High-density culture of Escherichia coli carrying recombinant plasmid in a membrane cell recycle fermenter

Muscle stem cells (MuSCs) isolated ex vivo are essential original cells to produce cultured meat. Currently, one of the main obstacles for cultured meat production derives from the limited capacity of large-scale amplification of MuSCs, especially under high-density culture condition. Here, we show that at higher cell densities, proliferation and differentiation capacities of porcine MuSCs are impaired. We investigate the roles of Hippo-YAP signaling, which is important regulators in response to cell contact inhibition. Interestingly, abundant but not functional YAP proteins are accumulated in MuSCs seeded at high density. When treated with lysophosphatidic acid (LPA), the activator of YAP, porcine MuSCs exhibit increased proliferation and elevated differentiation potential compared with control cells. Moreover, constitutively active YAP with deactivated phosphorylation sites, but not intact YAP, promotes cell proliferation and stemness maintenance of MuSCs. Together, we reveal a potential molecular target that enables massive MuSCs expansion for large-scale cultured meat production under high-density condition.

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Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

KnE Life Sciences ◽

10.18502/kls.v3i5.986 ◽

2017 ◽

Vol 3 (5) ◽

pp. 139

Author(s):

Mariana Wahjudi ◽

Catherina . ◽

Nita Marcelia Wangunhardjo ◽

Ernest Suryadjaja ◽

Xavier Daniel

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Recombinant Plasmid ◽

Sodium Dodecyl ◽

Xylanase Activity ◽

Cellular Protein ◽

Host Cells ◽

Chromosomal Dna ◽

Clear Zone ◽

Sds Page

The xynB gene of Bacillus subtilis subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the xynB gene from Bacillus subtilis subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the xynB gene in Escherichia coli Origami as host cells. The xynB gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The xynB gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for xynB gene and for the vector, then continued by restriction analyses. The result showed that transformants clone 9 and 10 bear the recombinant pMMB-xynB plasmid. The xylanase activity of xynB gene in Escherichia coli Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-xynB recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the xynB gene of Bacillus subtilis subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the Escherichia coli Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.

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Cloning and expression of Human Papilloma virus type 16 L1 capsid protein in bacteria

Health Science Journal of Indonesia ◽

10.22435/hsji.v12i2.2442 ◽

2019 ◽

Vol 10 (2) ◽

pp. 82-89

Author(s):

Silvia Tri Widyaningtyas ◽

Sofy Meilany ◽

Budiman Bela

Keyword(s):

Escherichia Coli ◽

Cervical Cancer ◽

Human Papillomavirus ◽

Recombinant Protein ◽

Capsid Protein ◽

Recombinant Plasmid ◽

Health Science ◽

Science Journal ◽

Hpv 16 ◽

L1 Protein

Latar belakang: Secara alamiah protein kapsid L1 Human Papillomavirus (HPV) tipe 16 dapat mengalami auto assembly untuk membentuk Viral like particle (VLP). Terkait dengan penelitian vaksin HPV, VLP dapat digunakan untuk berbagai keperluan seperti vaksin, pseudovirion atau SpyTag-Spycatcher. Penelitian ini ditujukan untuk mendapatkan plasmid rekombinan yang digunakan untuk produksi protein L1 HPV 16. Metode: Gen penyandi protein L1 HPV 16 diklona ke dalam vector pQE80L, suatu plasmid yang mengandung sistem ekspresi untuk prokariota. DNA penyandi HPV 16 L1 disisipkan pada situs restriksi BamHI dan Hind III plasmid pQE80L. Plasmid rekombinan yang mengandung gen L1 HPV 16dikonfirmasi menggunakan PCR dan analisis enzim restriksi. Lebih lanjut untuk memastikan bahwa gen rekombinan L1 HPV 16 dapat diekspresikan dalam prokariota, plasmid rekombinan ditransformasikan ke bakteri Escherichia coli BL21 (DE3). Bakteri diinduksi dengan Isopropyl β-D-1-thiogalactopyranoside (IPTG) dengan berbagai konsentrasi dan berbagai waktu inkubasi. Hasil: protein rekombinan L1, berat 56 kDa, telah berhasil diekspresikan dalam sistem prokariota. Protein rekombinan L1 dapat dimurnikan menggunakan TalonR dalam kondisi denaturasi. Kesimpulan: gen L1 HPV 16 telah dikloning ke dalam pQE80L dan berhasil diekspresikan dalam sistem prokariota. (Health Science Journal of Indonesia 2019;10(2):82-9) Kata kunci: L1, HPV 16, cervical cancer Abstract Background: Naturally Human Papillomavirus (HPV) type 16 L1 capsid protein can auto assemble to form Viral like particles (VLP). Concerning to vaccine development for HPV, VLP can be used for a variety of needs such as a vaccine, pseudovirion or SpyTag-Spycatcher. In this study, to obtain a vector expression that can be used in the production of HPV L1 protein, we cloned gene coding HPV 16 L1 protein into pQE80L a plasmid contains an expression system for prokaryote. Methods: The DNA coding HPV 16 L1 was inserted at BamHI and Hind III restriction sites of pQE80L plasmid. The recombinant plasmid containing the HPV L1 gene was confirmed using PCR colony and enzyme restriction. Further to ensure the recombinant HPV 16 L1 gene could be expressed in a prokaryote, the recombinant plasmid was transformed into bacteria Escherichia coli BL21 (DE3). The bacteria were induced with IPTG with various concentrations and various incubation time. Result: L1 recombinant protein, 56 kDa in weight, has successfully been expressed in prokaryote system. L1 recombinant protein can be purified using TalonR under denaturing conditions. Conclusion: L1 HPV 16 gene has been cloned into pQE80L and successfully expressed in prokaryote system. (Health Science Journal of Indonesia 2019;10(2):82-9) Keywords: L1, HPV 16, cervical cancer

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Hyper-intensive farming white shrimp Litopenaeus vannamei (Decapoda: Penaeidae) in a seawater tank under semi-controlled conditions

UNED Research Journal ◽

10.22458/urj.v5i1.249 ◽

2013 ◽

Vol 5 (1) ◽

pp. 69-74

Author(s):

A. A. Ortega-Salas ◽

L. A. Rendón M.

Keyword(s):

Litopenaeus Vannamei ◽

Maximum Likelihood Method ◽

High Density ◽

White Shrimp ◽

Likelihood Method ◽

Food Conversion ◽

High Density Culture ◽

Intensive Farming ◽

Controlled Conditions ◽

Commercial Size

Shrimp development to a commercial size in high density culture saves food and avoids predators and disease. Our study was conducted to calculate the growth of white shrimp Litopenaeus vannamei by hyper-intensive cultivation under semi-controlled conditions. We seeded at a density of 550 shrimp per m3 during the first cycle and 400 shrimp per m3 in the second cycle in an outdoor tank of 6m3or 6m2 covered with mesh, constant aeration. The shrimp were fed Artemia franciscana during the first two weeks and camaronina pellets (35% protein) as required, in food baskets, aftterwards. The temperature ranged from 22,3 to 31,3°C, pH 7,5-8,7, oxygen 4,26±1,43. The tanks are siphoned of debris every other day, and water was replaced according to a program. The food conversion ratio (FCR) was 1:1,3. The shrimp were measured weekly to calculate growth with the Bertalanffy model. Survival in the first cycle was 95,8 ( 97,9% for the second cycle). Population parameters (maximum likelihood method) for the first cycle were k=0,0301, L∞ =322,16 and t0 =-0,8852; second cycle: k=0,0203, L∞ =294,42 and t0 =-5,3771. There was rapid growth during the first 10 weeks. Biomass was 27kg for the first cycle (second: 16kg). KEY WORDSGrowth, high density, survival, biomass, semi-controlled conditions.

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