ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

Scientific Reports
David RichterStefan Schuster

Abstract

The recently evolved field of synthetic biology has revolutionized the way we think of biology as an "engineerable" discipline. The newly sprouted branch is constantly in need of simple, cost-effective and automatable DNA-assembly methods. We have developed a reliable DNA-assembly system, ZeBRα (Zero-Background Redα), for cloning multiple DNA-fragments seamlessly with very high efficiency. The hallmarks of ZeBRα are the greatly reduced hands-on time and costs and yet excellent efficiency and flexibility. ZeBRα combines a "zero-background vector" with a highly efficient in vitro recombination method. The suicide-gene in the vector acts as placeholder, and is replaced by the fragments-of-interest, ensuring the exclusive survival of the successful recombinants. Thereby the background from uncut or re-ligated vector is absent and screening for recombinant colonies is unnecessary. Multiple fragments-of-interest can be assembled into the empty vector by a recombinogenic E. coli-lysate (SLiCE) with a total time requirement of less than 48 h. We have significantly simplified the preparation of the high recombination-competent E. coli-lysate compared to the original protocol. ZeBRα is the least labor intensive among comparable state-of-...Continue Reading

References

Jun 5, 1983·Journal of Molecular Biology·D Hanahan
Nov 11, 1993·Nucleic Acids Research·J D OlinerB Vogelstein
Jul 25, 1993·Nucleic Acids Research·P BubeckW Bautsch
Jul 16, 2004·Journal of Proteome Research·Jodi R ParrishRussell L Finley
May 26, 2005·Protein Expression and Purification·F William Studier
Dec 31, 2005·Molecular Biotechnology·Junping WangYouming Zhang
Nov 14, 2006·PLoS Genetics·Darius BalciunasStephen C Ekker
Dec 7, 2007·Plant Physiology·Mansour KarimiPierre Hilson
Nov 6, 2008·PloS One·Carola EnglerSylvestre Marillonnet
May 1, 2009·Plant Physiology·Songbiao ChenGuo-Liang Wang
May 22, 2010·Science·Daniel G GibsonJ Craig Venter
Jul 28, 2010·Antimicrobial Agents and Chemotherapy·Renu SinghVincent H Tam
Oct 12, 2010·Nature Methods·Daniel G GibsonChuck Merryman
Jan 14, 2012·Nucleic Acids Research·Yongwei ZhangWinfried Edelmann
Feb 14, 2012·Methods in Molecular Biology·Mamie Z Li, Stephen J Elledge
Sep 12, 2012·Protein Expression and Purification·Buenafe T AracheaRonald E Viola
Feb 7, 2014·Molecular Biotechnology·Quirino ScheferCarsten Grötzinger
Jan 1, 2013·Frontiers in Bioengineering and Biotechnology·Adam B FisherGeorge H McArthur
Mar 17, 2015·PloS One·Ana Paula Jacobus, Jeferson Gross
Apr 10, 2015·ACS Synthetic Biology·Gur PinesRyan T Gill
Jun 13, 2015·Scientific Reports·Massimiliano ZampiniFinbarr Hayes
Aug 12, 2015·Nature Reviews. Genetics·Adrian D HaimovichFarren J Isaacs
Sep 12, 2015·PloS One·Hannes M BeyerMatias D Zurbriggen
Apr 14, 2016·PloS One·Roger M BenoitManfred Auer
Jun 7, 2016·Scientific Reports·Javier García-NafríaIngo H Greger
Jun 18, 2016·Analytical Biochemistry·Katrin MesserschmidtBernd Mueller-Roeber
Sep 29, 2017·Biochemistry and Biophysics Reports·Ken Motohashi
Dec 12, 2018·Journal of Bacteriology·Shingo Nozaki, Hironori Niki

❮ Previous
Next ❯

Citations

Dec 11, 2020·Metabolic Engineering·Rosanna YoungPaul S Freemont
Feb 26, 2021·Biotechnology Advances·Atrayee ChattopadhyayMrinal K Maiti
Jun 22, 2021·Access Microbiology·Ian R Monk, Timothy P Stinear
Jul 31, 2021·Frontiers in Bioengineering and Biotechnology·Wenfang WangYinhua Lu

❮ Previous
Next ❯

Datasets Mentioned

BETA
CP010816.1
CP017100.1

Methods Mentioned

BETA
in vitro transcription
phosphotransferase

Software Mentioned

Gateway®
iVEC
RapGene
NEBuilder HiFi DNA
SLiCE
OpenWetware
Fiji
Golden
ZeBRα
multisite

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

JAMA : the Journal of the American Medical Association
Richard J Rosenthal
The American Journal of Nursing
Janice A Habinski
Advance for Nurse Practitioners
Muhammad Saifudin Ibn Samuel
© 2022 Meta ULC. All rights reserved