Use of a counterselectable transposon to create markerless knockouts from a 18,432-clone ordered M. bovis BCG mutant resource

2019 ◽  
Author(s):  
Katlyn Borgers ◽  
Kristof Vandewalle ◽  
Annelies Van Hecke ◽  
Gitte Michielsen ◽  
Evelyn Plets ◽  
...  
Keyword(s):  
Vaccine Strain ◽  
Vaccine Development ◽  
Cost Effective ◽  
Essential Genes ◽  
Selection Marker ◽  
Marker Genes ◽  
Gene Effects ◽  
Genome Wide ◽  
Effective Manner ◽  
Slow Growing

AbstractMutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach; despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian Pooling-Coordinate Sequencing allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which is undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described, which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the M. tb complex (18,432 clones, mutating 83% of the non-essential M. tb homologues), from which clean knockouts can be generated.ImportanceWhile speeding up research for many fields of biology (e.g. yeast, plant, and C. elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner, and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of M. bovis BCG Danish targets 83% of all non-essential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g. drug resistance research, vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the M. tb complex. The stretch to a full collection of mutants in all non-essential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

scholarly journals Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Katlyn Borgers ◽  
Kristof Vandewalle ◽  
Annelies Van Hecke ◽  
Gitte Michielsen ◽  
Evelyn Plets ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Vaccine Strain ◽  
Marker Genes ◽  
Gene Effects ◽  
Content Type ◽  
Genome Wide ◽  
Effective Manner ◽  
Slow Growing ◽  
Nonessential Genes

ABSTRACT Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated. IMPORTANCE While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

scholarly journals Systematic comparison of experimental assays and analytical pipelines for identification of active enhancers genome-wide

2021 ◽  
Author(s):  
Li Yao ◽  
Jin Liang ◽  
Abdullah Ozer ◽  
Alden King-Yung Leung ◽  
John T. Lis ◽  
...  
Keyword(s):  
Large Scale ◽  
K562 Cells ◽  
Cost Effective ◽  
Regulatory Elements ◽  
Nascent Transcript ◽  
Transcription Start Sites ◽  
The Road ◽  
Genome Wide ◽  
Active Enhancer ◽  
Effective Manner

Mounting evidence supports the idea that transcriptional patterns serve as more specific identifiers of active enhancers than histone marks; however, the optimal strategy to identify active enhancers both experimentally and computationally has not been determined. In this study, we compared 13 genome-wide RNA sequencing assays in K562 cells and showed that the nuclear run-on followed by cap-selection assay (namely, GRO/PRO-cap) has significant advantages in eRNA detection and active enhancer identification. We also introduced a new analytical tool, Peak Identifier for Nascent-Transcript Sequencing (PINTS), to identify active promoters and enhancers genome-wide and pinpoint the precise location of the 5′ transcription start sites (TSSs) within these regulatory elements. Finally, we compiled a comprehensive enhancer candidate compendium based on the detected eRNA TSSs available in 120 cell and tissue types. To facilitate the exploration and prioritization of these enhancer candidates, we also built a user-friendly web server (https://pints.yulab.org) for the compendium with various additional genomic and epigenomic annotations. With the knowledge of the best available assays and pipelines, this large-scale annotation of candidate enhancers will pave the road for selection and characterization of their functions in a time-, labor-, and cost-effective manner in future.

scholarly journals Searching for the human genetic factors standing in the way of universally effective vaccines

2015 ◽  
Vol 370 (1671) ◽  
pp. 20140341 ◽  
Author(s):  
Alexander J. Mentzer ◽  
Daniel O'Connor ◽  
Andrew J. Pollard ◽  
Adrian V. S. Hill
Keyword(s):  
Association Studies ◽  
Significant Proportion ◽  
Cost Effective ◽  
Genome Wide Association Studies ◽  
Genetic Determinants ◽  
Vaccine Response ◽  
Genetic Studies ◽  
Genome Wide ◽  
Effective Manner ◽  
Genetic Techniques

Vaccines have revolutionized modern public health. The effectiveness of some vaccines is limited by the variation in response observed between individuals and across populations. There is compelling evidence that a significant proportion of this variability can be attributed to human genetic variation, especially for those vaccines administered in early life. Identifying and understanding the determinants of this variation could have a far-reaching influence upon future methods of vaccine design and deployment. In this review, we summarize the genetic studies that have been undertaken attempting to identify the genetic determinants of response heterogeneity for the vaccines against hepatitis B, measles and rubella. We offer a critical appraisal of these studies and make a series of suggestions about how modern genetic techniques, including genome-wide association studies, could be used to characterize the genetic architecture of vaccine response heterogeneity. We conclude by suggesting how the findings from such studies could be translated to improve vaccine effectiveness and target vaccination in a more cost-effective manner.

scholarly journals High-throughput CRISPRi phenotyping in Streptococcus pneumoniae identifies new essential genes involved in cell wall synthesis and competence development

2016 ◽  
Author(s):  
Xue Liu ◽  
Clement Gallay ◽  
Morten Kjos ◽  
Arnau Domenech ◽  
Jelle Slager ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Vaccine Development ◽  
Teichoic Acid ◽  
Competence Development ◽  
Essential Genes ◽  
Large Set ◽  
Genome Wide ◽  
Opportunistic Human Pathogen

AbstractGenome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae. However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon-sequencing (Tn-Seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD1416 and SPD1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD1198 and SPD1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clpproteolytic system in regulation of competence development and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.

Strategic planning in grassland farming: Principles andapplications

1997 ◽  
pp. 191-197
Author(s):  
W.J. Parker ◽  
N.M. Shadbolt ◽  
D.I. Gray
Keyword(s):  
Strategic Planning ◽  
Performance Indicators ◽  
Cost Effective ◽  
Key Performance Indicators ◽  
Strategic Plans ◽  
Effective Manner ◽  
Farm Business ◽  
Pastoral Farming ◽  
Financial Environment

Three levels of planning can be distinguished in grassland farming: strategic, tactical and operational. The purpose of strategic planning is to achieve a sustainable long-term fit of the farm business with its physical, social and financial environment. In pastoral farming, this essentially means developing plans that maximise and best match pasture growth with animal demand, while generating sufficient income to maintain or enhance farm resources and improvements, and attain personal and financial goals. Strategic plans relate to the whole farm business and are focused on the means to achieve future needs. They should be routinely (at least annually) reviewed and monitored for effectiveness through key performance indicators (e.g., Economic Farm Surplus) that enable progress toward goals to be measured in a timely and cost-effective manner. Failure to link strategy with control is likely to result in unfulfilled plans. Keywords: management, performance

scholarly journals Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1072
Author(s):  
Raquel Cid ◽  
Jorge Bolívar
Keyword(s):  
Infectious Diseases ◽  
Mammalian Cells ◽  
Vaccine Development ◽  
Low Cost ◽  
Cost Effective ◽  
Insect Larvae ◽  
Subunit Vaccines ◽  
Effective Strategies ◽  
Alternative Systems ◽  
Inactivated Vaccines

To date, vaccination has become one of the most effective strategies to control and reduce infectious diseases, preventing millions of deaths worldwide. The earliest vaccines were developed as live-attenuated or inactivated pathogens, and, although they still represent the most extended human vaccine types, they also face some issues, such as the potential to revert to a pathogenic form of live-attenuated formulations or the weaker immune response associated with inactivated vaccines. Advances in genetic engineering have enabled improvements in vaccine design and strategies, such as recombinant subunit vaccines, have emerged, expanding the number of diseases that can be prevented. Moreover, antigen display systems such as VLPs or those designed by nanotechnology have improved the efficacy of subunit vaccines. Platforms for the production of recombinant vaccines have also evolved from the first hosts, Escherichia coli and Saccharomyces cerevisiae, to insect or mammalian cells. Traditional bacterial and yeast systems have been improved by engineering and new systems based on plants or insect larvae have emerged as alternative, low-cost platforms. Vaccine development is still time-consuming and costly, and alternative systems that can offer cost-effective and faster processes are demanding to address infectious diseases that still do not have a treatment and to face possible future pandemics.

scholarly journals The mutL Gene as a Genome-Wide Taxonomic Marker for High Resolution Discrimination of Lactiplantibacillus plantarum and Its Closely Related Taxa

2021 ◽  
Vol 9 (8) ◽  
pp. 1570
Author(s):  
Chien-Hsun Huang ◽  
Chih-Chieh Chen ◽  
Yu-Chun Lin ◽  
Chia-Hsuan Chen ◽  
Ai-Yun Lee ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Target Genes ◽  
Marker Genes ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Discrimination Power ◽  
Sequence Identity ◽  
Genome Wide ◽  
A Genome

The current taxonomy of the Lactiplantibacillus plantarum group comprises of 17 closely related species that are indistinguishable from each other by using commonly used 16S rRNA gene sequencing. In this study, a whole-genome-based analysis was carried out for exploring the highly distinguished target genes whose interspecific sequence identity is significantly less than those of 16S rRNA or conventional housekeeping genes. In silico analyses of 774 core genes by the cano-wgMLST_BacCompare analytics platform indicated that csbB, morA, murI, mutL, ntpJ, rutB, trmK, ydaF, and yhhX genes were the most promising candidates. Subsequently, the mutL gene was selected, and the discrimination power was further evaluated using Sanger sequencing. Among the type strains, mutL exhibited a clearly superior sequence identity (61.6–85.6%; average: 66.6%) to the 16S rRNA gene (96.7–100%; average: 98.4%) and the conventional phylogenetic marker genes (e.g., dnaJ, dnaK, pheS, recA, and rpoA), respectively, which could be used to separat tested strains into various species clusters. Consequently, species-specific primers were developed for fast and accurate identification of L. pentosus, L. argentoratensis, L. plantarum, and L. paraplantarum. During this study, one strain (BCRC 06B0048, L. pentosus) exhibited not only relatively low mutL sequence identities (97.0%) but also a low digital DNA–DNA hybridization value (78.1%) with the type strain DSM 20314T, signifying that it exhibits potential for reclassification as a novel subspecies. Our data demonstrate that mutL can be a genome-wide target for identifying and classifying the L. plantarum group species and for differentiating novel taxa from known species.

scholarly journals Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 186
Author(s):  
Jia-Huan Qu ◽  
Karen Leirs ◽  
Remei Escudero ◽  
Žiga Strmšek ◽  
Roman Jerala ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Fluorescent Protein ◽  
Cost Effective ◽  
Nitrilotriacetic Acid ◽  
Antibody Fragments ◽  
Rapid Screening ◽  
Red Fluorescent Protein ◽  
Effective Manner ◽  
Surface Regeneration ◽  
Sensitivity Specificity

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

scholarly journals Postnatal management of children with antenatal hydronephrosis

2020 ◽  
Vol 26 (1) ◽  
Author(s):  
Mohammed S. ElSheemy
Keyword(s):  
Renal Function ◽  
Urinary Tract Obstruction ◽  
Cost Effective ◽  
Voiding Cystourethrography ◽  
Main Body ◽  
Antenatal Hydronephrosis ◽  
Postnatal Management ◽  
Effective Manner ◽  
Lower Urinary Tract Obstruction

Abstract Background Postnatal management of infants with antenatal hydronephrosis (ANH) is still one of the most controversial issues. The majority of infants with ANH are asymptomatic with only few children who develop renal insufficiency. Thus, the biggest challenge for pediatric urologists is to distinguish children who will require further investigations and possible intervention prior to the development of symptoms, complications or renal damage in a cost effective manner without exposing them to the hazards of unnecessary investigations. Main body In this review article, literature on ANH were reviewed to present the current suggestions, recommendations, guidelines and their rational for postnatal management of ANH. It is agreed that a large portion of infants with ANH will improve; thus, the protocol of management is based mainly on observation and follow-up by ultrasound to detect either resolution, stabilization or worsening of hydronephrosis. The first 2 years of life are critical for this follow-up as the final picture is mostly reached during that period. Advanced imaging using voiding cystourethrography or renal scintigraphy are required for children at risk. Then, surgical intervention is selected only for a subgroup of these infants who showed worsening of hydronephrosis or renal function. Conclusions The protocol of management is based mainly on observation and follow-up by US to detect either resolution, stabilization or worsening of hydronephrosis. Postnatal evaluation should be performed for any neonate with a history ANH at any stage during pregnancy even if it was resolved during third trimester. Exclusion of UTI should be performed by urinalysis for all cases followed by urine culture if indicated. Serum creatinine should be performed especially in patients with bilateral ANH. US is the initial standard diagnostic imaging technique. Other imaging modalities like VCUG and nuclear renal scans may be required according to the results of the US evaluation. The most important items in decision making are the presence of bilateral or unilateral hydronephrosis, presence or absence of hydroureter, presence of lower urinary tract obstruction and degree of hydronephrosis on the initial postnatal US. Then an intervention is selected only for a subgroup of these patients who showed deterioration in renal function or degree of hydronephrosis or were complicated by UTIs. All these recommendations are based on the available literature. However, management of ANH is still a controversial issue due to lack of high evidence-based recommendations. Randomised controlled studies are still needed to provide a high level evidence for different aspects of management.

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