Matching Items (20)

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Cloning Sulfate Transporters in Growth Inhibited Yeast S. Cerevisiae

Description

The yeast project studies the growth of yeast Saccharomyces Cerevisiae (S. Cerevisiae) in high and low sulfate environments and analyzes the potential for genetically mutated plasmids to facilitate sulfate uptake

The yeast project studies the growth of yeast Saccharomyces Cerevisiae (S. Cerevisiae) in high and low sulfate environments and analyzes the potential for genetically mutated plasmids to facilitate sulfate uptake in gene deficient yeast medias. The goal of the project was to transform the Sul1 and Sul2 transporters into the nutrient deficient yeast strain BY4743 and observe growth in conditions that would otherwise prohibit growth in order to create a model that can be used to study the effect of sulfate concentration on the transporters. The experimental results showed that expressing the sulfate transporters in the BY4743 strain provided the potential for the yeast to grow in nutrient-poor media. The growth potential model allows for further analysis on the sulfate transporters and will be used for research projects going forward.

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  • 2019-05

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Polymeric Micelle Characterization and Stability for Drug Delivery Systems

Description

The current methods of drug delivery prove to have inefficiencies as far as drug administration to the target site. Due to adverse factors that the drug faces within the body,

The current methods of drug delivery prove to have inefficiencies as far as drug administration to the target site. Due to adverse factors that the drug faces within the body, it can be broken down before the therapeutic can be applied. Polymeric micelles have shown promising results in the face of these circumstances, by being able to self-assemble into a core-shell structure to better house the medicine as it travels through blood stream upon intravenous injection. The triblock copolymer, PEG-PPG-PEG, uses it hydrophilic and hydrophobic components to form a spherical micelle at a nanoscale size allowing it cross barriers with greater ease and prolong dissociation. The resulting size of the micelle is measured by the use of a dynamic light scattering machine. Stability factors, such as, thermodynamic and kinetic stability, also aid in the formation of micelles, but are generally effected in drug delivery process by factors such as salt concentration and pH. Both these factors can cause a lack of stability resulting in aggregation of the micelles; therefore, their affects need to be prolonged in order to have sufficient drug delivery.

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Date Created
  • 2017-05

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Optimizing the Expression of Therapeutic Antigen-Binding Fragments in E. coli

Description

The C6T single-chain variable fragment (scFv) is an antibody fragment designed as a potential Alzheimer’s therapeutic protein. However, this protein has been shown to be unstable and difficult to express

The C6T single-chain variable fragment (scFv) is an antibody fragment designed as a potential Alzheimer’s therapeutic protein. However, this protein has been shown to be unstable and difficult to express in E. coli. In this project, the C6T scFv is converted into an antigen-binding fragment (Fab), a larger and more stable antibody fragment. A C6T Fab sequence was derived from the scFv sequence, and a plasmid containing genes to express the Fab was constructed. Due to the disulfide-bonded structure of Fabs, the protein needs to be exported to the periplasm to properly fold. Therefore, the stII post-translational periplasmic secretion signal sequence was built into the expression vector, preceding both the heavy and light chain of the C6T Fab. The plasmid was transformed and expressed in BW25113 E. coli cells. A polyhistidine-tag was added to the Fab and it was purified on a nickel bead column. Protein characterization demonstrated that the correct Fab was produced.
Efforts were then made to optimize the expression of the C6T Fab in E. coli. Both the periplasmic secretion pathway and the effect of trigger factor were tested. Four expression systems were tested, consisting of one of two signal sequences (either DsbA directing through the SRP-dependent co-translational pathway or stII directing through the sec-dependent post-translational pathway) and one of two expression strains (BW25113 (tig+) containing trigger factor and KTD101 (Δtig) lacking trigger factor). Plasmids were constructed allowing the C6T Fab to be expressed and secreted using both pathways, and transformed into both strains. It was predicted that the protein expression could be optimized by employing the co-translational pathway in cells lacking trigger factor (i.e. the Δtig-DsbA expression system). However, this system severely decreased cell growth post-induction. It was found that both the lack of trigger factor and the employment of the co-translational pathway both significantly decrease cell growth post-induction. It is theorized that the increase in protein expression and secretion rate stresses the cell to a point where it is unable to maintain normal cell function and growth.

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  • 2016-12

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Enhancing the Expression Levels of Fabs in Escherichia coli

Description

Enhancing the expression levels of Fabs (antigen-binding antibody fragments) in Escherichia coli is a difficult field that has a variety of potential exciting implications. The field has grown substantially in

Enhancing the expression levels of Fabs (antigen-binding antibody fragments) in Escherichia coli is a difficult field that has a variety of potential exciting implications. The field has grown substantially in the past twenty years. The main area of difficulty is facilitating the entry of the antibody fragments into the periplasm of E. Coli, where the antibody fragments can be successfully expressed. Entry into the periplasm is difficult for antibody fragments due to their inability to fold in any other section besides the periplasm. Therefore it is necessary for the antibody to enter the periplasm in an unfolded state. Background research was done into inspecting the three primary methods of periplasmic entry: the Sec-dependent pathway, the SRP-dependent pathway (signal recognition particle) and the TAT-dependent pathway (twin arginine translocase). The Sec-dependent and SRP-dependent pathways were deemed more viable for expressing antibodies due to their ability to transfer an unfolded protein into the periplasm, which the TAT-dependent pathway cannot do. Academic research showed that the Sec-dependent and SRP-dependent pathways were equally viable methods, with more research being done into the Sec-dependent pathway, particularly of the OmpA signal sequence. Physical experiments were done using typical cloning procedures with slight modifications to the ligation step (Gibson Assembly was performed instead of normal ligation). These physical experiments showed that the Sec-dependent and SRP-dependent pathways were equally viable methods of periplasmic entry. The A4 and C6 antibodies were successfully expressed using these pathways. These antibodies were expressed on an SDS gel using 10% SDS. It was hypothesized that with further experimental modifications, using different signal sequences, Fabs can be expressed at higher and more consistent level.

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Date Created
  • 2016-12

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Methods to Enhance Overexpression of Membrane Protein in Escherichia coli

Description

Membrane proteins (MPs) are an important aspect of cell survival that ensure structural integrity, signaling, and transportation of molecules. Since 2015, over 450 MPs have been studied to find their

Membrane proteins (MPs) are an important aspect of cell survival that ensure structural integrity, signaling, and transportation of molecules. Since 2015, over 450 MPs have been studied to find their functionalities and structure. Sufficient amounts of correctly folded MPs are needed to accurately study them through crystallography and other structural study methods. Use of recombinant technology is needed to overexpress MPs as natural abundance of MP is often too slow to provide the necessary amounts. However, an increase in toxicity and decrease in generation time deter the overexpression of MPs. The following report discusses two methods of enhancing overexpression in Escherchia coli, the use of T7 RNA polymerase (T7RNAP) and the reprogramming of chaperon pathways, that combats toxicity and promotes cell growth. Overall, both methods are proven to work effectively to overexpress MPs by regulating transcription rate of mRNA (T7RNAP) or folding and transporting of polypeptides to inner membrane (chaperon pathway). To further study the effectiveness of the two methods, they will need to be compared at the same conditions. In addition, a combination of two methods should also be studied to find out if the combination would have a great impact on the overexpression of the MPs.

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Date Created
  • 2018-05

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Modulating the Heat Shock Response in E. coli to Optimize Membrane Protein Expression

Description

Membrane proteins are essential for cell survival and show potential as pharmacological and therapeutic targets in the field of nanobiotechnology.[1,2] In spite of their promise in these fields, research surrounding

Membrane proteins are essential for cell survival and show potential as pharmacological and therapeutic targets in the field of nanobiotechnology.[1,2] In spite of their promise in these fields, research surrounding membrane proteins lags since their over-expression often leads to cell toxicity and death.[3,4] It was hypothesized that membrane protein expression could be regulated and optimized by modifying the heat shock response of Escherichia coli (E. coli). To test this hypothesis, the membrane protein expression pathway was reprogrammed using gene-blocks that were antisense to vital membrane protein DNA and RNA binding-site sequences and included an IbpA-σ32 heat shock promoter. Anti-PBAD and anti-HtdR gene-blocks were designed to have antisense sequences to the DNA of the arabinose PBAD promotor and Haloterrigena turkmenica deltarhodopsin (HtdR) transmembrane protein respectively. These sequences were then employed to be cloned into a pMM102 vector and grown in NEB-5α E. coli cells.

Stable glycerol stocks of the pIbpA-antiPBAD and pIbpA-antiHtdR in BW25113 cells with either a pBLN200 or pHtdR200 plasmid were created. Then after inducing the cells with L-arabinose and 10mM all-trans retinal to allow for membrane protein expression, spectrophotometry was used to test the optical density of the cells at an absorbance of 600nm. Although general trends showed that the pHtdR200-pMM102 and pHtdR200-pIbpA cells had lower optical densities than the pBLN200 cells of all types, the results were determined to be statistically insignificant. Continuing, the pHtdR200 cells of all types showed a purple phenotype when spun down, as expected, while the cells with the pBLN200 plasmid had a colorless phenotype in pellet form. Further work will include cloning a GFP gene-block to test the ability of the anti-PBAD sequence in tuning the transcription of the GFP protein.

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Date Created
  • 2018-05

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Protein-mediated Synthesis of Gold Nanoparticles

Description

Gold nanoparticles are valuable for their distinct properties and nanotechnology applications. Because their properties are controlled in part by nanoparticle size, manipulation of synthesis method is vital, since the chosen

Gold nanoparticles are valuable for their distinct properties and nanotechnology applications. Because their properties are controlled in part by nanoparticle size, manipulation of synthesis method is vital, since the chosen synthesis method has a significant effect on nanoparticle size. By aiding mediating synthesis with proteins, unique nanoparticle structures can form, which open new possibilities for potential applications. Furthermore, protein-mediated synthesis favors conditions that are more environmentally and biologically friendly than traditional synthesis methods. Thus far, gold particles have been synthesized through mediation with jack bean urease (JBU) and para mercaptobenzoic acid (p-MBA). Nanoparticles synthesized with JBU were 80-90nm diameter in size, while those mediated by p-MBA were revealed by TEM to have a size between 1-3 nm, which was consistent with the expectation based on the black-red color of solution. Future trials will feature replacement of p-MBA by amino acids of similar structure, followed by peptides containing similarly structured amino acids.

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Date Created
  • 2018-05

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Characterization of Designed Ankyrin Repeat Proteins for Neurodegenerative Disease Diagnostics

Description

The following paper discusses the potential for Designed Ankyrin Repeat Proteins (DARPin) use as a diagnostic tool for neurodegenerative diseases in particular Alzheimer's disease (AD) and Parkinson's disease (PD). The

The following paper discusses the potential for Designed Ankyrin Repeat Proteins (DARPin) use as a diagnostic tool for neurodegenerative diseases in particular Alzheimer's disease (AD) and Parkinson's disease (PD). The two structures investigated for AD and PD were ADC7 and PDC1. Plasmid transformation was performed in order to grow the DARPin in E. coli for simple expression. Following growth and purification the proteins were validated using SDS-PAGE, Western Blot, BCA and indirect sandwich ELISA using transgenic mouse brain tissue. Targeted functionality of the DARPin structure was utilized during characterization methods to ensure the efficacy of the protein as a diagnostic for the respective disease targets. Both the ADC7 and PDC1 demonstrated improved binding with transgenic mice compared to wild type with a maximum 1.8 and 1.7 relative ratio, respectively. Additionally, both of the proteins demonstrated exclusive binding to their disease target and did not provide false positive results.

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Date Created
  • 2016-12

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Plasmid construction for the overexpression of waste biomass-degrading enzymes

Description

Lignin is a naturally abundant source of aromatic carbon but is largely underutilized in industry because it is difficult to decompose. Under the current study we engineered Corynebacterium glutamicum for

Lignin is a naturally abundant source of aromatic carbon but is largely underutilized in industry because it is difficult to decompose. Under the current study we engineered Corynebacterium glutamicum for the depolymerization of lignin with the goal of using it as raw feed for the sustainable production of valuable chemicals. C. glutamicum is a standout candidate for the depolymerization and assimilation of lignin because of its performance as an industrial producer of amino acids, resistance to aromatic compounds in lignin, and low extracellular protease activity. Three different foreign and native ligninolytic enzymes were tested in combination with three signal peptides to assess lignin degradation efficacy. At this stage, six of the nine plasmid constructs have been constructed.

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Date Created
  • 2021-05

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S. Cerevisiae Sul1 and Sul2 Sulfate Transporters in Varying Sulfate Concentrations

Description

The primary objective of this project is to further the knowledge about SCL26 family of anion transporters. The goals of the experiment were to find the lowest sulfate concentration where

The primary objective of this project is to further the knowledge about SCL26 family of anion transporters. The goals of the experiment were to find the lowest sulfate concentration where the yeast without Sulp1 and Sulp2 is able to grow, but it grows very slowly, and to find a higher sulfate concentration where the yeast grows quickly, with or without the sulfate transporters. The lowest sulfate concentration where the yeast without the sulfate transporters is able to grow was determined to be 2-4 mM, however, this range can likely be refined by more quantitative analytical methods. At a sulfate concentration of 20 mM sulfate or higher, the yeast is able to grow quickly without high-affinity sulfate transporters. The next step in the project is to re-introduce the Sulp1 and Sulp2 genes into the yeast, so that growth in low and high sulfate conditions can be compared with and without the Sulp1 and Sulp2 proteins. The long-term goals of the project are to bring experience with yeast to Dr. Nannenga’s structural discovery lab, to determine if yeast sulfate transporters respond in the same way to drug candidates as human sulfate transporters, and to determine the structure of the proteins using cryo-electron microscopy.

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Date Created
  • 2019-05