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Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917

| SophiaSophia | 0 Comment

Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, generally employed to deal with sure gastrointestinal issues. It’s quick rising as an vital goal for the event of therapeutic engineered micro organism, benefiting from the wealth of data of E. coli biology and ease of manipulation. Bacterial artificial biology tasks generally make the most of engineered plasmid vectors, that are easy to engineer and might reliably obtain excessive ranges of protein expression. Nonetheless, plasmids usually require antibiotics for upkeep, and the administration of an antibiotic is commonly incompatible with in vivo experimentation or therapy.

EcN natively accommodates plasmids pMUT1 and pMUT2, which haven’t any identified operate however are secure inside the micro organism. Right here, we describe the event of the pMUT plasmids into a strong platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to take away the native plasmids. We systematically engineered each pMUT plasmids to include choice markers, fluorescent markers, temperature delicate expression, and curli secretion programs to export a customizable useful materials into the extracellular house.

We then show that the engineered plasmids have been maintained in micro organism because the engineered micro organism move by the mouse GI tract with out choice, and that the secretion system stays useful, exporting functionalized curli proteins into the intestine. Our plasmid system presents a platform for the speedy improvement of therapeutic EcN micro organism.

Amplification of EBNA-1 by a single-plasmid vector-based gene amplification system in HEK293 cells as an environment friendly transient gene expression system

Our earlier work confirmed that there’s a limitation in the usage of dihydrofolate reductase (dhfr)/methotrexate (MTX)-mediated gene amplification programs in dhfr-non-deficient HEK293 cells, as endogenous dhfr might intervene with the amplification course of. Within the current examine, we efficiently generated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-amplified HEK293 cells in a dhfr-non-deficient HEK293 cell background utilizing a single-plasmid vector-based gene amplification system with shRNA concentrating on the three’-UTR of endogenous dhfr. The introduction of this shRNA effectively downregulated the expression of endogenous dhfr within the HEK293 cells with out affecting exogenous dhfr expression.

The downregulation of endogenous dhfr improved the effectivity of EBNA-1 amplification, as evidenced by a comparability with the amplification extent in cells missing shRNA expression on the similar MTX focus. The EBNA-1 expression ranges from the EBNA-1-amplified clones chosen on this examine have been larger than these obtained from EBNA-1-amplified clones that have been generated utilizing the traditional amplification in our earlier examine. In step with earlier research, EBNA-1 amplification improved the manufacturing of the Fc-fusion protein by a selected protein productiveness (qp)-enhancing impact, slightly than by bettering cell progress or transfection effectivity.

As well as, the N-glycan profiles within the Fc-fusion protein produced utilizing this transient gene expression (TGE) system weren’t affected by EBNA-1 amplification. These outcomes point out the potential utility of EBNA-1-amplified mammalian cells, developed utilizing a single-plasmid vector-based gene amplification system, for environment friendly protein manufacturing.

Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917

Manufacturing of lentiviral vectors in suspension cells utilizing low proportion of supercoiled round plasmid DNA

The supercoiled round (SC) topology type of plasmid DNA has been regarded to be advantageous over open round or linearized analogue in transfection and expression effectivity, and subsequently are largely demanded within the biopharmaceutical manufacturing. Nonetheless, manufacturing of high-purity SC plasmid DNA would lead to excessive manufacturing price. The impact of SC proportion in plasmid DNA on the standard of packaged lentiviral vectors has by no means been reported. On this examine, we established an environment friendly system for manufacturing of high-titer lentiviral vectors utilizing suspension HEK293SF cells in serum-free media, and the lentiviral titer was not related to the proportion of SC plasmid DNA.

Plasmids DNA with totally different proportion of SC, open-circular, and linearized varieties have been ready utilizing the thermal denaturation technique, and have been transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, however not from HEK293SF cells, was considerably impaired when the proportion of SC plasmid DNA decreased from 60-80% to 30-40%. Additional lower of SC plasmid proportion to three% led to a dramatic discount of lentiviral titer irrespective of the packaging cell line was.

Nonetheless, lentiviral vectors from HEK293SF cells nonetheless confirmed a excessive titer even when the proportion of SC plasmid DNA was 3%. This examine demonstrated that extraordinarily excessive proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at the least underneath our manufacturing course of.

Cloning in Plasmid Vectors: Blunt-Finish Cloning

This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To acquire the utmost variety of “right” ligation merchandise when cloning blunt-ended goal fragments, the 2 parts of DNA within the ligation response should be current at an applicable ratio.

VAHTS Universal DNA Library Prep Kit for MGI

NDM607-02 Vazyme 96 rxn
EUR 2136

2K DNA Marker

20-abx098045 Abbexa
  • EUR 258.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

5K DNA Marker

20-abx098048 Abbexa
  • EUR 258.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

15K DNA Marker

20-abx098049 Abbexa
  • EUR 272.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

1Kb DNA Marker

20-abx098050 Abbexa
  • EUR 258.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

100bp DNA Marker

20-abx098052 Abbexa
  • EUR 272.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

100bp DNA Marker

D1100-050 GenDepot 50㎍
EUR 165

100bp DNA Marker

D1100-250 GenDepot 5X50㎍
EUR 550

1Kb DNA Marker

D1101-050 GenDepot 50㎍
EUR 131

1Kb DNA Marker

D1101-250 GenDepot 5X50㎍
EUR 300

DL5000 DNA Marker

MD102-01 Vazyme 250 μl
EUR 107

DL5000 DNA Marker

MD102-02 Vazyme 500 μl
EUR 113

DL15000 DNA Marker

MD103-01 Vazyme 250 μl
EUR 109

DL15000 DNA Marker

MD103-02 Vazyme 500 μl
EUR 115

VAHTS Universal DNA Library Prep Kit for Illumina V3

ND607-01 Vazyme 24 rxn
EUR 441

VAHTS Universal DNA Library Prep Kit for Illumina V3

ND607-02 Vazyme 96 rxn
EUR 1405

VAHTS Universal Pro DNA Library Prep Kit for Illumina

ND608-01 Vazyme 24 rxn
EUR 465

VAHTS Universal Pro DNA Library Prep Kit for Illumina

ND608-02 Vazyme 96 rxn
EUR 1549

VAHTS Universal Plus DNA Library Prep Kit for Illumina

ND617-01 Vazyme 24 rxn
EUR 492

VAHTS Universal Plus DNA Library Prep Kit for Illumina

ND617-02 Vazyme 96 rxn
EUR 1605

VAHTS Universal DNA Library Prep Kit for Ion Torrent

ND701-01 Vazyme 24 rxn
EUR 733

VAHTS Universal DNA Library Prep Kit for Ion Torrent

ND701-02 Vazyme 96 rxn
EUR 2136

VAHTS Universal Pro DNA Library Prep Kit for MGI

NDM608-01 Vazyme 24 rxn
EUR 756

VAHTS Universal Pro DNA Library Prep Kit for MGI

NDM608-02 Vazyme 96 rxn
EUR 2284

VAHTS Universal Plus DNA Library Prep Kit for MGI

NDM617-01 Vazyme 24 rxn
EUR 824

VAHTS Universal Plus DNA Library Prep Kit for MGI

NDM617-02 Vazyme 96 rxn
EUR 2408

UNIVERSAL PLATFORM

480144 CORNING 1/pk
EUR 269
Description: Lab Equipment; Constant Temperature Equipment

1Kb Plus DNA Marker

20-abx098051 Abbexa
  • EUR 300.00
  • EUR 203.00
  • 2.5 ml
  • 500 ul

Linear Kinetoplast DNA Marker

TG2018-1 TopoGen 10 ug
EUR 179

Decatenated Kinetoplast DNA Marker

TG2020-1 TopoGen 10 ug
EUR 190

Relaxed pRYG DNA Marker

TG2025-1 TopoGen 10 ug
EUR 268

Linear pRYG DNA Marker

TG2028-1 TopoGen 10 ug
EUR 179

DL2000 Plus DNA Marker

MD101-01 Vazyme 250 μl
EUR 107

DL2000 Plus DNA Marker

MD101-02 Vazyme 500 μl
EUR 113

1 Kb Ladder DNA Marker

FYD001-500UL Yeastern Biotech 20 µg/ 500 µl Ask for price

100 bp Ladder DNA Marker

FYD002-500UL Yeastern Biotech 30 µg/ 500 µl Ask for price

50 bp Ladder DNA Marker

FYD004-500UL Yeastern Biotech 32.5 µg/ 500 µl Ask for price

Universal qPCR Mixture

QPCR1000-UNIV Bio Basic 4X1.25ml
EUR 276.2

Universal Coating Solution

AC002 Neuromics 20 ml
EUR 297

Universal Detachment Solution

AD002 Neuromics 100 ml
EUR 148

Universal Neutralization Buffer

NB001 Neuromics 100 ml
EUR 135

UNIVERSAL BEER AGAR

U21-100-10kg Alphabiosciences 10 kg
EUR 1524

UNIVERSAL BEER AGAR

U21-100-2kg Alphabiosciences 2kg
EUR 370

UNIVERSAL BEER AGAR

U21-100-500g Alphabiosciences 500 g
EUR 137

UNIVERSAL PREENRICHMENT BROTH

U21-101-10kg Alphabiosciences 10 kg
EUR 749

UNIVERSAL PREENRICHMENT BROTH

U21-101-2Kg Alphabiosciences 2 Kg
EUR 202

UNIVERSAL PREENRICHMENT BROTH

U21-101-500g Alphabiosciences 500 g
EUR 91

Universal Spring Platform

H1000-P-SP Benchmark Scientific 1 PC
EUR 323.39

VAHTS Universal DNA Library Prep Kit for Illumina V3 (PCR-Free)

ND607-03 Vazyme 24 rxn
EUR 429

VAHTS Universal DNA Library Prep Kit for Illumina V3 (PCR-Free)

ND607-04 Vazyme 96 rxn
EUR 1354

50-500bp Low Range DNA Marker

GM305 Bio Basic 200loading, 200prep
EUR 114.82

500-10000bp DNA Marker, Original Form

M101O-1 Bio Basic 100loading, 100prep
EUR 91.76

500-10000bp DNA Marker, Original Form

M101O-2 Bio Basic 5x100loading, 500prep
EUR 219.65

100-1500bp DNA Marker, Original Form

M102O-1 Bio Basic 100loading, 100prep
EUR 90.02

100-1500bp DNA Marker, Original Form

M102O-2 Bio Basic 5x100loading, 500prep
EUR 215.3

100-1500bp DNA Marker, Original Form

M107O-1 Bio Basic 100loading, 100prep
EUR 91.76

100-1500bp DNA Marker, Original Form

M107O-2 Bio Basic 5x100loading, 500prep
EUR 219.65

Image Green? 100 bp DNA Marker

M1155-500 Biovision
EUR 294

Image Green? 1 kb DNA Marker

M1156-500 Biovision
EUR 294

2K Plus DNA Marker (8 linear double-stranded DNA bands)

20-abx098046 Abbexa
  • EUR 258.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

2K Plus DNA Marker (9 linear double-stranded DNA bands)

20-abx098047 Abbexa
  • EUR 258.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

100bp Plus DNA Marker (12 linear double-stranded DNA bands)

20-abx098053 Abbexa
  • EUR 272.00
  • EUR 189.00
  • 2.5 ml
  • 500 ul

100bp Plus DNA Marker (14 linear double-stranded DNA bands)

20-abx098054 Abbexa
  • EUR 300.00
  • EUR 203.00
  • 2.5 ml
  • 500 ul

Universal Protein Precipitating Reagent

BSP012 Bio Basic 100preps
EUR 81.32

PCR plate-foil universal

A28216 GeneOn 1x100 pcs
EUR 94

PCR plate-foil universal

A28216L GeneOn 5x100 pcs
EUR 310

Universal Full Growth Medium

TM001 Neuromics 500 ml
EUR 336

5min Universal Ligation Mix

C311-01 Vazyme 50 rxn
EUR 214

5min Universal Ligation Mix

C311-02 Vazyme 100 rxn
EUR 283

Universal Casein Diluent/Blocker

85R-108 Fitzgerald 250 ml
EUR 230
Description: Ready-to-use biotin free reagent applicable for blocking ELISA plates

100-1200bp DNA Marker, Ready-to-use

GM333 Bio Basic 50loading, 50prep
EUR 67.4

100-1200bp DNA Marker, Ready-to-use

GM334 Bio Basic 5x50loading, 250prep
EUR 128.3

100-2000bp DNA Marker, Ready-to-use

GM335 Bio Basic 50loading, 50prep
EUR 67.4

100-2000bp DNA Marker, Ready-to-use

GM336 Bio Basic 5x50loading, 250prep
EUR 128.3

200-1500bp DNA Marker, Ready-to-use

GM337 Bio Basic 50loading, 50prep
EUR 67.4

200-1500bp DNA Marker, Ready-to-use

GM338 Bio Basic 5x50loading, 250prep
EUR 128.3

200-2000bp DNA Marker, Ready-to-use

GM339 Bio Basic 50loading, 50prep
EUR 67.4

200-2000bp DNA Marker, Ready-to-use

GM340 Bio Basic 5x50loading, 250prep
EUR 128.3

300-2500bp DNA Marker, Ready-to-use

GM341 Bio Basic 50loading, 50prep
EUR 67.4

300-2500bp DNA Marker, Ready-to-use

GM342 Bio Basic 5x50loading, 250prep
EUR 128.3

100-1000bp DNA Marker, Ready-to-use

GM343 Bio Basic 50loading, 50prep
EUR 80.45

100-1000bp DNA Marker, Ready-to-use

GM345 Bio Basic 50loading, 50prep
EUR 84.8

500-10000bp DNA Marker, Ready-to-use

M101R-1 Bio Basic 100loading, 100prep
EUR 91.76

500-10000bp DNA Marker, Ready-to-use

M101R-2 Bio Basic 5x100loading, 500prep
EUR 219.65

100-1500bp DNA Marker, Ready-to-use

M102R-1 Bio Basic 100loading, 100prep
EUR 90.02

100-1500bp DNA Marker, Ready-to-use

M102R-2 Bio Basic 5x100loading, 500prep
EUR 215.3

Lambda DNA/HindIII Marker, Ready-to-use

M104R-1 Bio Basic 100loading, 100prep
EUR 65.66

Lambda DNA/HindIII Marker, Ready-to-use

M104R-2 Bio Basic 5x100loading, 500prep
EUR 124.82

100-1500bp DNA Marker, Ready-to-use

M107R-1 Bio Basic 100loading, 100prep
EUR 91.76

100-1500bp DNA Marker, Ready-to-use

M107R-2 Bio Basic 5x100loading, 500prep
EUR 219.65

50-1500bp DNA Marker, Ready-to-use

M109-A Bio Basic 100loading, 100prep
EUR 91.76

50-1500bp DNA Marker, Ready-to-use

M109-B Bio Basic 5x100loading, 500prep
EUR 219.65
×

If the molar ratio of plasmid vector to focus on DNA is simply too excessive, then the ligation response might generate an undesirable variety of round empty plasmids, each monomeric and polymeric; if too low, the ligation response might generate an extra of linear and round homopolymers and heteropolymers of various sizes, orientations, and compositions. Because of this, the orientation of the international DNA and the variety of inserts in every recombinant clone should all the time be validated by restriction endonuclease mapping or another means.

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anti cre antibody cgmp elisa granzyme b elisa hmgb1 elisa kit il 18 elisa ldh cytotoxicity assay kit lentivirus titer leptin test kit loading control antibody mpo elisa nephrin antibody paav mcs Pcdna3.1 Sequence Pcdna3.1 Zeo Pcdna6 2 pdk4 antibody pegfp n3 Pet28A pgl3 promoter Plenti Puro plko 1 ppar alpha antibody prl tk Pshuttle Pspax2 Ptripz Ptripz Vector Puc18 Puc 19 Puc Vector Rfp Plasmid Sequence Analysis Software socs1 antibody Strepii Tag Sequence Sumo Sequence Ta Clone Taq Dna Polymerase Taq Polymerase Is Tet Operator Sequence Tev Sequence Topo Pcr4 Turborfp V5 Sequence Wpre Plasmid Xhoi Restriction Site

Categories

  • alas1
  • anti tdtomato antibody
  • Blog
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  • EIA
  • electrophoresis
  • glut2 antibody
  • klk7 max antibody
  • luciferase kit
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  • Percp
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Recent Posts

  • Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays-Protein and Buffer Composition Contribution
  • Combinatorial nanodot stripe assay to systematically study cell haptotaxis
  • Inter- and Intrapopulational Heterogeneity of Characteristic Markers in Adult Human Neural Crest-derived Stem Cells
  • Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique.
  • Protein Kinase A Detection in Human Urine Samples

Tags

anti cre antibody cgmp elisa granzyme b elisa hmgb1 elisa kit il 18 elisa ldh cytotoxicity assay kit lentivirus titer leptin test kit loading control antibody mpo elisa nephrin antibody paav mcs Pcdna3.1 Sequence Pcdna3.1 Zeo Pcdna6 2 pdk4 antibody pegfp n3 Pet28A pgl3 promoter Plenti Puro plko 1 ppar alpha antibody prl tk Pshuttle Pspax2 Ptripz Ptripz Vector Puc18 Puc 19 Puc Vector Rfp Plasmid Sequence Analysis Software socs1 antibody Strepii Tag Sequence Sumo Sequence Ta Clone Taq Dna Polymerase Taq Polymerase Is Tet Operator Sequence Tev Sequence Topo Pcr4 Turborfp V5 Sequence Wpre Plasmid Xhoi Restriction Site
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