA molecules suitable for field applications

The use of mRNA in biotechnology has expanded with novel applications such as vaccines and therapeutic mRNA delivery recently demonstrated. For mRNA to be used in patients, quality control assays will need to be routinely established. Currently, there is a gap between the highly sophisticated RNA integrity tests available and broader application of mRNA-based products by non-specialist users, e.g. in mass vaccination campaigns. Therefore, the aim of this work was to develop a low-cost biosensor able to test the integrity of a mRNA molecule with low technological requirements and easy end-user application. The biosensor is based on a bi-functional fusion protein, composed by the λN peptide that recognizes its cognate aptamer encoded on the 5’ end of the RNA under study and β-lactamase, which is able to produce a colorimetric response through a simple test. We propose two different mechanisms for signal processing adapted to two levels of technological sophistication, one based on spectrophotometric measurements and other on visual inspection. We show that the proposed λN-βLac chimeric protein specifically targets its cognate RNA aptamer, boxB, using both gel shift and biolayer interferometry assays. More importantly, the results presented confirm the biosensor performs reliably, with a wide dynamic range and a proportional response at different percentages of full-length RNA, even when gene-sized mRNAs were used. Thus, the features of the proposed biosensor would allow to end-users of products such as mRNA vaccines to test the integrity of the product before its application in a low-cost fashion, enabling a more reliable application of these products

Designing an artificial Golgi reactor for cell-free glycosylation

Glycosylation of therapeutically relevant proteins such as monoclonal antibodies (mAbs), is critical as it can offer increased drug efficiency, efficacy and half-life. Therefore, the production of modern biotherapeutics focuses on controlling the protein glycosylation profile using various methods. Currently, the dominating method is the traditional cell-line engineering of host cells such as mammalian cells. The main goal is to produce mAbs with a human-like glycosylation pattern. However, this approach often struggles due to high sensitivity to the fermentation environment making it difficult to scale up and control. The latter can lead to structural heterogeneity amongst the products which can be immunogenic. In addition to the in vivo methods, there are many in vitro techniques such as chemoselective or enzymatic glycosylation. However, they are often limited by the difficult implementation and, as before, product heterogeneity due to lack of control over the enzymatic reactions. In line with the need to control glycosylation in the production of therapeutic proteins, we propose an artificial Golgi reactor for in vitro glycosylation. By expressing selected glycosyltransferases and immobilizing them on solid supports we can achieve sequential enzymatic reactions required for protein glycosylation. The spatial separation will allow strict control over the reaction conditions while addressing enzyme promiscuity. Both should enhance product quality. Furthermore, we aim to perform a single-step glycosyltransferases purification/immobilization. Thanks to that, as well as the modularity of our design, the proposed system would be more sustainable and easily tailored for each application, thus producing any desired glycoform to homogeneity. A detailed mathematical approach to design and optimisation of the proposed artificial Golgi reactor focusing on mAb therapeutics has been published [1]. The authors report an optimisation of the reactor design and operational parameters that directs the whole process towards the desired glycan structure. In this research project, we have achieved expression and in vivo biotinylation of Nicotiana Tabacum GnTI (NtGnTI) and human GalT in E. coli. The biotinylated enzymes were successfully bound to streptavidin beads and used for artificial glycan synthesis. NtGnTI and GalT reacted in a sequential fashion to produce the glycan GalGlcNAcMan5GlcNAc2, as confirmed with MALDI/TOF MS analysis. In the future, we aim in extending the pathway of immobilized enzymes thus demonstrating the importance of this novel platform for in vitro glycosylation. References: [1] Klymenko, O. V., Shah, N., Kontoravdi, C., Royle, K. E. & Polizzi, K. M. Designing an Artificial Golgi reactor to achieve targeted glycosylation of monoclonal antibodies. AIChE J. 62, 2959–2973 (2016)

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology

Tools for Maximizing the Efficiency of Protein Engineering

Biocatalysts offer advantages over their chemical counterparts in terms of their high enantioselectivity and the opportunity to develop more environmentally friendly processes. However, the widespread adoption of biocatalytic processes is hampered by the long development times for enzymes with novel and sufficient activity and adequate stability under operating conditions. Protein engineering, while extremely useful for modifying the properties of protein catalysts in select cases, still cannot be performed rapidly enough for many applications. In order for biocatalysts to become a competitive alternative to chemical catalysts, new tools to make the tailoring of biocatalysts by protein engineering methods speedier and more efficient are necessary. The aim of this work was to develop methods to aid in the faster production of novel biocatalysts. Protein engineering involves two steps: the generation of diversity and the screening or selection of variants with the desired properties. Both of these must be targeted to create a faster protein engineering process. In the case of the former, this work sought to clone and overexpress some template enzymes which would create smaller, more manageable libraries of mutants with a higher likelihood of function by the manipulation of a few focused amino acid residues. For the latter, this work developed and validated a Monte-Carlo simulation model of pooling to increase screening throughput and created a set of vectors to aid in high-throughput screening by eliminating unwanted mutants from the assay procedure entirely.Ph.D.Committee Chair: Bommarius, Andrea

Intracellular delivery of biologic therapeutics by bacterial secretion systems

Biologics are a promising new class of drugs based on complex macromolecules such as proteins and nucleic acids. However, delivery of these macromolecules into the cytoplasm of target cells remains a significant challenge. Here we present one potential solution: bacterial nanomachines that have evolved over millions of years to efficiently deliver proteins and nucleic acids across cell membranes and between cells. In this review, we provide a brief overview of the different bacterial systems capable of direct delivery into the eukaryotic cytoplasm and the medical applications for which they are being investigated, along with a perspective on the future directions of this exciting field

EcoFlex: A Multifunctional MoClo Kit for <i>E. coli</i> Synthetic Biology

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as <i>Escherichia coli</i> has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in <i>E. coli</i>, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize <i>in vivo</i> and <i>in vitro</i> library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in <i>E. coli</i> synthetic biology

Handbook of Cell Biosensors

Handbook of Cell Biosensor