Matching Items (24)

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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 functionalities and structure. Sufficient amounts of correctly folded MPs are

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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2018-05

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Purification and Characterization of TRI 05 I13S M6I

Description

2,2’ bipyridine (Bpy) can form metal complexes with divalent metals in the form of [M(Bpy-ala)¬3]+2 where M is any divalent metal. These [M(Bpy-ala)¬3]+2 complexes can have very interesting photochemical and redox potentials that can be useful in more complex systems.

2,2’ bipyridine (Bpy) can form metal complexes with divalent metals in the form of [M(Bpy-ala)¬3]+2 where M is any divalent metal. These [M(Bpy-ala)¬3]+2 complexes can have very interesting photochemical and redox potentials that can be useful in more complex systems. The use of (2,2′-bipyridin-5yl)alanine (Bpy-ala) as a Noncanonical Amino Acid (NCAA) has allowed Bpy to be incorporated into an amino acid sequence which can now function in a protein scaffold. Previous studies have utilized that power of Bpy-ala to design a protein that can assemble a homotrimeric protein complex in the presence of a divalent metal. However, the issue with this design was that when the homotrimer was formed and the divalent was removed, the protein complex would not dissemble indicating that it was not metal dependent. Point mutations were made to disrupt the protein-protein interactions to favor disassembly in the absence of a divalent metal. Successfully, a mutation was made that allowed the designed protein to be metal dependent for self-assembly. Nevertheless, an issue with this design is that it poorly incorporated ruthenium(II) into the tris Bpy complex forming [Ru(Bpy-ala)¬3]+2, which was one of the main goals of the original design. This thesis sets out to form TRI 05 I13S M6I which should uphold the same metal-dependence as its predecessor and should combine ruthenium (II) into the protein complex forming [Ru(Bpy-ala)¬3]+2. The thesis shows the success of formation and expression of TRI 05 I13S M6I in Escherichia coli cells. This thesis also reports several purification steps and procedures to not only purify TRI 05 I13S M6I but also removing both the His-tag sequence and Fe(II) from the protein. The thesis also shows that TRI 05 I13S M6I does not behave like its predecessor in that it is not metal dependent for self-assembly. While this may be true, this paper also reports the incorporation of ruthenium (II) in the protein structure. Though this may be the first time that ruthenium (II) has been recorded to be in the TRI 05 protein complex with a significant signal, it is still nowhere near the optimal fluorescence that small molecule Bpy can achieve by itself. The thesis reports potential conditions and a plan of attack that should drive this project forward into achieving an optimal signal of the [Ru(Bpy-ala)¬3]+2 complex in a TRI 05 protein scaffold.

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2019-05

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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 in gene deficient yeast medias. The goal of the project

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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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 yeast without Sulp1 and Sulp2 is able to grow,

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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2019-05

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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 lags since their over-expression often leads to cell

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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2018-05

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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, it can be broken down before the therapeutic can be

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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2017-05

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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 synthesis method has a significant effect on nanoparticle size. By

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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2018-05

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Mass Transfer Phenomena for Novel Carbon Dioxide Adsorbents in Biocompatible Media

Description

Improvement in carbon capture percentage was calculated as most effective in 10 mg/L-MEA BG-11 media, with improvement in carbon capture of 1.012% over the control. In studying the effect of agitation at 150 revolutions-per-minute (RPM) with a magnetic stir bar,

Improvement in carbon capture percentage was calculated as most effective in 10 mg/L-MEA BG-11 media, with improvement in carbon capture of 1.012% over the control. In studying the effect of agitation at 150 revolutions-per-minute (RPM) with a magnetic stir bar, it was found that mass transfer actually decreased. Future investigations are warranted to fully characterize the effect of different alkanolamine types, concentrations, and mixing regimens on mass transfer of CO2. In this thesis, emphasis was placed on experimental setup to allow for a discussion of the unexpected characteristics of the findings of the mass transfer experiments. Understanding the effect of experimental setup on mass transfer will be important in designing more effective methods of CO2 absorption for improving growth of cyanobacteria.

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2019-05

Gasification of Municipal Solid Waste for Hydrogen Production

Description

A Study of the gasification of municipal solid waste (MSW) for hydrogen production was completed through research and statistical design of experiment. The study was done for general syngas production with conditions of high temperature and pressure. Waste samples from

A Study of the gasification of municipal solid waste (MSW) for hydrogen production was completed through research and statistical design of experiment. The study was done for general syngas production with conditions of high temperature and pressure. Waste samples from kitchen waste including rice, avocado, and egg shells were used. Dry orange blossom tree leaves were included and a very minimal fraction of used paper and Styrofoam. One of the components of the syngas predicted was hydrogen, but this study does not discuss techniques for the separation of the hydrogen from the syngas. A few suggestions, however, such as the use of gas chromatography and membranes are made for the study of the syngas and separation of the hydrogen from the syngas. A three level, three factors-half factorial design was used to analyze the impact of pressure, residence time and temperature on the gasification of MSW through a hydrothermal gasification approach. A series 4590 micro stirred reactor of 100mL was used to gasify MSW, but first, it was established through a TGA approach that the waste was about 5% moisture content and 55% organic content (OC). The TGA device used was the TG 209 F1 Libra. Results of the gasification indicated that the most important factor in the gasification of MSW is temperature, followed by residence time and that the syngas yield increases with a decreasing pressure of the system. A thermodynamic model relating the three factors and the syngas yield was developed.

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2019-05

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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 the past twenty years. The main area of difficulty is

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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2016-12