Reliable handling of highly A/T-rich genomic DNA for efficient generation of knockin strains of Dictyostelium discoideum.

Abstract:

BACKGROUND:Social amoeba, Dictyostelium discoideum, is a well-established model organism for studying cellular physiology and developmental pattern formation. Its haploid genome facilitates functional analysis of genes by a single round of mutagenesis including targeted disruption. Although the efficient generation of knockout strains based on an intrinsically high homologous recombination rate has been demonstrated, successful reports for knockin strains have been limited. As social amoeba has an exceptionally high adenine and thymine (A/T)-content, conventional plasmid-based vector construction has been constrained due to deleterious deletion in E. coli. RESULTS:We describe here a simple and efficient strategy to construct GFP-knockin cassettes by using a linear DNA cloning vector derived from N15 bacteriophage. This allows reliable handling of DNA fragments whose A/T-content may be as high as 85 %, and which cannot be cloned into a circular plasmid. By optimizing the length of recombination arms, we successfully generate GFP-knockin strains for five genes involved in cAMP signalling, including a triple-colour knockin strain. CONCLUSIONS:This robust strategy would be useful in handling DNA fragments with biased A/T-contents such as the genome of lower organisms and the promoter/terminator regions of higher organisms.

journal_name

BMC Biotechnol

journal_title

BMC biotechnology

authors

Mukai A,Ichiraku A,Horikawa K

doi

10.1186/s12896-016-0267-8

subject

Has Abstract

pub_date

2016-04-14 00:00:00

pages

37

issn

1472-6750

pii

10.1186/s12896-016-0267-8

journal_volume

16

pub_type

杂志文章
  • A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

    abstract:BACKGROUND:Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity. RESULTS:Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-014-0093-9

    authors: Jiang W,Chen L,Hu N,Yuan S,Li B,Liu Z

    更新日期:2014-11-14 00:00:00

  • Efficient production of heat-stable antifungal factor through integrating statistical optimization with a two-stage temperature control strategy in Lysobacter enzymogenes OH11.

    abstract:BACKGROUND:Heat-stable antifungal factor (HSAF) is a newly identified broad-spectrum antifungal antibiotic from the biocontrol agent Lysobacter enzymogenes and is regarded as a potential biological pesticide, due to its novel mode of action. However, the production level of HSAF is quite low, and little research has re...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-018-0478-2

    authors: Tang B,Sun C,Zhao Y,Xu H,Xu G,Liu F

    更新日期:2018-10-24 00:00:00

  • Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding.

    abstract:BACKGROUND:Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane do...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-11-60

    authors: Kato T,Suzuki F,Park EY

    更新日期:2011-06-02 00:00:00

  • Antibody degradation in tobacco plants: a predominantly apoplastic process.

    abstract:BACKGROUND:Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem. RESULTS:In this study, we investigated the dynamics of the assem...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-11-128

    authors: Hehle VK,Paul MJ,Drake PM,Ma JK,van Dolleweerd CJ

    更新日期:2011-12-30 00:00:00

  • Keratinocyte cytoskeletal roles in cell sheet engineering.

    abstract:BACKGROUND:There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragil...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-13-17

    authors: Wei Q,Reidler D,Shen MY,Huang H

    更新日期:2013-02-26 00:00:00

  • The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris.

    abstract:BACKGROUND:Copper is an essential chemical element for life as it is a part of prosthetic groups of enzymes including super oxide dismutase and cytochrome c oxidase; however, it is also toxic at high concentrations. Here, we present the trade-off of copper availability and growth inhibition of a common host used for co...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-016-0251-3

    authors: Balakumaran PA,Förster J,Zimmermann M,Charumathi J,Schmitz A,Czarnotta E,Lehnen M,Sudarsan S,Ebert BE,Blank LM,Meenakshisundaram S

    更新日期:2016-02-20 00:00:00

  • Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations.

    abstract:BACKGROUND:The cattle ticks, Boophilus spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant B. microplus Bm86 protective antigen has been shown to protect cattle against tick i...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-9-29

    authors: Canales M,Almazán C,Naranjo V,Jongejan F,de la Fuente J

    更新日期:2009-03-31 00:00:00

  • Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance.

    abstract:BACKGROUND:Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of start...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-5-24

    authors: Bergen AW,Qi Y,Haque KA,Welch RA,Chanock SJ

    更新日期:2005-09-16 00:00:00

  • The case for well-conducted experiments to validate statistical protocols for 2D gels: different pre-processing = different lists of significant proteins.

    abstract:BACKGROUND:The proteomics literature has seen a proliferation of publications that seek to apply the rapidly improving technology of 2D gels to study various biological systems. However, there is a dearth of systematic studies that have investigated appropriate statistical approaches to analyse the data from these expe...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-5-7

    authors: Meleth S,Deshane J,Kim H

    更新日期:2005-02-11 00:00:00

  • Resveratrol alleviates lipopolysaccharide-induced inflammation in PC-12 cells and in rat model.

    abstract:BACKGROUND:Spinal cord injury (SCI) remains a huge medical problem nowadays as there is no hospital providing the versatile strategies for repairing central nervous system and restoring function. Herein, we focused on PC-12 cells as an important research tool and studied the potential role of resveratrol (RSV) in infla...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-019-0502-1

    authors: Zhang G,Liu Y,Xu L,Sha C,Zhang H,Xu W

    更新日期:2019-01-28 00:00:00

  • Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase.

    abstract:BACKGROUND:Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse tran...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-8-60

    authors: Moldt B,Staunstrup NH,Jakobsen M,Yáñez-Muñoz RJ,Mikkelsen JG

    更新日期:2008-08-09 00:00:00

  • A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli.

    abstract:BACKGROUND:The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (beta2GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are criti...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-6-8

    authors: Ioannou Y,Giles I,Lambrianides A,Richardson C,Pearl LH,Latchman DS,Isenberg DA,Rahman A

    更新日期:2006-02-10 00:00:00

  • Degradation or excretion of quantum dots in mouse embryonic stem cells.

    abstract:BACKGROUND:Quantum dots (QDs) have been considered as a new and efficient probe for labeling cells non-invasively in vitro and in vivo, but fairly little is known about how QDs are eliminated from cells after labeling. The purpose of this study is to investigate the metabolism of QDs in different type of cells. RESULT...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-10-36

    authors: Pi QM,Zhang WJ,Zhou GD,Liu W,Cao Y

    更新日期:2010-05-06 00:00:00

  • Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines.

    abstract:BACKGROUND:As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-020-00604-3

    authors: Michno JM,Virdi K,Stec AO,Liu J,Wang X,Xiong Y,Stupar RM

    更新日期:2020-02-24 00:00:00

  • Different genome-wide transcriptome responses of Nocardioides simplex VKM Ac-2033D to phytosterol and cortisone 21-acetate.

    abstract:BACKGROUND:Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and oth...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-021-00668-9

    authors: Shtratnikova VY,Sсhelkunov MI,Fokina VV,Bragin EY,Shutov AA,Donova MV

    更新日期:2021-01-13 00:00:00

  • Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers.

    abstract:BACKGROUND:Mesenchymal stem cells (MSCs) are of great interest in bone regenerative medicine due to their osteogenic potential and trophic effects. However, challenges to large-scale production of MSCs can hinder the translation of MSC therapies. 3D Microcarrier (MC)-based MSC culture presents a scalable and cost-effec...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-015-0219-8

    authors: Shekaran A,Sim E,Tan KY,Chan JK,Choolani M,Reuveny S,Oh S

    更新日期:2015-10-31 00:00:00

  • Selection of oleaginous yeasts for fatty acid production.

    abstract:BACKGROUND:Oleaginous yeast species are an alternative for the production of lipids or triacylglycerides (TAGs). These yeasts are usually non-pathogenic and able to store TAGs ranging from 20 % to 70 % of their cell mass depending on culture conditions. TAGs originating from oleaginous yeasts can be used as the so-call...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-016-0276-7

    authors: Lamers D,van Biezen N,Martens D,Peters L,van de Zilver E,Jacobs-van Dreumel N,Wijffels RH,Lokman C

    更新日期:2016-05-27 00:00:00

  • Identification and characterization of an Endo-glucanase secreted from cellulolytic Escherichia coli ZH-4.

    abstract:BACKGROUND:In the previous study, the cellulolytic Escherichia coli ZH-4 isolated from bovine rumen was found to show extracellular cellulase activity and could degrade cellulose in the culture. The goal of this work was to identify and characterize the secreted cellulase of E. coli ZH-4. It will be helpful to re-under...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-019-0556-0

    authors: Pang J,Wang J,Liu Z,Zhang Q,Qi Q

    更新日期:2019-08-27 00:00:00

  • A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid.

    abstract:BACKGROUND:The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient qua...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-12-54

    authors: Sinah N,Williams CA,Piper RC,Shields SB

    更新日期:2012-08-23 00:00:00

  • Selective permeation and organic extraction of recombinant green fluorescent protein (gfpuv) from Escherichia coli.

    abstract:BACKGROUND:Transformed cells of Escherichia coli DH5-alpha with pGFPuv, induced by IPTG (isopropyl-beta-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-p...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-2-7

    authors: Vessoni Penna TC,Ishii M

    更新日期:2002-04-24 00:00:00

  • Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

    abstract:BACKGROUND:Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discover...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-017-0335-8

    authors: Ziemons S,Koutsantas K,Becker K,Dahlmann T,Kück U

    更新日期:2017-02-16 00:00:00

  • Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration.

    abstract:BACKGROUND:Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether at...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-018-0452-z

    authors: Watanabe Y,Matsuba T,Nakanishi M,Une M,Hanajima R,Nakashima K

    更新日期:2018-06-11 00:00:00

  • Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts.

    abstract:BACKGROUND:Rapid, robust and reversible induction of transgene expression would significantly facilitate cancer gene therapy as well as allow the in vivo functional study of newly discovered genes in tumor formation and progression. The popularity of the ecdysone inducible gene switch system has led us to investigate w...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-1-11

    authors: Karns LR,Kisielewski A,Gulding KM,Seraj JM,Theodorescu D

    更新日期:2001-01-01 00:00:00

  • Practical and reliable FRET/FLIM pair of fluorescent proteins.

    abstract:BACKGROUND:In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings. RE...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-9-24

    authors: Shcherbo D,Souslova EA,Goedhart J,Chepurnykh TV,Gaintzeva A,Shemiakina II,Gadella TW,Lukyanov S,Chudakov DM

    更新日期:2009-03-25 00:00:00

  • Thin films of Type 1 collagen for cell by cell analysis of morphology and tenascin-C promoter activity.

    abstract:BACKGROUND:The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-6-14

    authors: Langenbach KJ,Elliott JT,Tona A,McDaniel D,Plant AL

    更新日期:2006-03-06 00:00:00

  • Production of recombinant VP1-derived virus-like particles from novel human polyomaviruses in yeast.

    abstract:BACKGROUND:Eleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly int...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-015-0187-z

    authors: Norkiene M,Stonyte J,Ziogiene D,Mazeike E,Sasnauskas K,Gedvilaite A

    更新日期:2015-08-04 00:00:00

  • Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability.

    abstract:BACKGROUND:The recent H1N1 influenza pandemic illustrated the shortcomings of the vaccine manufacturing process. The A/California/07/2009 H1N1 pandemic influenza vaccine or A(H1N1)pdm09 was available late and in short supply as a result of delays in production caused by low yields and poor antigen stability. Recombinan...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-12-77

    authors: Feshchenko E,Rhodes DG,Felberbaum R,McPherson C,Rininger JA,Post P,Cox MM

    更新日期:2012-10-30 00:00:00

  • Purification of the recombinant green fluorescent protein from tobacco plants using alcohol/salt aqueous two-phase system and hydrophobic interaction chromatography.

    abstract:BACKGROUND:The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. RESULT...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/s12896-019-0590-y

    authors: Dong J,Ding X,Wang S

    更新日期:2019-12-09 00:00:00

  • A novel carboxyl-terminal protease derived from Paenibacillus lautus CHN26 exhibiting high activities at multiple sites of substrates.

    abstract:BACKGROUND:Carboxyl-terminal protease (CtpA) plays essential functions in posttranslational protein processing in prokaryotic and eukaryotic cells. To date, only a few bacterial ctpA genes have been characterized. Here we cloned and characterized a novel CtpA. The encoding gene, ctpAp (ctpA of Paenibacillus lautus), wa...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-13-89

    authors: Li Y,Pan Y,She Q,Chen L

    更新日期:2013-10-25 00:00:00

  • A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination.

    abstract:BACKGROUND:Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the...

    journal_title:BMC biotechnology

    pub_type: 杂志文章

    doi:10.1186/1472-6750-11-14

    authors: Vasconcelos EA,Santana CG,Godoy CV,Seixas CD,Silva MS,Moreira LR,Oliveira-Neto OB,Price D,Fitches E,Filho EX,Mehta A,Gatehouse JA,Grossi-De-Sa MF

    更新日期:2011-02-07 00:00:00