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.
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.
Methylamp Universal Methylated DNA Kit |
P-1011 |
EpiGentek |
-
Ask for price
-
Ask for price
-
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|
- 20 Modifications
- 10 Modifications
- 20 Modifications
|
Allprep DNA/RNA/miRNA Universal Kit |
2148A |
Unibiotest |
50 Tests |
EUR 500 |
Allprep DNA/RNA/miRNA Universal Kit |
2148B |
Unibiotest |
4*50 Tests |
EUR 1800 |
VAHTS Universal DNA Library Prep Kit for MGI |
NDM607-01 |
Vazyme |
24 rxn |
EUR 879.6 |
VAHTS Universal DNA Library Prep Kit for MGI |
NDM607-02 |
Vazyme |
96 rxn |
EUR 2563.2 |
Universal Solution for DNA isolation (GeDI) |
E3760-01 |
EURx |
25ml |
EUR 30.52 |
Universal Solution for DNA isolation (GeDI) |
E3760-02 |
EURx |
100ml |
EUR 95.92 |
VAHTS Universal DNA library Prep Kit for MGI V4 |
NDM610-01 |
Vazyme |
24 rxns |
EUR 235.2 |
VAHTS Universal DNA library Prep Kit for MGI V4 |
NDM610-02 |
Vazyme |
96 rxns |
EUR 880 |
VAHTS Universal DNA library Prep Kit for MGI V3 |
NDM607-00-6rxns |
Vazyme |
6 rxns |
EUR 1.09 |
VAHTS Universal DNA library Prep Kit for MGI V3 |
NDM607-01-24rxns |
Vazyme |
24 rxns |
EUR 304.66 |
VAHTS Universal DNA library Prep Kit for MGI V3 |
NDM607-02-96rxns |
Vazyme |
96 rxns |
EUR 1139.05 |
GeneMATRIX UNIVERSAL DNA/RNA/Protein Purification Kit |
E3597-01 |
EURx |
25preps |
EUR 81.75 |
Description: Kit for isolation of total DNA/RNA/Protein from the same biological sample(bacteria, tissue, plant, yeast, cell culture) |
GeneMATRIX UNIVERSAL DNA/RNA/Protein Purification Kit |
E3597-02 |
EURx |
100preps |
EUR 299.75 |
Description: Kit for isolation of total DNA/RNA/Protein from the same biological sample(bacteria, tissue, plant, yeast, cell culture) |
Allprep RNA/DNA/miRNA/protein Universal Kit |
2149A |
Unibiotest |
50 Tests |
EUR 600 |
Allprep RNA/DNA/miRNA/protein Universal Kit |
2149B |
Unibiotest |
4*50 Tests |
EUR 2200 |
Methylamp Universal Methylated DNA Preparation Kit |
P-1019 |
EpiGentek |
40 µg |
EUR 416.7 |
VAHTS Universal DNA Library Prep Kit for Illumina V3 |
ND607-01 |
Vazyme |
24 rxn |
EUR 529.2 |
VAHTS Universal DNA Library Prep Kit for Illumina V3 |
ND607-02 |
Vazyme |
96 rxn |
EUR 1686 |
VAHTS Universal DNA Library Prep Kit for Illumina V4 |
ND610-01 |
Vazyme |
24 rxns |
EUR 294 |
VAHTS Universal DNA Library Prep Kit for Illumina V4 |
ND610-02 |
Vazyme |
96 rxns |
EUR 1100 |
VAHTS Universal DNA Library Prep Kit for Ion Torrent |
ND701-01 |
Vazyme |
24 rxn |
EUR 879.6 |
VAHTS Universal DNA Library Prep Kit for Ion Torrent |
ND701-02 |
Vazyme |
96 rxn |
EUR 2563.2 |
VAHTS Universal DNA Library Prep Kit for Illumina V4 |
ND610-00-6rxns |
Vazyme |
6 rxns |
EUR 1.09 |
VAHTS Universal DNA Library Prep Kit for Illumina V4 |
ND610-01-24rxns |
Vazyme |
24 rxns |
EUR 304.44 |
VAHTS Universal DNA Library Prep Kit for Illumina V4 |
ND610-02-96rxns |
Vazyme |
96 rxns |
EUR 1139.05 |
VAHTS Universal DNA Library Prep Kit for Illumina V4 |
ND610-03-24rxns |
Vazyme |
24 rxns |
EUR 269.23 |
VAHTS Universal DNA Library Prep Kit for Illumina V4 |
ND610-04-96rxns |
Vazyme |
96 rxns |
EUR 999.53 |
AFTSpin Universal Plant Fast DNA Extraction Kit |
RK30111 |
Abclonal |
50RXN |
EUR 69 |
EpiMag High Throughput DNA Isolation Universal Kit |
P-1022-96 |
EpiGentek |
96 Samples |
EUR 227.7 |
EpiMag High Throughput DNA Isolation Universal Kit |
P-1022 |
EpiGentek |
96 Samples |
EUR 215 |
Universal Genomic DNA Purification kit(Spin Column) |
2546A |
Unibiotest |
50 Tests |
EUR 100 |
Universal Genomic DNA Purification kit(Spin Column) |
2546B |
Unibiotest |
100 Tests |
EUR 150 |
Universal-1 |
OD308-1PK |
EWC Diagnostics |
1 unit |
EUR 2.65 |
Description: Universal-1 |
Universal-1 |
OD308R-1PK |
EWC Diagnostics |
1 unit |
EUR 2.65 |
Description: Universal-1 |
VAHTS Universal DNA Library Prep Kit for Ion Torrent V2 |
ND702-01 |
Vazyme |
24 rxns |
EUR 294 |
VAHTS Universal DNA Library Prep Kit for Ion Torrent V2 |
ND702-02 |
Vazyme |
96 rxns |
EUR 1100 |
Universal Kit for DNA isolation with GeDI Solution |
E3765-01 |
EURx |
25preps |
EUR 59.95 |
Universal Kit for DNA isolation with GeDI Solution |
E3765-02 |
EURx |
100preps |
EUR 210.37 |
VAHTS Universal Pro DNA Library Prep Kit for MGI |
NDM608-01 |
Vazyme |
24 rxn |
EUR 907.2 |
VAHTS Universal Pro DNA Library Prep Kit for MGI |
NDM608-02 |
Vazyme |
96 rxn |
EUR 2740.8 |
VAHTS Universal Plus DNA Library Prep Kit for MGI |
NDM617-01 |
Vazyme |
24 rxn |
EUR 988.8 |
VAHTS Universal Plus DNA Library Prep Kit for MGI |
NDM617-02 |
Vazyme |
96 rxn |
EUR 2889.6 |
VAHTS Universal Pro DNA Library Prep Kit for Illumina |
ND608-01 |
Vazyme |
24 rxn |
EUR 558 |
VAHTS Universal Pro DNA Library Prep Kit for Illumina |
ND608-02 |
Vazyme |
96 rxn |
EUR 1858.8 |
VAHTS Universal Plus DNA Library Prep Kit for MGI V2 |
NDM627-01 |
Vazyme |
24 rxns |
EUR 312.5 |
VAHTS Universal Plus DNA Library Prep Kit for MGI V2 |
NDM627-02 |
Vazyme |
96 rxns |
EUR 1150 |
VAHTS Universal Plus DNA Library Prep Kit for MGI V2 |
NDM627-00-6rxns |
Vazyme |
6 rxns |
EUR 1.09 |
VAHTS Universal Plus DNA Library Prep Kit for MGI V2 |
NDM627-01-24rxns |
Vazyme |
24 rxns |
EUR 323.59 |
VAHTS Universal Plus DNA Library Prep Kit for MGI V2 |
NDM627-02-96rxns |
Vazyme |
96 rxns |
EUR 1190.83 |
VAHTS Universal Plus DNA Library Prep Kit for Illumina |
ND617-01 |
Vazyme |
24 rxn |
EUR 590.4 |
VAHTS Universal Plus DNA Library Prep Kit for Illumina |
ND617-02 |
Vazyme |
96 rxn |
EUR 1926 |
Universal Holder |
LBI10201 |
Westburg |
each |
EUR 161.87 |
Hexa Universal-2 |
HX038-1PK |
EWC Diagnostics |
1 unit |
EUR 2.51 |
Description: Hexa Universal-2 |
VAHTS Universal DNA Library Prep Kit for Illumina V3 (PCR-Free) |
ND607-03 |
Vazyme |
24 rxn |
EUR 514.8 |
VAHTS Universal DNA Library Prep Kit for Illumina V3 (PCR-Free) |
ND607-04 |
Vazyme |
96 rxn |
EUR 1624.8 |
Universal Indicator |
I013-100ML |
EWC Diagnostics |
1 unit |
EUR 2.65 |
Description: Universal Indicator |
Universal Indicator |
I013-500ML |
EWC Diagnostics |
1 unit |
EUR 9.01 |
Description: Universal Indicator |
VAHTS Universal Plus DNA Library Prep Kit for Illumina V2 |
ND627-01 |
Vazyme |
24 rxns |
EUR 312.5 |
VAHTS Universal Plus DNA Library Prep Kit for Illumina V2 |
ND627-02 |
Vazyme |
96 rxns |
EUR 1150 |
VAHTS Universal Plus DNA Library Prep Kit for Illumina V2 |
ND627-00-6rxns |
Vazyme |
6 rxns |
EUR 1.09 |
VAHTS Universal Plus DNA Library Prep Kit for Illumina V2 |
ND627-01-24rxns |
Vazyme |
24 rxns |
EUR 323.59 |
VAHTS Universal Plus DNA Library Prep Kit for Illumina V2 |
ND627-02-96rxns |
Vazyme |
96 rxns |
EUR 1190.83 |
Hexa Universal - 1 |
HX032-1PK |
EWC Diagnostics |
1 unit |
EUR 2.51 |
Description: Hexa Universal - 1 |
Hexa Universal - 4 |
HX715-1PK |
EWC Diagnostics |
1 unit |
EUR 2.51 |
Description: Hexa Universal - 4 |
Hexa Universal - 5 |
HX716-1PK |
EWC Diagnostics |
1 unit |
EUR 2.51 |
Description: Hexa Universal - 5 |
Icosa Universal - 1 |
IC001-1PK |
EWC Diagnostics |
1 unit |
EUR 9.32 |
Description: Icosa Universal - 1 |
Icosa Universal - 2 |
IC006-1PK |
EWC Diagnostics |
1 unit |
EUR 9.32 |
Description: Icosa Universal - 2 |
Dodeca Universal -XI |
DE026-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal -XI |
Dodeca - Universal XV, |
DE042-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca - Universal XV, |
Universal V1-Puck |
M-CP-111-021 |
MiTeGen |
1 PUCK |
EUR 350 |
Description: Universal V1-Puck |
Dodeca Universal-I |
DE001-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-I |
Dodeca Universal-V |
DE013-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-V |
Dodeca Universal -XII |
DE027-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal -XII |
Dodeca Universal XIV |
DE035-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal XIV |
Universal WB Toolkit |
KTD106-EN-each |
Abbkine |
each |
Ask for price |
Universal WB Toolkit |
KTD106-EN-Null |
Abbkine |
Null |
Ask for price |
Universal WB Toolkit |
KTD106-EN-20T |
Abbkine |
20 T |
EUR 59 |
Description: Abbkine has developed a Universal WB Toolkit. After careful design and research, one-stop operation can meet the needs of routine WB experiments and obtain excellent experimental results. |
Universal WB Toolkit |
MBS9719335-20Tests |
MyBiosource |
20Tests |
EUR 195 |
Universal WB Toolkit |
MBS9719335-5x20Tests |
MyBiosource |
5x20Tests |
EUR 885 |
Dodeca Universal-II |
DE007-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-II |
Dodeca Universal-IV |
DE012-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-IV |
Dodeca Universal-VI |
DE014-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-VI |
Dodeca Universal-IX |
DE017-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-IX |
Dodeca Universal- XIII |
DE028-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal- XIII |
Dodeca Universal-III |
DE008-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-III |
Dodeca Universal-VII |
DE015-1PK |
EWC Diagnostics |
1 unit |
EUR 3.96 |
Description: Dodeca Universal-VII |
APP Antibody (universal) |
GWB-C298C6 |
GenWay Biotech |
0.1 ml |
Ask for price |
Universal qPCR Mixture |
QPCR1000-UNIV |
Bio Basic |
4X1.25ml |
EUR 331.44 |
|
*Universal Liquid Medium |
M1332-500G |
EWC Diagnostics |
1 unit |
EUR 25.78 |
Description: *Universal Liquid Medium |
Universal vessel holder |
LBI10230 |
Westburg |
each |
EUR 156.96 |
Universal Heating System |
LBI10501 |
Westburg |
each |
EUR 11281.5 |
0.1-10µL Universal Fit |
2040 |
Biotix |
1,000/PACK, 20/CASE |
EUR 240.16 |
0.1-10µL Universal Fit |
2040.N |
Biotix |
1,000/PACK, 20/CASE |
EUR 255.24 |
0.1-10µL Universal Fit |
2042 |
Biotix |
10/PACK, 5/CASE |
EUR 127.04 |
0.1-10µL Universal Fit |
2042.N |
Biotix |
10/PACK, 5/CASE |
EUR 122.98 |
0.1-10µL Universal Fit |
2042.NS |
Biotix |
10/PACK, 5/CASE |
EUR 127.04 |
0.1-10µL Universal Fit |
2042.S |
Biotix |
10/PACK, 5/CASE |
EUR 134.58 |
0.1-10µL Universal Fit |
2047 |
Biotix |
20/PACK, 4/CASE |
EUR 137.48 |
0.1-10µL Universal Fit |
2047.N |
Biotix |
20/PACK, 4/CASE |
EUR 138.64 |
20-300µL Universal Fit |
2090 |
Biotix |
1,000/PACK, 10/CASE |
EUR 105 |
20-300µL Universal Fit |
2090.N |
Biotix |
1,000/PACK, 10/CASE |
EUR 89.92 |
20-300µL Universal Fit |
2092 |
Biotix |
10/PACK, 5/CASE |
EUR 127.04 |
20-300µL Universal Fit |
2092.N |
Biotix |
10/PACK, 5/CASE |
EUR 122.98 |
20-300µL Universal Fit |
2092.NS |
Biotix |
8/PACK, 4/CASE |
EUR 127.04 |
20-300µL Universal Fit |
2092.S |
Biotix |
10/PACK, 5/CASE |
EUR 134.58 |
20-300µL Universal Fit |
2097 |
Biotix |
10/PACK, 10/CASE |
EUR 137.48 |
20-300µL Universal Fit |
2097.N |
Biotix |
10/PACK, 10/CASE |
EUR 174.03 |
20-200µL Universal Fit |
2100 |
Biotix |
1,000/PACK, 10/CASE |
EUR 105 |
20-200µL Universal Fit |
2100.N |
Biotix |
1,000/PACK, 10/CASE |
EUR 89.92 |
20-200µL Universal Fit |
2102 |
Biotix |
10/PACK, 5/CASE |
EUR 127.04 |
20-200µL Universal Fit |
2102.N |
Biotix |
10/PACK, 5/CASE |
EUR 122.98 |
1-20µL Universal Fit |
2102.NS |
Biotix |
10/PACK, 5/CASE |
EUR 127.04 |
20-200µL Universal Fit |
2102.S |
Biotix |
10/PACK, 5/CASE |
EUR 134.58 |
20-200µL Universal Fit |
2107 |
Biotix |
10/PACK, 10/CASE |
EUR 180.41 |
20-200µL Universal Fit |
2107.N |
Biotix |
10/PACK, 10/CASE |
EUR 174.03 |
20-280µL Universal Fit |
2152.96.N |
Biotix |
8/PACK, 4/CASE |
EUR 519.77 |
10-280µL Universal Fit |
2152.96.NS |
Biotix |
8/PACK, 4/CASE |
EUR 555.74 |
100-1000µL Universal Fit |
2160 |
Biotix |
1,000/PACK, 4/CASE |
EUR 45.25 |
100-1000µL Universal Fit |
2162.96 |
Biotix |
8/PACK, 4/CASE |
EUR 130.52 |
100-1000µL Universal Fit |
2162.96.S |
Biotix |
8/PACK, 4/CASE |
EUR 108.48 |
100-1000µL Universal Fit |
2167.96 |
Biotix |
10/PACK, 5/CASE |
EUR 92.82 |
100-1250µL Universal Fit |
2370 |
Biotix |
1,000/PACK, 4/CASE |
EUR 120.08 |
100-1250µL Universal Fit |
2370.N |
Biotix |
1,000/PACK, 4/CASE |
EUR 45.25 |
100-1250µL Universal Fit |
2372 |
Biotix |
8/PACK, 4/CASE |
EUR 108.48 |
100-1250µL Universal Fit |
2372.N |
Biotix |
8/PACK, 4/CASE |
EUR 103.84 |
100-1250µL Universal Fit |
2372.NS |
Biotix |
8/PACK, 4/CASE |
EUR 108.48 |
100-1250µL Universal Fit |
2372.S |
Biotix |
8/PACK, 4/CASE |
EUR 115.44 |
100-1250µL Universal Fit |
2377 |
Biotix |
10/PACK, 5/CASE |
EUR 120.08 |
100-1250µL Universal Fit |
2377.N |
Biotix |
10/PACK, 5/CASE |
EUR 116.02 |
0.1-10µL Universal Fit |
BT10.N |
Biotix |
10/PACK, 5/CASE |
EUR 224.5 |
100-1000µL Universal Fit |
BT1000.96.N |
Biotix |
8/PACK, 4/CASE |
EUR 195.49 |
100-1250µL Universal Fit |
BT1250.N |
Biotix |
8/PACK, 4/CASE |
EUR 202.46 |
10-230µL Universal Fit |
BT200XLT |
Biotix |
8/PACK, 4/CASE |
EUR 718.16 |
Universal Coating Solution |
MBS556488-20mL |
MyBiosource |
20mL |
EUR 310 |
Universal Coating Solution |
MBS556488-5x20mL |
MyBiosource |
5x20mL |
EUR 1235 |
Universal Detachment Solution |
AD002 |
Neuromics |
100 ml |
EUR 177.6 |
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.