Obtaining a fluorescently labeled endophytic strain of bacteria Herbaspirillum lusitanum P6-12 for their detection in vivo and in vitro

The Herbaspirillum lusitanum P6-12 strain containing the vector plasmid pJN105TurboGFP, which encodes the synthesis of the green fluorescent protein GFP, and which has resistance to the antibiotic gentamicin, was obtained by electroporation. The constructed strain of H. lusitanum P6-12 in cultural, morphological and biochemical properties did not differ from the original typical natural strain of H. lusitanum P6-12. On solid growth media, the recombinant strain formed yellow-green colonies, fluorescent under UV irradiation. Upon inoculation with the resulting culture of plant objects, a green glow of the marked H. lusitanum P6-12 cells, actively colonizing the internal tissues of the host plant, was observed. The created strain can be used as a model strain for studying the patterns and characteristics of the behaviour of organisms in integrated systems, including for tracking bacterial cells during interaction with plants, assessing their survival, competitiveness, etc.

Influenza D pseudotyped lentiviruses: production, neutralisation assay and serological surveillance

Influenza D virus (IDV) has been reported in many animal species and potentially humans worldwide. Cattle are considered the major reservoir. There are currently three main lineages based on the haemagglutinin-esterase (HEF) gene: D/OK, D/660 and D/Japan. We performed pilot surveillance for IDV by using pseudotyped lentivirus (PVs) to generate a cell-based test to identify prior-exposure to IDV in animals. The expression plasmids of the HEF genes, D/swine/Italy/2015, D/bovine/France/2014, and D/bovine/Ibaraki/2016, were constructed. The HEF plasmid was co-transfected with lentiviral vector plasmid expressing luciferase, lentiviral Gag-Pol plasmid, and HAT protease plasmid in producer cells (HEK293T/17). Three days post-transfection, supernatants were collected and used for titration on various cell lines and in micro-neutralisation tests. Sera from pigs vaccinated with D/swine/Italy/2015 and D/swine/Oklahoma/2011 were used to undertake a preliminary validation of the micro-neutralisation assay. All pig sera have neutralising activity to influenza D (Italy) pseudotyped lentiviruses. Cow and sheep sera, 145 and 114 specimens, respectively, collected from UK farms were screened using the micro-neutralisation test. We found 97 bovine sera (66.9%) were influenza D antibody positive. Collectively, pseudotyped lentivirus technology opens up opportunities for serological surveillance of influenza D viruses.

TRANSFORMASI PLASMID YANG MENGANDUNG GEN merB PADA BAKTERI Escherichia coli TOP-10

ABSTRACT DNA transformation is the process of inserting recombinant DNA into host cells via vector plasmid. The host cell that is often used is TOP-10 Escherichia coli. The transformation method is widely used to transfer plasmids containing genetic material. This study aimed to evaluate the results of plasmid transformation containing the merB gene in the Escherichia coli TOP-10 bacteria. This study initiated with identification of the microbiology of host cells to be used, namely Escherichia coli TOP-10. Escherichia coli TOP-10 host cells were made into competent cells by transforming plasmids containing merB gene into Escherichia coli TOP-10 host cells using the heat shock method. The transformation results were evaluated by observing at the growth of Escherichia coli TOP-10 colonies on agar LB media containing ampicillin antibiotics. Plasmids on Escherichia coli TOP-10 were isolated and analyzed by 1% agarose gel electrophoresis. The results showed that the transformation of plasmids containing merB in Escherichia coli TOP-10 bacteria was successfully carried out as indicated by the growth of Escherichia coli TOP-10 bacteria on LB media agar containing ampicillin and the visualization on agarose gel resulted that the plasmid which carried the merB gene could be transformed in to the E. coli TOP-10 bacteria cell. Keywords: Transformation, Plasmids, merB genes, heat shocks, E. coli TOP-10ABSTRAK Transformasi DNA merupakan proses memasukkan DNA kedalam sel bakteri. Metode transformasi dipakai secara luas untuk mantransfer plasmid yang mengandung bahan genetika. Penelitian ini bertujuan untuk mengevaluasi hasil transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli TOP-10. Penelitian ini didahului dengan identifikasi secara mikrobiologi bakteri Escherichia coli TOP-10. Bakteri Escherichia coli TOP-10 dibuat menjadi sel kompeten yang digunakan sebagai inang. Selanjutnya dilakukan transformasi plasmid yang mengandung gen merB kedalam sel inang E. coli TOP-10 menggunakan metode heatshock. Hasil transformasi dievaluasi dengan melihat adanya koloni E. coli TOP-10 pada media LB agar yang mengandung antibiotik ampisilin. Plasmid pada E. coli TOP-10 diisolasi dan dianalisis dengan elektroforesis gel agarose 1%. Hasil penelitian menunjukkan bahwa transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli TOP-10 berhasil dilakukan, ditunjukkan dengan adanya pertumbuhan bakteri E. coli TOP-10 pada media LB agar yang mengangandung ampisilin dan hasil visualisasi pada agarose gel terlihat bahwa plasmid yang membawa gen merB dapat ditransformasikan ke dalam sel bakteri E. coli TOP-10. Kata kunci : Transformasi, Plasmid, gen merB, heatshock, E. coli TOP-10

Konstruksi Kandidat Gen AV1 Begomovirus pada pBI121 dan Introduksinya ke dalam Tembakau Menggunakan Vektor Agrobacterium tumefaciens

<p>Construction of Begomovirus AV1 Gene Candidate into<br />pBI121 and Its Introduction into Tobacco by using<br />Agrobacterium tumefaciens Vector. Tri J. Santoso,<br />Muhammad Herman, Sri H. Hidayat, Hajrial<br />Aswidinnoor, and Sudarsono. Infection of Begomovirus<br />has caused leaf curl disease in tomato. This infection has<br />significantly impact on yield losses of tomato production.<br />Recently, in Indonesia there was no effectively way to<br />control this disease. The use of resistant tomato variety is<br />one of strategies to control this virus. Genetic engineering<br />technology gives an opportunity to develop the transgenic<br />tomato resistant to Begomovirus through pathogen derived<br />resistance (PDR) approach. The objectives of this study<br />were to construct the Begomovirus AV1 candidate gene in<br />the pBI121 and to introduce the construct into tobacco plant<br />genome through Agrobacterium tumefaciens vector. A series<br />activites in gene construct have been conducted include<br />PCR amplification of AV1 gene using a pair of specific<br />primer, cloning the gene into pGEM-T easy, transformation of<br />the clone into Escherichia coli DH5α competent cell,<br />construct the gene into pBI121, and transform the construct<br />into A. tumefaciens. Leaf segments of in vitro tobacco plant<br />were transformed by co-cultivation with A. tumefaciens<br />containing ToLCV-AV1 construct. In the research activitiy,<br />Indonesian Begomovirus AV1 gene was successfully<br />amplified and inserted in expression vector plasmid pBI121.<br />Tobacco transformants carrying kanamycin-resistant gene<br />(nptII gene) were regenerated and established in the<br />glasshouse. Those transformant plants are expected<br />containing the AV1 gene.</p>