scholarly journals ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes

2018 ◽  
Author(s):  
Kenan C. Murphy ◽  
Samantha J. Nelson ◽  
Subhalaxmi Nambi ◽  
Kadamba Papavinasasundaram ◽  
Christina E. Baer ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Genome Engineering ◽  
Target Genes ◽  
Dna Recombination ◽  
Single Step ◽  
Recombination Site ◽  
New Paradigm ◽  
Double Stranded Dna ◽  
Simultaneous Integration ◽  
A Site

ABSTRACTCurrent methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic “targeting oligonucleotide” is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a “payload plasmid” that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT‐ and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes inM. tuberculosisandM. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target‐ or modification-specific double-stranded DNA recombination substrates.

scholarly journals ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Kenan C. Murphy ◽  
Samantha J. Nelson ◽  
Subhalaxmi Nambi ◽  
Kadamba Papavinasasundaram ◽  
Christina E. Baer ◽  
...  
Keyword(s):  
Drug Targets ◽  
Single Step ◽  
Selection Marker ◽  
Recombination Site ◽  
New Paradigm ◽  
Content Type ◽  
Simultaneous Integration ◽  
Bacterial Chromosomes ◽  
Recombinant Formation ◽  
A Site

ABSTRACTTwo efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic “targeting oligonucleotide” is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a “payload plasmid” that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for “oligonucleotide-mediatedrecombineering followed byBxb1integrasetargeting”) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this “drag and drop” strategy by the construction of insertions or deletions in over 100 genes inMycobacteriumtuberculosisandM. smegmatis.IMPORTANCEWe sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use inM. smegmatisandM. tuberculosis. By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1attPsite into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries anattBsite and a drug selection marker, we show both formation of a chromosomalattPsite and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

scholarly journals Tandem paired nicking promotes precise genome editing with scarce interference by p53

2019 ◽  
Author(s):  
Toshinori Hyodo ◽  
Md Lutfur Rahman ◽  
Sivasundaram Karnan ◽  
Takuji Ito ◽  
Atsushi Toyoda ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Dna Recombination ◽  
Genetic Changes ◽  
Double Stranded Dna ◽  
Cas9 Nuclease ◽  
Target Sites ◽  
P53 Activation ◽  
Additional Nucleotide

SummaryTargeted knock-in mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knock-in. Here, we demonstrate that tandem paired nicking, a method for targeted knock-in involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knock-in largely equivalent to that of the Cas9 nuclease-based approach. Tandem paired nicking seems to accomplish targeted knock-in via DNA recombination analogous to Holliday’s model, and creates intended genetic changes in the genome without introducing additional nucleotide changes such as silent mutations. Targeted knock-in through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

scholarly journals Enhanced mating-type switching and sexual hybridization in heterothallic yeast Yarrowia lipolytica

2020 ◽  
Vol 20 (2) ◽  
Author(s):  
Changpyo Han ◽  
Heeun Kwon ◽  
Gyuyeon Park ◽  
Minjeong Jang ◽  
Hye-Jeong Lee ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Mating Type ◽  
Genome Engineering ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Industrial Applications ◽  
Single Step ◽  
Knockout Mutant ◽  
Wide Range ◽  
Mating Type Switching

ABSTRACT Yarrowia lipolytica is a non-conventional, heterothallic, oleaginous yeast with wide range of industrial applications. Increasing ploidy can improve advantageous traits for industrial applications including genetic stability, stress resistance, and productivity, but the construction of knockout mutant strains from polyploid cells requires significant effort due to the increased copy numbers of target genes. The goal of this study was to evaluate the effectiveness of a mating-type switching strategy by single-step transformation without a genetic manipulation vestige, and to optimize the conventional method for increasing ploidy (mating) in Y. lipolytica. In this study, mating-type genes in haploid Y. lipolytica cells were scarlessly converted into the opposite type genes by site-specific homologous recombination, and the resulting MATB-type cells were mated at low temperature (22°C) with addition of sodium citrate with each MATA-type haploid cell to yield a MATA/MATB-type diploid strain with genetic information from both parental strains. The results of this study can be used to increase ploidy and for whole genome engineering of a yeast strain with unparalleled versatility for industrial application.

scholarly journals In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1649-1663
Author(s):  
Oliver Z Nanassy ◽  
Kelly T Hughes
Keyword(s):  
Amino Acids ◽  
Biochemical Analysis ◽  
Mutational Analysis ◽  
Recombination Reaction ◽  
Recombination Site ◽  
Dna Inversion ◽  
Hin Recombinase ◽  
One Step ◽  
A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

scholarly journals Structural studies on M. tuberculosis O6-methylguanine methyltransferase

2014 ◽  
Vol 70 (a1) ◽  
pp. C832-C832
Author(s):  
Menico Rizzi ◽  
Riccardo Miggiano ◽  
Samarpita Lahiri ◽  
Giuseppe Perugino ◽  
Maria Ciaramella ◽  
...  
Keyword(s):  
Excision Repair ◽  
Single Step ◽  
Double Stranded Dna ◽  
Nucleotide Excision ◽  
Enzymatic Systems ◽  
Methylguanine Methyltransferase ◽  
Mutagenic Effects ◽  
Wild Type Form

Mycobacterium tuberculosis (MTB) is an extremely well adapted human pathogen capable to survive for decades inside the hostile environment represented by the host's infected macrophages despite exposure to multiple potential DNA-damaging stresses. In order to maintain a remarkable low level of genetic diversity, MTB deploys different strategies of DNA repair, including multi-enzymatic systems, such as Nucleotide Excision Repair, and single-step repair. In particular, to counteract the mutagenic effects of DNA alkylation, MTB performs the direct alkylated-base reversal by sacrificing one molecule of a DNA-protein alkyltransferase, such as O6-methylguanine methyltransferase (OGT; orf: Rv1316c). We present here the biochemical and structural characterization of recombinant mycobacterial OGT (MtOGT) in its wild-type form along with its mutated variants mimicking the ones occurring in relevant clinical strains (i.e. MtOGT-T15S and MtOGT-R37L). Our studies reveal that MtOGT-R37L is severely impaired in its activity as consequence of its ten-fold lower affinity for modified double-stranded DNA (dsDNA) (1). Further investigations on a new structure-based panel of OGT versions, designed to explore different molecular environment at position 37, allowed us a better understanding of the functional role of the MtOGT Arg37-bearing loop during catalysis. Moreover, we solved the crystal structure of MtOGT in covalent complex with modified dsDNA that reveals an unprecedented MtOGT::DNA architecture, suggesting that the MtOGT monomer performing the catalysis needs assisting unreacted subunits during cooperative DNA binding. This work is supported by European Community FP7 program SYSTEMTB (Health-F4-2010-241587)

scholarly journals Development of oriC-Based Plasmids for Mesoplasma florum

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Dominick Matteau ◽  
Marie-Eve Pepin ◽  
Vincent Baby ◽  
Samuel Gauthier ◽  
Mélissa Arango Giraldo ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Genetic Engineering ◽  
Genome Engineering ◽  
Antibiotic Resistance Genes ◽  
Viable Cell ◽  
Pathogenic Potential ◽  
Strong Basis ◽  
Content Type ◽  
Basic Biology

ABSTRACT The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10−6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10−6 and 8.44 × 10−7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

Urban Transformation in Ancient Molise

2021 ◽  
Author(s):  
Elizabeth C. Robinson
Keyword(s):  
Social Transformation ◽  
Local Level ◽  
Urban Transformation ◽  
Gradual Change ◽  
New Paradigm ◽  
Complex Processes ◽  
Global Movements ◽  
Social War ◽  
A Site ◽  
Cultural Choices

This book uses all the available evidence to create a site biography of Larinum from 400 bce to 100 ce, with a focus on the urban transformation that occurs there during the Roman conquest. Larinum, a pre-Roman town in the modern region of Molise, undergoes a unique transition from independence to municipal status when it receives Roman citizenship in the 80s bce shortly after the Social War. Its trajectory illuminates complex processes of cultural, social, and political change associated with the Roman conquest throughout the Italian peninsula in the first millennium bce. This work highlights the importance of local isolated variability in studies of the Roman conquest and provides a narrative that supplements larger works on this theme. Through a focus on local-level agency, it demonstrates strong local continuity in Larinum and its surrounding territory. This continuity is the key to Larinum’s transition into the Roman state, which is spearheaded by the local elites. They participate in the broader cultural choices of the Hellenistic koiné and strive to be part of a Mediterranean-wide dialog that, over time, will come to be dominated by Rome. The case is made for advancing the field of Roman conquest studies under a new paradigm of social transformation that focuses on a history of gradual change, continuity, connectivity, and local isolated variability that is contingent on highly specific issues rather than global movements.

Personalization in the Information Era

2011 ◽  
pp. 3059-3064
Author(s):  
José Juan Pazos-Arias ◽  
Martín López-Nores
Keyword(s):  
Information Overload ◽  
Large Body ◽  
Communication Technologies ◽  
Digital Subscriber Line ◽  
New Paradigm ◽  
Entertainment Education ◽  
Open Problems ◽  
Amount Of Information ◽  
A Site ◽  
Watching Tv

We are witnessing the development of new communication technologies (e.g., DTV networks [digital TV], 3G [thirdgeneration] telephony, and DSL [digital subscriber line]) and a rapid growth in the amount of information available. In this scenario, users were supposed to benefit extensively from services delivering news, entertainment, education, commercial functionalities, and so forth. However, the current situation may be better referred to as information overload; as it frequently happens that users are faced with an overwhelming amount of information. A similar situation was noticeable in the 1990s with the exponential growth of the Internet, which made users feel disoriented among the myriad of contents available through their PCs. This gave birth to search engines (e.g., Google and Yahoo) that would retrieve relevant Web pages in response to user-entered queries. These tools proved effective, with millions of people using them to find pieces of information and services. However, the advent of new devices (DTV receivers, mobile phones, media players, etc.) introduces consumption and usage habits that render the search-engine paradigm insufficient. It is no longer realistic to think that users will bother to visit a site, enter queries describing what they want, and select particular contents from among those in a list. The reasons may relate to users adopting a predominantly passive role (e.g., while driving or watching TV), the absence of bidirectional communication (as in broadcasting environments), or users feeling uneasy with the interfaces provided. To tackle these issues, a large body of research is being devoted nowadays to the design and provision of personalized information services, with a new paradigm of recommender systems proactively selecting the contents that match the interests and needs of each individual at any time. This article describes the evolution of these services, followed by an overview of the functionalities available in diverse areas of application and a discussion of open problems. Background The development of personalized information

scholarly journals Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L)

2020 ◽  
Vol 2 ◽  
Author(s):  
Limin Hu ◽  
Olalekan Amoo ◽  
Qianqian Liu ◽  
Shengli Cai ◽  
Miaoshan Zhu ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Molecular Breeding ◽  
Target Genes ◽  
Agronomic Traits ◽  
Crop Improvement ◽  
Brassica Napus L ◽  
Editing Efficiency ◽  
Base Editing ◽  
Expected Gain ◽  
Genome Wide

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

scholarly journals Capture of a Transition State Using Molecular Dynamics: Creation of an Intercalation Site in dsDNA with Ethidium Cation

2010 ◽  
Vol 2010 ◽  
pp. 1-4 ◽  
Author(s):  
Regina R. Monaco
Keyword(s):  
External Surface ◽  
Computational Study ◽  
Helical Axis ◽  
Base Pairs ◽  
Bond Rotation ◽  
Double Stranded Dna ◽  
Dna Backbone ◽  
Interaction Measure ◽  
The Creation ◽  
A Site

The mechanism of intercalation and the ability of double stranded DNA (dsDNA) to accommodate a variety of ligands in this manner has been well studied. Proposed mechanistic steps along this pathway for the classical intercalator ethidium have been discussed in the literature. Some previous studies indicate that the creation of an intercalation site may occur spontaneously, with the energy for this interaction arising either from solvent collisions or soliton propagation along the helical axis. A subsequent 1D diffusional search by the ligand along the helical axis of the DNA will allow the ligand entry to this intercalation site from its external, electrostatically stabilized position. Other mechanistic studies show that ethidium cation participates in the creation of the site, as a ligand interacting closely with the external surface of the DNA can cause unfavorable steric interactions depending on the ligands' orientation, which are relaxed during the creation of an intercalation site. Briefly, such a site is created by the lengthening of the DNA molecule via bond rotation between the sugars and phosphates along the DNA backbone, causing an unwinding of the dsDNA itself and separation between the adjacent base pairs local to the position of the ligand, which becomes the intercalation site. Previous experimental measurements of this interaction measure the enthalpic cost of this part of the mechanism to be about −8 kcal/mol. This paper reports the observation, during a computational study, of the spontaneous opening of an intercalation site in response to the presence of a single ethidium cation molecule in an externally bound configuration. The concerted motions between this ligand and the host, a dsDNA decamer, are clear. The dsDNA decamer AGGATGCCTG was studied; the central site was the intercalation site.

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