Kloning Gen Coat Protein (CP) Carnation Mottle Virus (CarMV) pada Vektor Ekspresi [Cloning of Carnation Mottle Virus (CarMV) Coat Protein Gene into Expression Vector]

<p>Carnation mottle virus (CarMV) termasuk anggota genus Carmovirus dalam famili Tombusviridae. Virus ini banyak ditemukan menginfeksi tanaman anyelir di Jawa Barat dan menyebabkan gejala mottle. Sebagai langkah awal untuk memproduksi antiserum melalui teknik ekspresi gen CP perlu diklon pada vektor yang sesuai. Penelitian ini bertujuan mendapatkan klon CarMV yang berfungsi melalui kloning dan subkloning gen CP CarMV ke dalam vektor ekspresi yang sesuai. Penelitian dilakukan dalam beberapa tahap, yaitu ekstraksi RNA total dan amplifikasi cDNA CarMV dengan RT-PCR, menggunakan primer spesifik CarMVF dan CarMVR yang mengandung situs enzim restriksi XhoI dan BamHI, kloning dan subkloning DNA sisipan, serta konfirmasi transforman. Rekombinan gen sisipan CP CarMV dalam bakteri dikonfirmasi dengan koloni PCR. Gen CP CarMV berhasil dikloning ke dalam TA vektor pTZ57R/T dan disubkloning ke vektor ekspresi pET28a. Sekuen rekombinan CP CarMV berhasil dikonfirmasi melalui perunutan DNA. Penelitian lebih lanjut diperlukan untuk mendapatkan produksi antigen rekombinan yang melimpah pada bakteri ekspresi dan kondisi yang sesuai.</p><p><strong>Keywords</strong></p><p>Dianthus caryophillus L.; Carmovirus; Kloning; Subkloning; Bakteri ekspresi</p><p><strong>Abstract</strong></p><p>Carnation mottle virus (CarMV) is a type member of Carmovirus genus in family of Tombusvirus. The virus infects carnation plants in the centre area production of West Java and it cause mottle symptoms. The research aimed to obtain functional clone(s) of CarMV CP gene in suitable expression kloning vector. The research was carried out through several steps, namely total RNA extraction and amplification of cDNA of CP CarMV by RT-PCR using specific primer pairs CarMVF and CarMVR containing restriction enzyme sites XhoI and BamHI, respectively, TA cloning, and subcloning into expression vector pET28a and confirmation of recombinant plasmids by colony PCR. CarMV CP gen was successfully cloned into TA cloning vector pTZ57R/T and subcloned into vector pET28a, alsowere confirmed by DNA sequencing. Future experiment is necessary to be conducted to obtain abundance recombinant antigen production of CarMV CP in suitable expression condition and bacterial host.</p>

So you want to express your protein in Escherichia coli?

Recombinant proteins have been extensively employed as therapeutics for the treatment of various critical and life-threatening diseases and as industrial enzymes in high-value industrial processes. Advances in genetic engineering and synthetic biology have broadened the horizon of heterologous protein production using multiple expression platforms. Selection of a suitable expression system depends on a variety of factors ranging from the physicochemical properties of the target protein to economic considerations. For more than 40 years, Escherichia coli has been an established organism of choice for protein production. This review aims to provide a stepwise approach for any researcher embarking on the journey of recombinant protein production in E. coli. We present an overview of the challenges associated with heterologous protein expression, fundamental considerations connected to the protein of interest (POI) and designing expression constructs, as well as insights into recently developed technologies that have contributed to this ever-growing field.

Język tekstów legislacyjnych i jego ocena normatywna – na przykładzie wybranych aktów prawnych

The article presents the results of a linguistic-stylistic analysis of selected three acts, posted on the official website of the Journal of the Polish Republic in November and December 2016. As pointed out by the author, in spite of the specific terminology and phraseology, legal language can not be considered as a separate code (which is, for example, the Polish ethnic language), because it has not developed its own grammar. Therefore, if the author speaks of the legal language, she means rather the legal style, understood as a variety of the special (functional) Polish general language, assessed from the perspective of the normative rules of a general language. The analysis has shown that the examined legislative texts contain primarily the syntax errors (57% of all errors), followed (in terms of the number of occurrences) by the errors regarding the other subsystems of language. In a total of 39 pages the author recorded 292 violations of the linguistic correctness. Despite revealing numerous imperfections of the studied writings, the results of the overall analysis proved them satisfactory. Negligible number of grammatical shortcomings, flawless spelling, small number of mistakes concerning the government and isolated cases of syntactic homonymy demonstrate good quality of the studied texts. However, the authors need to ensure greater accuracy in terms of formation, reduce the number of the troublesome participle clauses and multi-nominal constructions, use more suitable expression, and avoid the heavy style.

Transport engineering for improving production and secretion of valuable alkaloids in Escherichia coli

Metabolic engineering of microorganisms to produce specialized plant metabolites has been established. However, these methods are limited by low productivity and the intracellular accumulation of metabolites. Here, we aimed to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. We established a reticuline-producing Escherichia coli strain and introduced a multidrug and toxic compound extrusion transporter, Arabidopsis AtDTX1, into it. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 significantly enhanced reticuline production by 11-fold; produced reticuline was secreted into the medium. AtDTX1 expression conferred high plasmid stability, and up- or downregulated genes associated with biological processes including metabolic pathways for reticuline biosynthesis, leading to a high production and secretion of reticuline. The successful application of a transporter for alkaloid production suggests that the transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

A chromatogram-simplified Streptomyces albus host for heterologous production of natural products

Cloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign biosynthetic gene clusters by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.

A chromatogram-simplified Streptomyces albus host for heterologous production of natural products

Cloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign BGCs by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.

Sampling Associated with a Unitary Representation of a Semi-Direct Product of Groups: A Filter Bank Approach

An abstract sampling theory associated with a unitary representation of a countable discrete non abelian group G, which is a semi-direct product of groups, on a separable Hilbert space is studied. A suitable expression of the data samples, the use of a filter bank formalism and the corresponding frame analysis allow for fixing the mathematical problem to be solved: the search of appropriate dual frames for ℓ 2 ( G ) . An example involving crystallographic groups illustrates the obtained results by using either average or pointwise samples.

Pseudomonas aeruginosaesterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization

The human membrane-bound α/β-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein fromPseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence ofn-octyl β-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified fromEscherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space groupI4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.

STRATEGIES IN THE TRANSLATION OF REFUSAL EXPESSIONS IN JOHN GREEN’S PAPER TOWNS

This article examined about translation from English to Indonesian related to non-equivalence words on expression of refusal either directly or indirectly, and the using of translation strategies in adjusting non-equivalence. This study focused to expression of refusal either directly or indirectly in English on Paper Towns novel by John Green (2008) as a source of data. There are 33 total data, consists of 22 of expression of indirect refusal and 11 expression of direct refusal. Those data are analysed by descriptive qualitative method. The result shows that strategy used for solving non equivalence problem in this translation are seven (7): (1) translation by more general word, (2) translation by more neutral word, (3) translation by cultural substitution, (4) translation using a loan word, (5) translation by paraphrasing using a related word, (6) translation by paraphrasing using an unrelated word, and (7) translation by omission. From all those strategies, strategies five (5) is often used in the translation because there is no suitable expression in Indonesian from English.

Ethics and Literature

Moral philosophers typically assume that literary texts have no significance for ethical reflection beyond providing one possible source of examples of moral problems. This is in line with philosophy’s long-standing hostility towards literature; it is described as an ancient quarrel even by Plato, whose expulsion of the poets from his republic is often cited as a founding gesture of the discipline. However, some recent writers on ethics contest this assumption. Neo-Aristotelian theorists see the novel as a medium in which their approach finds its most suitable expression. Proponents of moral perfectionism see literary techniques as indispensable in achieving their desired relation to their readers. Others of no particular theoretical affiliation see literary texts as exemplifying the internal relation of reason to imagination, feeling and sensibility in fundamental modes of moral thought. Taken as a whole, these arguments suggest that a moral philosopher’s evaluation of the ethical significance of literature reveals fundamental features of their conception of ethics in general, of the relation between moral philosophy and other philosophical concerns or questions, and hence of philosophy itself.