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Biomedical Engineering and Bioengineering Commons™
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- Clostridium thermocellum (6)
- Genetics (4)
- Cellulosome (3)
- Ethanol (3)
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- Metabolism (3)
- Adenosine triphosphate (2)
- Anaerobic (2)
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- Thermoanaerobacterium (2)
- 4-β-xylanase (1)
- Alcohol dehydrogenase (1)
- Alpha-Mannosidase (1)
- Artificial evolution (1)
- Aspergillus (1)
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- Bacterial proteins (1)
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Articles 1 - 16 of 16
Full-Text Articles in Biomedical Engineering and Bioengineering
Using Paired-Agent Imaging To Track Changes In Head And Neck Cancers After Undergoing Photodynamic Therapy Treatment, Reeham M. Choudhury
Using Paired-Agent Imaging To Track Changes In Head And Neck Cancers After Undergoing Photodynamic Therapy Treatment, Reeham M. Choudhury
Dartmouth College Master’s Theses
Head and neck cancers affect thousands of people across the world, and photodynamic therapy (PDT) has been shown to have great potential to treat said cancers in a noninvasive manner. However, imaging head and neck cancers has been difficult, and molecular changes caused by PDT are not well-understood. Therefore, we propose the use of paired-agent imaging (PAI) to track changes in these cancers after after PDT treatment. For these studies, we primarily used benzoporphyrin derivative monoacid (BPD) for our photosensitizer, which is activated by a 690 nm laser. We first looked at changes in EGFR expression in vitro, and …
Modeling The Effect Of Igg Subclasses And Specificity In The Translocation Of Monoclonal Antibodies Across The Placental Barrier, Sayuri Tais Miyamoto Magnabosco
Modeling The Effect Of Igg Subclasses And Specificity In The Translocation Of Monoclonal Antibodies Across The Placental Barrier, Sayuri Tais Miyamoto Magnabosco
ENGS 88 Honors Thesis (AB Students)
Infections are responsible for over half a million neonatal deaths every year (Lawn et al., 2014). Thus, there is huge interest in leveraging maternal immunization against infectious diseases to grant fetal protection during its development through the vertical transferring of IgG antibodies, the only Ig subclass that can significantly cross the placental barrier. Studies about vertical immunization rely on in-vitro models to extrapolate physiological conditions of the human placenta. The BeWo Transwell model (Bode et al., 2006) presents itself as a reliable model to mimic the transplacental transport mechanism of antibodies (Ellinger et al., 1999; Poulsen et al., 2009) …
Bio Circuits For Evolutionary Biotech, Camilo Toruno
Bio Circuits For Evolutionary Biotech, Camilo Toruno
ENGS 86 Independent Projects (AB Students)
The field of bioengineering has much promise for renewable chemical production, bioremediation, and of course medical applications. Developing new useful microorganisms is extremely time and capital intensive, typically taking 50 million USD and eight years. This is due in large part to the low throughput techniques that are characteristic of the field of metabolic engineering. Here we describe the modification of an existing synthetic biosensor to measure the pharmaceutical dopamine, and the use of a circuit simulator Cadence to predict improvements to the biosensor. This biosensor paired with directed evolution techniques could reach throughputs of 5 million cells per day …
Developing A Control System To Better Understand The Effects Of Pyruvate Decarboxylase Activity On Clostridium Thermocellum Metabolism, Nicholas Cervenka
Developing A Control System To Better Understand The Effects Of Pyruvate Decarboxylase Activity On Clostridium Thermocellum Metabolism, Nicholas Cervenka
ENGS 88 Honors Thesis (AB Students)
In order for cellulosic biofuels from Clostridium thermocellum to be commercially viable, the ethanol yield and titer of the microbe must be increased. To accomplish this, it has been suggested to introduce the Pyruvate Decarboxylase (PDC) enzyme into C. thermocellum. In order to demonstrate effects on ethanol production by PDC prior to genetic modification, a cell free system (CFS) has been developed. A purified enzyme system was developed with the CFS to function as a control. Using the purified enzyme system, PDC from Saccharomyces cerevisiae was demonstrated to be a good candidate for further testing in the CFS.
Atypical Glycolysis In Clostridium Thermocellum, Jilai Zhou, Daniel G. Olson, D. Aaron Argyros, Yu Deng, Walter M. Van Gulik, Johannes P. Van Dijken, Lee R. Lynd
Atypical Glycolysis In Clostridium Thermocellum, Jilai Zhou, Daniel G. Olson, D. Aaron Argyros, Yu Deng, Walter M. Van Gulik, Johannes P. Van Dijken, Lee R. Lynd
Dartmouth Scholarship
Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did …
Characterization Of Xylan Utilization And Discovery Of A New Endoxylanase In Thermoanaerobacterium Saccharolyticum Through Targeted Gene Deletions, Kara K. Podkaminer, Adam M. Guss, Heather L. Trajano, David A. Hogsett, Lee R. Lynd
Characterization Of Xylan Utilization And Discovery Of A New Endoxylanase In Thermoanaerobacterium Saccharolyticum Through Targeted Gene Deletions, Kara K. Podkaminer, Adam M. Guss, Heather L. Trajano, David A. Hogsett, Lee R. Lynd
Dartmouth Scholarship
The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C5 and C6 sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of …
Ethanol And Anaerobic Conditions Reversibly Inhibit Commercial Cellulase Activity In Thermophilic Simultaneous Saccharification And Fermentation (Tssf), Kara K. Podkaminer, William R. Kenealy, Christopher D. Herring, David A. Hogsett, Lee R. Lynd
Ethanol And Anaerobic Conditions Reversibly Inhibit Commercial Cellulase Activity In Thermophilic Simultaneous Saccharification And Fermentation (Tssf), Kara K. Podkaminer, William R. Kenealy, Christopher D. Herring, David A. Hogsett, Lee R. Lynd
Dartmouth Scholarship
A previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study.
Computational Design And Characterization Of A Temperature-Sensitive Plasmid Replicon For Gram Positive Thermophiles, Daniel G. Olson, Lee R. Lynd
Computational Design And Characterization Of A Temperature-Sensitive Plasmid Replicon For Gram Positive Thermophiles, Daniel G. Olson, Lee R. Lynd
Dartmouth Scholarship
Temperature-sensitive (Ts) plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out …
Complete Genome Sequence Of Clostridium Clariflavum Dsm 19732, Javier A. Izquierdo, Lynne Goodwin, Karen W. Davenport, Hazuki Teshima
Complete Genome Sequence Of Clostridium Clariflavum Dsm 19732, Javier A. Izquierdo, Lynne Goodwin, Karen W. Davenport, Hazuki Teshima
Dartmouth Scholarship
Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 protein-coding and 98 RNA genes, for the type strain DSM 19732.
Enhanced Microbial Utilization Of Recalcitrant Cellulose By An Ex Vivo Cellulosome-Microbe Complex, Chun You, Xiao-Zhou Zhang, Noppadon Sathitsuksanoh, Lee R. Lynd
Enhanced Microbial Utilization Of Recalcitrant Cellulose By An Ex Vivo Cellulosome-Microbe Complex, Chun You, Xiao-Zhou Zhang, Noppadon Sathitsuksanoh, Lee R. Lynd
Dartmouth Scholarship
A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high accessibility regenerated amorphous cellulose (RAC). The cellbound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely …
Mutant Alcohol Dehydrogenase Leads To Improved Ethanol Tolerance In Clostridium Thermocellum, Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks
Mutant Alcohol Dehydrogenase Leads To Improved Ethanol Tolerance In Clostridium Thermocellum, Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks
Dartmouth Scholarship
Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. …
Deletion Of The Cel48s Cellulase From Clostridium Thermocellum, Daniel G. Olson, Shital A. Tripathi, Richard J. Giannone, Jonathan Lo, Nicky C. Caiazza, David A. Hogsett, Robert L. Hettich, Adam M. Guss, Genia Dubrovsky, Lee R. Lynd
Deletion Of The Cel48s Cellulase From Clostridium Thermocellum, Daniel G. Olson, Shital A. Tripathi, Richard J. Giannone, Jonathan Lo, Nicky C. Caiazza, David A. Hogsett, Robert L. Hettich, Adam M. Guss, Genia Dubrovsky, Lee R. Lynd
Dartmouth Scholarship
Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose with the aid of a multienzyme cellulosome complex. Creation of knockout mutants for Cel48S (also known as CelS, S(S), and S8), the most abundant cellulosome subunit, was undertaken to gain insight into its role in enzymatic and microbial cellulose solubilization. Cultures of the Cel48S deletion mutant (S mutant) were able to completely solubilize 10 g/L crystalline cellulose. The cellulose hydrolysis rate of the S mutant strain was 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. The cellulosome produced …
Development Of Pyrf-Based Genetic System For Targeted Gene Deletion In Clostridium Thermocellum And Creation Of A Pta Mutant, Shital A. Tripathi, Daniel G. Olson, D. Aaron Argyros, Bethany B. Miller, Trisha F. Barrett, Daniel M. Murphy, Jesse D. Mccool, Anne K. Warner, Vineet B. Rajgarhia, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza
Development Of Pyrf-Based Genetic System For Targeted Gene Deletion In Clostridium Thermocellum And Creation Of A Pta Mutant, Shital A. Tripathi, Daniel G. Olson, D. Aaron Argyros, Bethany B. Miller, Trisha F. Barrett, Daniel M. Murphy, Jesse D. Mccool, Anne K. Warner, Vineet B. Rajgarhia, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza
Dartmouth Scholarship
We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes …
Metabolic Engineering Of A Thermophilic Bacterium To Produce Ethanol At High Yield, A. Joe Shaw, Kara K. Podkaminer, Sunil G. Desai, John S. Bardsley, Stephen R. Rogers, Philip G. Thorne, David A. Hogsett, Lee R. Lynd
Metabolic Engineering Of A Thermophilic Bacterium To Produce Ethanol At High Yield, A. Joe Shaw, Kara K. Podkaminer, Sunil G. Desai, John S. Bardsley, Stephen R. Rogers, Philip G. Thorne, David A. Hogsett, Lee R. Lynd
Dartmouth Scholarship
We report engineering Thermoanaerobacterium saccharolyticum, a thermophilic anaerobic bacterium that ferments xylan and biomass-derived sugars, to produce ethanol at high yield. Knockout of genes involved in organic acid formation (acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase) resulted in a strain able to produce ethanol as the only detectable organic product and substantial changes in electron flow relative to the wild type. Ethanol formation in the engineered strain (ALK2) utilizes pyruvate:ferredoxin oxidoreductase with electrons transferred from ferredoxin to NAD(P), a pathway different from that in previously described microbes with a homoethanol fermentation. The homoethanologenic phenotype was stable for >150 generations …
N-Glycan Modification In Aspergillus Species, Elke Kainz, Andreas Gallmetzer, Christian Hatzl, Juergen H. Nett, Huijuan Li, Thorsten Schinko, Robert Pachlinger, Harald Berger, Yazmid Reyes-Dominguez, Andreas Bernreiter, Tillmann Gerngross, Stefan Wildt, Joseph Strauss
N-Glycan Modification In Aspergillus Species, Elke Kainz, Andreas Gallmetzer, Christian Hatzl, Juergen H. Nett, Huijuan Li, Thorsten Schinko, Robert Pachlinger, Harald Berger, Yazmid Reyes-Dominguez, Andreas Bernreiter, Tillmann Gerngross, Stefan Wildt, Joseph Strauss
Dartmouth Scholarship
The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. . This strategy allowed the isolation of a strain with a functional -1,2-mannosidase producing increased amounts of N-glycans of the Man 5 GlcNAc 2 type. This strain was further engineered by the introduction of …
Cellulose Utilization By Clostridium Thermocellum: Bioenergetics And Hydrolysis Product Assimilation, Yi-Heng P. Zhang, Lee R. Lynd
Cellulose Utilization By Clostridium Thermocellum: Bioenergetics And Hydrolysis Product Assimilation, Yi-Heng P. Zhang, Lee R. Lynd
Dartmouth Scholarship
The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, Y(X/ATP)True = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for G(ATP)P-T (ATP from phosphorolytic beta-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed beta-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-2)/n [corrected] (n = mean cellodextrin DP assimilated). A mean G(ATP)(P-T) value of 0.52 +/- 0.06 was calculated …