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Optimizing Recombinant Protein Production for Domain Antibodies: Proof-of-Concept

Description

Recent studies in traumatic brain injury (TBI) have found a temporal window where therapeutics on the nanometer scale can cross the blood-brain barrier and enter the parenchyma. Developing protein-based therapeutics is attractive for a number of reasons, yet, the production

Recent studies in traumatic brain injury (TBI) have found a temporal window where therapeutics on the nanometer scale can cross the blood-brain barrier and enter the parenchyma. Developing protein-based therapeutics is attractive for a number of reasons, yet, the production pipeline for high yield and consistent bioactive recombinant proteins remains a major obstacle. Previous studies for recombinant protein production has utilized gram-negative hosts such as Escherichia coli (E. coli) due to its well-established genetics and fast growth for recombinant protein production. However, using gram-negative hosts require lysis that calls for additional optimization and also introduces endotoxins and proteases that contribute to protein degradation. This project directly addressed this issue and evaluated the potential to use a gram-positive host such as Brevibacillus choshinensis (Brevi) which does not require lysis as the proteins are expressed directly into the supernatant. This host was utilized to produce variants of Stock 11 (S11) protein as a proof-of-concept towards this methodology. Variants of S11 were synthesized using different restriction enzymes which will alter the location of protein tags that may affect production or purification. Factors such as incubation time, incubation temperature, and media were optimized for each variant of S11 using a robust design of experiments. All variants of S11 were grown using optimized parameters prior to purification via affinity chromatography. Results showed the efficiency of using Brevi as a potential host for domain antibody production in the Stabenfeldt lab. Future aims will focus on troubleshooting the purification process to optimize the protein production pipeline.

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Date Created
2019-05

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Characterizing Primary Mesothelioma Cell Lines by Exome Sequencing

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Malignant Pleural Mesothelioma is a type of lung cancer usually discovered at an advanced stage at which point there is no cure. Six primary MPM cell lines were used to conduct in vitro research to make conclusions about specific gene

Malignant Pleural Mesothelioma is a type of lung cancer usually discovered at an advanced stage at which point there is no cure. Six primary MPM cell lines were used to conduct in vitro research to make conclusions about specific gene mutations associated with Mesothelioma. DNA exome sequencing, a time efficient and inexpensive technique, was used for identifying specific DNA mutations. Computational analysis of exome sequencing data was used to make conclusions about copy number variation among common MPM genes. Results show a CDKN2A gene heterozygous deletion in Meso24 cell line. This data is validated by a previous CRISPR-Cas9 outgrowth screen for Meso24 where the knocked-out gene caused increased Meso24 growth.

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Date Created
2020-05

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Comprehensive Analysis of Volatile Biomarkers for Female Fertility

Description

One out of ten women has a difficult time getting or staying pregnant in the United States. Recent studies have identified aging as one of the key factors attributed to a decline in female reproductive health. Existing fertility diagnostic methods

One out of ten women has a difficult time getting or staying pregnant in the United States. Recent studies have identified aging as one of the key factors attributed to a decline in female reproductive health. Existing fertility diagnostic methods do not allow for the non-invasive monitoring of hormone levels across time. In recent years, olfactory sensing has emerged as a promising diagnostic tool for its potential for real-time, non-invasive monitoring. This technology has been proven promising in the areas of oncology, diabetes, and neurological disorders. Little work, however, has addressed the use of olfactory sensing with respect to female fertility. In this work, we perform a study on ten healthy female subjects to determine the volatile signature in biological samples across 28 days, correlating to fertility hormones. Volatile organic compounds (VOCs) present in the air above the biological sample, or headspace, were collected by solid phase microextraction (SPME), using a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coated fiber. Samples were analyzed, using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). A regression model was used to identify key analytes, corresponding to the fertility hormones estrogen and progesterone. Results indicate shifts in volatile signatures in biological samples across the 28 days, relevant to hormonal changes. Further work includes evaluating metabolic changes in volatile hormone expression as an early indicator of declining fertility, so women may one day be able to monitor their reproductive health in real-time as they age.

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Agent

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Date Created
2018

Investigating the Role of the Perivascular Niche on Glioma Stem Cell Invasion in a Three-Dimensional Microfluidic Tumor Microenvironment Model

Description

Glioblastoma Multiforme (GBM) is a grade IV astrocytoma and the most aggressive form of cancer that begins within the brain. The two-year average survival rate of GBM in the United States of America is 25%, and it has a higher

Glioblastoma Multiforme (GBM) is a grade IV astrocytoma and the most aggressive form of cancer that begins within the brain. The two-year average survival rate of GBM in the United States of America is 25%, and it has a higher incidence in individuals within the ages of 45 - 60 years. GBM Tumor formation can either begin as normal brain cells or develop from an existing low-grade astrocytoma and are housed by the perivascular niche in the brain microenvironment. This niche allows for the persistence of a population of cells known as glioma stem cells (GSC) by supplying optimum growth conditions that build chemoresistance and cause recurrence of the tumor within two to five years of treatment. It has therefore become imperative to understand the role of the perivascular niche on GSCs through in vitro modelling in order to improve the efficiency of therapeutic treatment and increase the survival rate of patients with GBM.

In this study, a unique three dimensional (3D) microfluidic platform that permitted the study of intercellular interactions between three different cell types in the perivascular niche of the brain was developed and utilized for the first time. Specifically, human endothelial cells were embedded in a fibrin matrix and introduced into the vascular layer of the microfluidic platform.

After spontaneous formation of a vascular layer, Normal Human Astrocytes and Patient derived GSC were embedded in a Matrigel® matrix and incorporated in the stroma and tumor regions of the microfluidic device respectively.

Using the established platform, migration, proliferation and stemness of GSCs studies were conducted. The findings obtained indicate that astrocytes in the perivascular niche significantly increase the migratory and proliferative properties of GSCs in the tumor microenvironment, consistent with previous in vivo findings.

The novel GBM tumor microenvironment developed herein, could be utilized for further

in-depth cellular and molecular level studies to dissect the influence of individual factors within the tumor niche on GSCs biology, and could serve as a model for developing targeted therapies.

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Agent

Created

Date Created
2020

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Engineering of Synthetic DNA/RNA Modules for Manipulating Gene Expression and Circuit Dynamics

Description

Gene circuit engineering facilitates the discovery and understanding of fundamental biology and has been widely used in various biological applications. In synthetic biology, gene circuits are often constructed by two main strategies: either monocistronic or polycistronic constructions. The Latter architecture

Gene circuit engineering facilitates the discovery and understanding of fundamental biology and has been widely used in various biological applications. In synthetic biology, gene circuits are often constructed by two main strategies: either monocistronic or polycistronic constructions. The Latter architecture can be commonly found in prokaryotes, eukaryotes, and viruses and has been largely applied in gene circuit engineering. In this work, the effect of adjacent genes and noncoding regions are systematically investigated through the construction of batteries of gene circuits in diverse scenarios. Data-driven analysis yields a protein expression metric that strongly correlates with the features of adjacent transcriptional regions (ATRs). This novel mathematical tool helps the guide for circuit construction and has the implication for the design of synthetic ATRs to tune gene expression, illustrating its potential to facilitate engineering complex gene networks. The ability to tune RNA dynamics is greatly needed for biotech applications, including therapeutics and diagnostics. Diverse methods have been developed to tune gene expression through transcriptional or translational manipulation. Control of RNA stability/degradation is often overlooked and can be the lightweight alternative to regulate protein yields. To further extend the utility of engineered ATRs to regulate gene expression, a library of RNA modules named degradation-tuning RNAs (dtRNAs) are designed with the ability to form specific 5’ secondary structures prior to RBS. These modules can modulate transcript stability while having a minimal interference on translation initiation. Optimization of their functional structural features enables gene expression level to be tuned over a wide dynamic range. These engineered dtRNAs are capable of regulating gene circuit dynamics as well as noncoding RNA levels and can be further expanded into cell-free system for gene expression control in vitro. Finally, integrating dtRNA with synthetic toehold sensor enables improved paper-based viral diagnostics, illustrating the potential of using synthetic dtRNAs for biomedical applications.

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Agent

Created

Date Created
2020

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Investigating the Mechanism of a Multi-State Model of WNT Signaling

Description

The WNT signaling pathway plays numerous roles in development and maintenance of adult homeostasis. In concordance with it’s numerous roles, dysfunction of WNT signaling leads to a variety of human diseases ranging from developmental disorders to cancer. WNT signaling is

The WNT signaling pathway plays numerous roles in development and maintenance of adult homeostasis. In concordance with it’s numerous roles, dysfunction of WNT signaling leads to a variety of human diseases ranging from developmental disorders to cancer. WNT signaling is composed of a family of 19 WNT soluble secreted glycoproteins, which are evolutionarily conserved across all phyla of the animal kingdom. WNT ligands interact most commonly with a family of receptors known as frizzled (FZ) receptors, composed of 10 independent genes. Specific interactions between WNT proteins and FZ receptors are not well characterized and are known to be promiscuous, Traditionally canonical WNT signaling is described as a binary system in which WNT signaling is either off or on. In the ‘off’ state, in the absence of a WNT ligand, cytoplasmic β-catenin is continuously degraded by the action of the APC/Axin/GSK-3β destruction complex. In the ‘on’ state, when WNT binds to its Frizzled (Fz) receptor and LRP coreceptor, this protein destruction complex is disrupted, allowing β-catenin to translocate into the nucleus where it interacts with the DNA-bound T cell factor/lymphoid factor (TCF/LEF) family of proteins to regulate target gene expression. However in a variety of systems in development and disease canonical WNT signaling acts in a gradient fashion, suggesting more complex regulation of β-catenin transcriptional activity. As such, the traditional ‘binary’ view of WNT signaling does not clearly explain how this graded signal is transmitted intracellularly to control concentration-dependent changes in gene expression and cell identity. I have developed an in vitro human pluripotent stem cell (hPSC)-based model that recapitulates the same in vivo developmental effects of the WNT signaling gradient on the anterior-posterior (A/P) patterning of the neural tube observed during early development. Using RNA-seq and ChIP-seq I have characterized β-catenin binding at different levels of WNT signaling and identified different classes of β-catenin peaks that bind cis-regulatory elements to influence neural cell fate. This work expands the traditional binary view of canonical WNT signaling and illuminates WNT/β-catenin activity in other developmental and diseased contexts.

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Agent

Created

Date Created
2019

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Control of Tissue Homeostasis, Tumorigenesis, and Degeneration by Coupled Bidirectional Bistable Switches

Description

The Hippo-YAP/TAZ signaling pathway plays a critical role in tissue homeostasis, tumorigenesis, and degeneration disorders. The regulation of YAP/TAZ levels is controlled by a complex regulatory network, where several feedback loops have been identified. However, it remains elusive how these

The Hippo-YAP/TAZ signaling pathway plays a critical role in tissue homeostasis, tumorigenesis, and degeneration disorders. The regulation of YAP/TAZ levels is controlled by a complex regulatory network, where several feedback loops have been identified. However, it remains elusive how these feedback loops contain the YAP/TAZ levels and maintain the system in a healthy physiological state or trap the system into pathological conditions. Here, a mathematical model was developed to represent the YAP/TAZ regulatory network. Through theoretical analyses, three distinct states that designate the three physiological and pathological outcomes were found. The transition from the physiological state to the two pathological states is mechanistically controlled by coupled bidirectional bistable switches, which are robust to parametric variation and stochastic fluctuations at the molecular level. This work provides a mechanistic understanding of the regulation and dysregulation of YAP/TAZ levels in tissue state transitions.

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Date Created
2021