Rekayasa Bakteri Untuk Ternak Dan Manusia: Pembuatan Mutan Escherichia Coli Penghasil Protein Rekombinan

Protein rekombinan seperti vaksin, antibodi, hormon, dan obat-obatan, semakin dibutuhkan oleh ternak dan manusia. Hambatan utama untuk menghasilkan protein rekombinan pada Escherichia coli sebagai inang yang digunakan paling luas adalah degradasi oleh enzim proteolitik. Hal ini disebabkan karena E. coli memiliki sejumlah enzim proteolitik yang tersebar di dalam sitoplasmanya. Untuk itu, lebih dari 90% degradasi protein terjadi di dalam sitoplasmanya. Pada penelitian ini, peneliti telah menghasilkan mutant E. coli BW25113 yang tidak memiliki gen penyandi enzim protease dengan menggunakan kombinasi metode pengerusakan kromosom dan metode transduksi phage P1. Pembuatan mutan tersebut dimulai dengan pengerusakan gen penyandi enzim protease pada kromosom bakteri dengan produk PCR yang memiliki bagian yang homolog dengan gen target. Mutan-mutan yang dihasilkan kemudian digunakan untuk menghasilkan mutan ganda dengan metode Transduksi phage P1. Analisis fenotif dan genotif menunjukkan bahwa kombinasi kedua metode tersebut sangat efektif untuk membuat lebih dari satu mutasi pada E. coli. Untuk itu, mutan E. coli yang telah diperoleh akan sangat bermanfaat untuk menghasilkan aneka protein rekombinan untuk ternak dan manusia

The expression of the ubiquitin ligase SIAH2 (seven in absentia homolog 2) is mediated through gene copy number in breast cancer and is associated with a basal-like phenotype and p53 expression

Introduction: The seven in absentia homolog 2 (SIAH2) protein plays a significant role in the hypoxic response by regulating the abundance of hypoxia-inducible factor-α; however, its role in breast carcinoma is unclear. We investigated the frequency and expression pattern of SIAH2 in two independent cohorts of sporadic breast cancers.Methods: Immunohistochemical evaluation of SIAH2protein expression was conducted in normal breast tissues and in tissue microarrays comprising ductal carcinoma in situ (DCIS) and a cohort of invasive breast carcinomas. Correlation analysis was performed between SIAH2 and clinicopathological variables and intrinsic breast cancer subgroups and validated in a cohort of 293 invasive ductal carcinomas. Promoter methylation, gene copy number and mRNA expression of SIAH2 were determined in a panel of basal-like tumors and cell lines.Results: There was a significant increase in nuclear SIAH2 expression from normal breast tissues through to DCIS and progression to invasive cancers. A significant inverse correlation was apparent between SIAH2 and estrogen receptor and progesterone receptor and a positive association with tumor grade, HER2, p53 and an intrinsic basal-like subtype. Logistic regression analysis confirmed the significant positive association between SIAH2 expression and the basal-like phenotype. No SIAH2 promoter methylation was identified, yet there was a significant correlation between SIAH2 mRNA and gene copy number. SIAH2-positive tumors were associated with a shorter relapse-free survival in univariate but not multivariate analysis.Conclusions: SIAH2 expression is upregulated in basal-like breast cancers via copy number changes and/or transcriptional activation by p53 and is likely to be partly responsible for the enhanced hypoxic drive through abrogation of the prolyl hydroxylases

Underwater Application of Quantitative PCR on an Ocean Mooring

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions

Thin-plate rectangular weir with inlet shaft

Diplomová práce je rozdělena na literární rešerši a praktickou část (vlastní měření). Obsahem literární rešerše je popis přepadu přes tenkostěnný pravoúhlý přeliv. V praktické části je popsán model, měřicí přístroje a vlastní měření. Výsledkem je vyhodnocení průběhu dolní i horní obálky paprsku, stanovení součinitele přepadu, součinitele relativní délky šachty a určení mezní hodnoty relativní délky šachty pro neovlivnění tvaru přepadového paprsku.The thesis is divided into literary research and practical part (own measurement). The content of the literary research is a description of the flow over thin-plate rectangular weir. The practical part is focused on a description of the model, measuring equipment and measurement. The results are evaluation of lower and upper surface of nappe, determination of discharge coefficient, coefficient of relative length of shaft, limit value of relative length of shaft for uninfluenced shape of nappe.

Development of a Deep-sea Hydrogen Sulfide Ion Sensor and its Application for Submarine Hydrothermal Plume Exploration

http://www.godac.jamstec.go.jp/darwin/cruise/yokosuka/yk09-08/

Single-molecule enzymatic analysis in a droplet-based microfluidic system

The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtolitre carriers to achieve single-molecule encapsulation. The tiny droplets (f~2.5-3 μm) generated from this fluidic system were highly monodisperse, beneficial for an analysis of single enzyme activity. Single-enzyme kinetics has previously been demonstrated in the microfluidic format in PDMS containers [1], surface-immobilized droplets [2], or liposomes [3]. Single- enzyme analysis in the droplet-based microfluidics was reported before [4] by encapsulating highly-diluted enzyme solution (110 fM) into large droplets (f~40 μm). But to our knowledge this is the first demonstration of the direct method of single enzyme encapsulation and analysis at high enzyme concentration in tiny droplets in a microfluidic system

Electrochemical analysis of microdroplet formation

This paper reports an electrochemical measurement system with a high-speed camera for observation of molecular transport phenomena at a water-oil (W/O) interface during microfluidic droplet formation. For demonstration of the system, currents corresponding to the transport of electrolyte ions to form the electrical double layer at the liquid interface were measured. Additionally, the high-speed camera observation revealed charge-effect on droplet stability during and/or just after the formation. This measurement system is expected to facilitate a full understanding of the droplet formation process