The field of protein engineering has been greatly augmented by the expansion of the genetic code using unnatural amino acids as well as the development of cell-free synthesis systems with high protein yield. Cell-free synthesis systems have improved considerably since they were first described almost 40 years ago. Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines high protein expression yields with a high level of analog substitution in the target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo format. We use this amino acid depleted cell-free synthesis system for the directed evolution of streptavidin, a protein that finds wide application in molecular biology and biotechnology. We evolve streptavidin using in vitro compartmentalization in emulsions to bind to desthiobiotin and find, at the conclusion of our experiment, that our evolved streptavidin variants are capable of binding to both biotin and desthiobiotin equally well. We also discover a set of mutations for streptavidin that are potentially powerful stabilizing mutations that we believe will be of great use to the greater research community. / text

abstract: In vitro selection technologies allow for the identification of novel biomolecules endowed with desired functions. Successful selection methodologies share the same fundamental requirements. First, they must establish a strong link between the enzymatic function being selected (phenotype) and the genetic information responsible for the function (genotype). Second, they must enable partitioning of active from inactive variants, often capturing only a small number of positive hits from a large population of variants. These principles have been applied to the selection of natural, modified, and even unnatural nucleic acids, peptides, and proteins. The ability to select for and characterize new functional molecules has significant implications for all aspects of research spanning the basic understanding of biomolecules to the development of new therapeutics. Presented here are four projects that highlight the ability to select for and characterize functional biomolecules through in vitro selection. Chapter one outlines the development of a new characterization tool for in vitro selected binding peptides. The approach enables rapid screening of peptide candidates in small sample volumes using cell-free translated peptides. This strategy has the potential to accelerate the pace of peptide characterization and help advance the development of peptide-based affinity reagents. Chapter two details an in vitro selection strategy for searching entire genomes for RNA sequences that enhance cap-independent initiation of translation. A pool of sequences derived from the human genome was enriched for members that function to enhance the translation of a downstream coding region. Thousands of translation enhancing elements from the human genome are identified and the function of a subset is validated in vitro and in cells. Chapter three discusses the characterization of a translation enhancing element that promotes rapid and high transgene expression in mammalian cells. Using this ribonucleic acid sequence, a series of full length human proteins is expressed in a matter of only hours. This advance provides a versatile platform for protein synthesis and is espcially useful in situations where prokaryotic and cell-free systems fail to produce protein or when post-translationally modified protein is essential for biological analysis. Chapter four outlines a new selection strategy for the identification of novel polymerases using emulsion droplet microfluidics technology. With the aid of a fluorescence-based activity assay, libraries of polymerase variants are assayed in picoliter sized droplets to select for variants with improved function. Using this strategy a variant of the 9°N DNA polymerase is identified that displays an enhanced ability to synthesize threose nucleic acid polymers. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

Philosophiae Doctor - PhD / The estimated 5 × 10³⁰ prokaryotic cells inhabiting our planet sequester some 350–550 Petagrams (1 Pg = 1015 g) of carbon, 85–130 Pg of nitrogen, and 9–14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio- rospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). The results obtained here show that the R. erythropolis cell-free system was over thirty-fold more effective than the E. coli based system based on the number of hits obtained per million double emulsions (dE) droplets screened. Six beta-xylosidase encoding genes were isolated and confirmed from twenty-eight positive dE droplets. Most of the droplets that were isolated from the same gate encoded the same enzyme, indicating that this technique is highly selective. A comparison of the hit rate of this screening approach with the traditional E. coli based fosmid library method shows that mIVC-FACS is at least 2.5 times more sensitive. Although only a few hits from the mIVC-FACS screening were selected for confirmation of beta-xylosidase activity, the proposed hit rate suggests that a significant number of positive hits are left un-accessed through the traditional clone library screening system. In addition, these results also suggest that E. coli expression system might be intrinsically sub-optimal for screening for hemicellulases from environmental genomes compared to R. erythropolis system. The workflow required for screening one million clones in a fosmid library was estimated to be about 320 hours compared to 144 hours required via the mIVC-FACS screening platform. Some of the gene products obtained in both screening platforms show multiple substrate activities, suggesting that the microbial consortia of composting material consist of microorganisms that produce enzymes with multiple lignocellulytic activities. While this platform still requires optimisation, we have demonstrated that this technique can be used to isolate genes encoding enzymes from mixed microbial genomes. mIVC-FACS is a promising technology with the potential to take metagenomic studies to the second generation of novel natural products bio-prospecting. The astonishing sensitivity and ultra-high throughput capacity of this technology offer numerous advantages in metagenomic bio-prospecting. / National Research Foundation (NRF)

Blood flow through microcirculation in both simple and complex geometry has been difficult to predict due to the composition and complex behavior of blood at the microscale. Blood is a dense suspension of deformable red blood cells that is comparable in dimensions to the microchannels that it flows through. As a result, rheological properties at the microscale can vastly differ from bulk rheological properties due to non-continuum effects. To further develop our understanding of blood microflow; experimental techniques should be explored. In this work, we explore micro-particle image velocimetry (μPIV) and two-beam fluorescence cross-correlation spectroscopy (2bFCCS) in the application of characterizing blood in microflow conditions. For the development of the μPIV analysis, a polydimethylsiloxane co-flow channel is used to observe blood flow in controlled conditions. Flow conditions (velocity profile and blood layer thickness) are selected based on an analytical model and compared to experimental measurement. The experimental results presented indicate that current flow conditions are inadequate in providing a controlled rate of shear on the blood layer in the co-flow channel and further optimization are required to improve the measurement of the velocity profile. For the development of the 2bFCCS application for blood flow analysis, a wide glass capillary microfluidic device is used to complete the verification of fluorescence fluid admissibility, the effect of laser intensity on inducing photobleaching and the velocity measurement performance. The experimental measurement of the velocity profile is validated against the theoretical profile for a rectangular channel. Results of the velocity profile of high concentration red blood cells show promise in the technique’s ability to measure blood microflows closer to physiological conditions.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21995号 / 医博第4509号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸井 雅和, 教授 万代 昌紀, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

abstract: The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

Bacteriocins are antimicrobial protein produced by certain Gram positive and negative bacteria as a defense mechanism against closely related bacteria competing for the same nutrient or in the same niche. The competition for the same nutrient is supported by the fact that bacteriocins have narrow range of effect and only likely to be effective against closely related bacteria for the same scares resources hence a bacteriocin produced by a Gram positive bacteria will be active against a Gram positive pathogens and a bacteriocin produced by a Gram negative bacteria will be active against Gram negative pathogens. This is due to the difference in cell wall composition, they are either bacteriocidal or bacteriostatic Bacteriocins have been used for thousands of years for food preservation unknowingly to man, they are considered advantageous not only to the producing bacteria, but it's now been used by the food industry as a tool to control both spoilage and pathogenic bacteria in food, in a natural manner which is acceptable to the consumer. With a lot of research been carried out on bacteriocins produced by Gram positive bacteria, antagonist to Gram positive food borne pathogens, little is known about bacteriocins produced by Gram negative bacteria which would be active against Gram negative food borne pathogens that predominate in produce. The objective of my research therefore is to screen for antimicrobial antagonist to Gram negative food borne pathogens (Escherichia coli O157:H7 and Salmonella Enteritidis) from produce, to determine an appropriate screening method, to carry out a preliminary characterization of antagonist discovered and also to determine antimicrobial spectrum of antagonist found. Lettuce was screened for antimicrobial antagonist against Gram negative pathogen (Escherichia coli O157:H7 and Salmonella Enteritidis) which were used as indicator strains With over 5000 colonies screen, 1 colony (Serratia plymuthica) was discovered to be antagonistic against these indicator strain. Further screening of cell free extract using the spot test method showed that extract from Serratia plymuthica grown alone in TSBYE showed antagonist activity against indicator strain with a little clearing on the spot of extract dropped. But extract of a co-culture of Serratia plymuthica and either Escherichia coli O157:H7 or Salmonella Enteritidis showed a more obvious clearing around spotted zone, which further indicates antagonism against indicator strains. Preliminary heat test indicates antagonist compound to be heat stable at 60oC for 30mins, 100oC for 30minutes and 60mins and 121oC for 20minites, and antagonist compound possessed antagonist activity against other strains of Escherichia coli when tested.

The demand for economical, efficient protein production, reuse, and recovery has never been greater due to their versatility in a large variety of applications ranging from industrial chemical manufacturing to pharmaceutical drug production. The applications for naturally and artificially produced proteins include protein drugs and other pharmaceutical products, as biocatalysts in environmentally friendly chemical manufacturing, as enzymes for food processing purposes, and as an essential component in many biomedical devices. However, protein production suffers from many challenges, which include the cost of production, protein stability especially under harsh conditions, and recoverability and reusability of the proteins. The combination of two developing technologies, cell-free protein synthesis systems (CFPS) and unnatural amino acid incorporation, provides solutions to these protein production challenges.This dissertation reports on the use of cell-free protein synthesis systems and unnatural amino acid incorporation to develop new proteins and enzyme immobilization techniques that significantly increase activity and stability while simplifying recoverability and reuse.

Cell-free protein synthesis (CFPS) using crude lysates has developed into a robust platform technology over the last 60 years to express numerous types of recombinant proteins. The open-nature, elimination of reliance on cell viability, and focus of all energy towards production of the protein of interest represent substantial advantages of CFPS over in vivo protein expression methods. CFPS has provided new opportunities across a series of research fields that include metabolic engineering, therapeutic and vaccine development, education, biosensors, and many more. In recent years, optimizations of CFPS have even allowed the platform to reach the industrial level of protein production. Although there have been many advancements toward CFPS development, the democratization of the platform to a wide variety of educational, research, and industrial institutions has lacked due to an absence of resources for new users as well as a limited number of developments toward redesigning the tedious and time-consuming protocols to generate robust cell extract. To address these challenges to CFPS implementation, a comprehensive review spanning numerous cell lines with their respective applications, methodologies, and reaction formats were provided in addition to detailed protocols outlining the process of going from E. coli cells to a completed CFPS reaction. Together, these resources provide the scientific community with easily accessible resources for CFPS implementation. Moreover, the aforementioned protocols were redesigned from a four-day process into one that may be completed in under 24-hour’s time with very little researcher oversight. The resulting workflow maintained the robustness of prior methods but generated 400% more extract compared to traditional methods via a set-it-and-forget-it approach. To date, the works presented herein have garnered tremendous viewership from the CFPS research community with a substantial following among all three of the articles. Moving forward, I anticipate that these works will continue to bring new users into the CFPS field through the ease of access to these resources and through the advance of the simplistic and reproducible new workflow for preparation of robust E. coli cell extract.

Liquid biopsy and next-generation sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offer a minimally-invasive solution to detect tumor cell genomic information to aid real-time clinical decision-making. Reliability and sensitivity in the detection of genomic alterations is crucial for patient management and it is particularly relevant in the context of targeted therapies. However, biological and technical factors, such as low cfDNA tumor fraction and sequencing errors, affect the correct interpretation of genomic data limiting the utility of non-invasive cfDNA-based tumor profiling. To address these issues, we designed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation of patients’ tumor. The framework also implements ABEMUS, an ad-hoc computational procedure we specifically designed for cfDNA samples to accurately detect somatic point mutations that could emerge under treatment pressure and as drug resistance mechanism. When applied on serial plasma samples from three patients receiving PARP inhibition harboring DNA repair gene aberrations, PCF_SELECT demonstrated high sensitivity in detecting BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. As a step towards a more sensitive detection of tumor features in circulation of cancer patients, we next hypothesized that recent WGS-based approaches exploiting cfDNA fragments characteristics could be extrapolated for targeted sequencing data and that gene-region specific measures could improve detection metrics. Preliminary results suggest an increased sensitivity compared to copy-number-based methods supporting the integration at no extra cost in our targeted assay. Overall, this work is relevant to the context of precision oncology. It provides an innovative platform for the management of cancer patients, delivering detailed patient-specific molecular information which could be applied to guide treatment and improve clinical outcomes.