Biotechnology Commons^™

Screening For The Suitable Signal Peptide For Secreted Nattokinase Expression In E. Coli Using An In Silico Approach: Precursor Vs. Mature Forms, Dewi Purwani Caya Ningsih, Kartika Sari Dewi, I Wayan Martadi Santika, Ade Andriani, Joko Pebrianto Trinugroho, Muhammad Yusuf

Makara Journal of Science

Nattokinase (NK), an enzyme with thrombolytic activity, has been widely used in treating cardiovascular disease. The expression of NK in the E. coli cell host is favored due to its several benefits, though it often results in inclusion bodies. Appropriate signal peptides can be attached to the N-terminus of a protein to initiate a secretory expression system, thus preventing the nascent protein from aggregating in the cytoplasm. This study aimed to identify suitable signal peptides for the secretory expression of active NK in E. coli and to investigate the impact of pre-protein in NK secretion using an in silico approach. …

Comparative Antibacterial Efficacy Of Povidone-Iodine, Citric Acid, And Starch: A Study Of Combined Activity And Interference Effects Against Clinically Relevant Bacteria, Falah Hasan Obayes Al-Khikani, Mustafa Mutar Trrad, Ali Abedulameer Alhusayni

AUIQ Complementary Biological System

Povidone-iodine and citric acid are widely used antimicrobial agents. However, data on their comparative efficacy and the functional outcomes of their combination, particularly regarding the influence of common excipients like starch, is limited. Understanding these interactions is crucial for optimizing antiseptic formulations. This study aimed to evaluate and compare the antibacterial activity of povidone-iodine, citric acid (5% and 20%), and starch (5% and 20%), both individually and in combination, against clinical isolates of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The antibacterial activity of the agents was assessed using an agar well diffusion assay. Mean inhibition zone …

Detection Method For Escherichia Coli Using Real-Time Polymerase Chain Reaction Targeting The Yhav Gene, Muktiningsih Nurjayadi, Anisa Fitriyanti, Royna Rahma Musie, Gusti Angieta Putri, Puan Aqila Azizah, Helzi Angelina, Grace Grace, Ananda Indah Putri Sihombing, Agus Setiawan, Jefferson Lynford Declan, Gladys Indira Putri, Dandy Akbar Juliansyah, Siti Fatimah, Ayu Berkahingrum, Irma Ratna Kartika, Fera Kurniadewi, Vira Saamia, Shyi-Tien Chen, Bassam Aboemolak, Hesham Ali El Enshasy

Makara Journal of Science

Escherichia coli is a foodborne pathogenic bacterium that can cause diarrhea, while yhaV is a virulence-associated gene linked to the toxin–antitoxin system in E. coli. This study was aimed at evaluating the confirmation, specificity, and sensitivity of a yhaV gene primer using real-time polymerase chain reaction. The yhaV-targeting PCR successfully amplified a DNA fragment with an amplicon length of 207 bp (base pairs) under an annealing temperature optimized to a range of 54 °C to 62 °C via gradient PCR. The PCR using the primer pair produced a consistent Ct (cycle threshold) of 14.14 ± 0.05 and showed …

Metabolite Analysis To Understand Factors Limiting Microbial Fermentation At High Substrate Concentrations, Bishal D. Sharma

Dartmouth College Ph.D Dissertations

Clostridium thermocellum, a thermophilic anaerobic bacterium, holds promise as a biocatalyst for conversion of lignocellulosic biomass into biofuels. This microbe has been subjected to various metabolic engineering strategies, including the deletion of genes encoding secondary fermentation products such as H₂, formate, acetate, and lactate, as well as the expression of heterologous genes to redirect carbon flux toward ethanol production. Despite these efforts, achieving economically viable ethanol titers remains a challenge. We hypothesized that studying how intracellular metabolite concentrations change as fermentation stops could reveal metabolic bottlenecks or regulatory mechanisms limiting ethanol production at high titers. However, metabolomics studies at …

Impact Of Some Parameters On The Survival And Proliferation Of Foodborne Pathogens: Escherichia Coli, Bacillus Subtilis, Staphylococcus Aureus, And Streptococcus Pyogenes, Hassan Agha, Ahmed Bashir Sanaalla, Muhanad Abdullah Salim Abdulsamad, Maroua Abderrahmane Salhi, Ghada Abdu-Razzaq Mohammad, Abdulmutalib Alabeed Alkamil Allaq

AUIQ Complementary Biological System

Food poisoning caused by foodborne microorganisms is a significant public health concern. This study aims to investigate the impact of several nutritional and environmental parameters on the growth of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Streptococc pyogenes in both model systems and food. The specific impact of temperature, pH, and UV light stress on pathogens.Applying physical stress on microorganisms is a standard method to induce cell inactivation and promote food stability.This study found that the optimal temperature for the growth of these pathogens is between 30°C and 50°C. No growth was observed beyond 50°C, indicating temperature sensitivity; low temperatures …

One-Step Production Of Fully Biotinylated And Glycosylated Human Fc Gamma Receptors, Minhyo Kang, Zening Wang, Xin Ge

Faculty, Staff and Student Publications

Initiating and regulating humoral immunity, Fc gamma receptors (FcγRs) have been identified both as therapeutics and as drug targets, and thus production of biologically active FcγRs is highly demanded for biopharmaceutical development. Focusing on low-affinity FcγRs IIA (131H/R allotypes), IIB, and IIIA (176F/V), this study used human 293-F cells to achieve correct post-translational modifications (PTMs) including biotinylation, N-glycosylation, and disulfides. Approaches involving co-expression of FcγR-AviTag and Escherichia coli biotin ligase BirA, endoplasmic reticulum retention, stable and transient transfections, and optimization of transgene ratio were investigated. Protein electrophoresis under reducing and non-reducing conditions, enzymatic deglycosylation, streptavidin pull-down assays, and binding kinetic …

Physiological Evidence For Isopotential Tunneling In The Electron Transport Chain Of Methane-Producing Archaea, Nikolas Duszenko, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per …

Contribution Of A Putative Up Element Dna Sequence To The Activity Of A Newly Identified Phage Promoter, Courtney Hamilton

Mahurin Honors College Capstone Experience/Thesis Projects

In transcription, a universal step in gene expression, information from a DNA sequence is copied into RNA. A key component in gene expression is the promoter sequence, a region of DNA to which RNA polymerase binds during the initiation of transcription of downstream genes. Most bacterial promoters contain a -10 and a -35 sequence that are bound by the RNA polymerase. Some promoters also contain an Upstream Promoter (UP) element. UP elements have been shown to boost promoter activity. We recently identified a new promoter in a mutant bacteriophage that grows on a bacterial host that prevents antitermination of phage …

Assessing Stress Responses To Atmospheric Cold Plasma Exposure Using Escherichia Coli Knock Out Mutants, Lu Han, Daniela Boehm, Sonal Patil, Patrick J. Cullen, Paula Bourke

Articles

Aim: This study investigated the effect of Atmospheric cold plasma (ACP) exposure induced stress on microbial inactivation patterns and the regulation of genes involved in the microbial stress response in conjunction with key processing parameters of exposure time and post treatment storage time.

Methods and Results: Cell suspensions of Escherichia coli BW 25113 and its isogenic knock-out mutants in rpoS, soxR, soxS, oxyR and dnaK genes were treated with high voltage ACP in a sealed package for 1, 3 and 5 min followed by 0, 1 and 24 h post-treatment storage. ROS densities and colony formation were …

Dcm Methylation Is Detrimental To Plasmid Transformation In Clostridium Thermocellum, Adam M. Guss, Daniel G. Olson, Nicky C. Caiazza, Lee R. Lynd

Dartmouth Scholarship

Background: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. Results: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam + dcm + E. coli strain, pAMG206 transforms C. thermocellum 100-fold better …

High-Temperature Fluorescent In Situ Hybridization For Detecting Escherichia Coli In Seawater Samples, Using Rrna-Targeted Oligonucleotide Probes And Flow Cytometry, Ying Zhong Tang, Karina Yew Hoong Gin, Tok Hoon Lim

OES Faculty Publications

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E. coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with …

Comparison Of Methods For Dna Isolation From Food Samples For Detection Of Shiga Toxin-Producing Escherichia Coli By Real-Time Pcr, Loree C. Heller, Carisa R. Davis, K. Kealy Peak, David Wingfield, Andrew C. Cannons, Philip T. Amuso, Jacqueline Cattani

Bioelectrics Publications

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Estimation Of Diversity And Community Structure Through Restriction-Fragment-Length-Polymorphism Distribution Analysis Of Bacterial 16s Ribosomal-Rna Genes From A Microbial Mat At An Active, Hydrothermal Vent System, Loihi Seamount, Hawaii, Craig L. Moyer, Fred C. Dobbs, David M. Karl

OES Faculty Publications

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete …