ISOLASI DNA PARSIAL GEN Tyrosinase-Related Protein-1 (TYRP1) PADA IKAN GURAME (Osphronemus gouramy)

Tyrosinase-Related Protein-1 (TYRP1) merupakan salah satu gen yang berperan dalam proses sintesis melanin. Gen TYRP1 memiliki peranan penting dalam menginstruksikan pembentukan enzim tyrosinase-related protein-1. Ekspresi gen TYRP1 dapat digunakan sebagai penanda kondisi stres pada ikan yang dapat disebabkan oleh faktor internal dan eskternal. Namun informasi genetik mengenai gen TYRP1 pada ikan gurame (Osphronemus gouramy) sangat minim informasi, sehingga pada penelitian ini bermaksud untuk mengisolasi dan mengarakterisasi gen TYRP1 pada ikan gurame. Amplifikasi gen TYRP1 pada DNA genom ikan gurame di lakukan dengan menggunakan dua pasang primer degenerate yang di rancang berdasarkan konsensus sikuen gen TYRP1 dari beberapa ikan dari infraclass Teleostei. Perancangan primer dilakukan secara online pada laman Primaclade dan Beacon Designer Free Edition. Primer terdiri dari set outer-primer dan inner-primer (nested), dimana outer-primer digunakan untuk memulai proses amplifikasi, kemudian produk amplifikasi yang tidak diharapkan dapat diminimalisir dengan keberadaan inner-primer. Analisa hasil sequencing dilakukan dengan BLAST, dan didapat hasil homologi pada ikan Takifugu rubripes (AF397401.1) dengan nilai kesamaan (Ident.) sebesar 76%. Sikuen DNA parsial gen TYRP1 ikan gurame dengan panjang 525 pb yang terdiri dari 36 pb daerah intron dan 489 pb daerah exon didapatkan dari hasil pensejajaran sikuen sampel dengan outer-primer dengan sikuen sampel dengan inner-primer menggunakan software Bioedit versi 7.2.5. Gen TYRP1 berhasil teramplifikasi dari DNA genom ikan gurame menggunakan dua pasang primer degenerate yang di rancang berdasarkan sikuen sekerabat ikan gurame dari infraclass Teleostei. Di lakukan perancangan primer spesifik gen TYRP1 ikan gurame dari sikuen daerah exon gen TYRP1 ikan gurame yang telah didapatkan. ---------- The Tyrosinase-Related Protein-1 (TYRP1) gene provides instructions for making an enzyme called tyrosinase-related protein-1 in the melanin biosynthesis pathway. TYRP1 enzyme function is to assist in the process of melanogenesis or synthesis melanin. TYRP1 gene expression can be used as a sign that the fish (and other vetebrates) under stress condition, either due to environmental disturbances or diseases. However the genetic information of the TYRP1 genes doesn’t exist in goramy (Osphronemus gouramy) or minimum shared information. In this study focus to isolate and characterize the TYRP1 genes on gouramy. Amplification of TYRP1 genes in the gouramy DNA genome performed using degenerate primers were designed based on sequence consensus TYRP1 genes of some Teleostei fish. Primer designed by Primaclade and to determining the secondary structure of primer using Beacon Designer Free Edition. First, used outer-primer for initial phase of amplification, and then non specifics products can be eliminated by nested-primer (inner-primer). Four sequences based on the results of sequencing performed contig and then obtained length 525 bp of DNA genom region. Intron and exon are exist in sequence. The identities percentage of sequence is 76 % with Takifugu rubripes (AF397401.1) in BLAST result. Gene sequences analysis obtained TYRP1 goramy exon region with sequence length 489 bp and 36 bp intron region. TYRP1 gene successfully amplified from the DNA genome of goramy using degenerate primers were designed based on sequences of Telesotei infraclass. Design of TYRP1 gene spesific primer on goramy was performed using exon region sequence

Direct measurement of single-molecule dynamics and reaction kinetics in confinement using time-resolved transmission electron microscopy

We report experimental methodologies utilising transmission electron microscopy (TEM) as an imaging tool for reaction kinetics at the single molecule level, in direct space and with spatiotemporal continuity. Using reactions of perchlorocoronene (PCC) in nanotubes of different diameters and at different temperatures, we found a period of molecular movement to precede the intermolecular addition of PCC, with a stronger dependence of the reaction rate on the nanotube diameter, controlling the local environments around molecules, than on the reaction temperature (−175, 23 or 400 °C). Once initiated, polymerisation of PCC follows zero-order reaction kinetics with the observed reaction cross section σobs of 1.13 × 10⁻⁹ nm2 (11.3 ± 0.6 barn), determined directly from time-resolved TEM image series acquired with a rate of 100 frames per second. Polymerisation was shown to proceed from a single point, with molecules reacting sequentially, as in a domino effect, due to the strict conformational requirement of the Diels–Alder cycloaddition creating the bottleneck for the reaction. The reaction mechanism was corroborated by correlating structures of reaction intermediates observed in TEM images, with molecular weights measured by using mass spectrometry (MS) when the same reaction was triggered by UV irradiation. The approaches developed in this study bring the imaging of chemical reactions at the single-molecule level closer to traditional concepts of chemistry

Subethnic interpersonal dynamic in diasporic community : a study on Chinese immigrants in Vancouver

201909 bcrcVersion of RecordPublishe

Counting molecules in nano test tubes: a method for determining the activation parameters of thermally driven reactions through direct imaging

A methodology for measuring activation parameters of a thermally driven chemical reaction by direct imaging and counting reactant molecules has been developed. The method combines the use of single walled carbon nanotubes (SWNTs) as a nano test tube, transmission electron microscopy (TEM) as an imaging tool, and a heating protocol that decouples the effect of the electron beam from the thermal activation. Polycyclic aromatic perchlorocoronene molecules are stable within SWNTs at room temperature, allowing imaging of individual molecules before and after each heating cycle between 500–600 °C. Polymerisation reaction rates can be determined at different temperatures simply by counting the number of molecules, resulting in an enthalpy of activation of 104 kJ mol−1 and very large entropic contributions to the Gibbs free energy of activation. This experimental methodology provides a link between reactions at the single-molecule level and macroscopic chemical kinetics parameters, through filming the chemical reaction in direct space

Single-molecule imaging and kinetic analysis of intermolecular polyoxometalate reactions

We induce and study reactions of polyoxometalate (POM) molecules, [PW12O40]3− (Keggin) and [P2W18O62]6− (Wells–Dawson), at the single-molecule level. Several identical carbon nanotubes aligned side by side within a bundle provided a platform for spatiotemporally resolved imaging of ca. 100 molecules encapsulated within the nanotubes by transmission electron microscopy (TEM). Due to the entrapment of POM molecules their proximity to one another is effectively controlled, limiting molecular motion in two dimensions but leaving the third dimension available for intermolecular reactions between pairs of neighbouring molecules. By coupling the information gained from high resolution structural and kinetics experiments via the variation of key imaging parameters in the TEM, we shed light on the reaction mechanism. The dissociation of W–O bonds, a key initial step of POM reactions, is revealed to be reversible by the kinetic analysis, followed by an irreversible bonding of POM molecules to their nearest neighbours, leading to a continuous tungsten oxide nanowire, which subsequently transforms into amorphous tungsten-rich clusters due to progressive loss of oxygen atoms. The overall intermolecular reaction can therefore be described as a step-wise reductive polycondensation of POM molecules, via an intermediate state of an oxide nanowire. Kinetic analysis enabled by controlled variation of the electron flux in TEM revealed the reaction to be highly flux-dependent, which leads to reaction rates too fast to follow under the standard TEM imaging conditions. Although this presents a challenge for traditional structural characterisation of POM molecules, we harness this effect by controlling the conditions around the molecules and tuning the imaging parameters in TEM, which combined with theoretical modelling and image simulation, can shed light on the atomistic mechanisms of the reactions of POMs. This approach, based on the direct space and real time chemical reaction analysis by TEM, adds a new method to the arsenal of single-molecule kinetics techniques