Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

Liu, Yilan; Chen, Jinjin; Thygesen, Anders

doi:10.1007/s12033-017-0050-7

Cited by 6 publications

(6 citation statements)

References 15 publications

(27 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The addition of a fourth primer (in this case, 27F) not only enables the production of 16S rRNA gene fragments of noncarrier cells, but also increases the yield of the fusion product (data not shown). Higher outer primer concentration (compared to inner primers) may enhance the production of fusion concatemer (Liu et al 2018). Thus, we tested several primer ratios and total concentrations in order to achieve the most efficient production of bla_16S concatemer.…”

Section: Discussionmentioning

confidence: 99%

Single-cell resolution genetic association analysis of heterogeneous bacterial communities by utilizing droplet digital PCR

Dutra

Jalasvuori

Franz

et al. 2022

Preprint

View full text Add to dashboard Cite

Microbial communities often respond to various challenges, such as the presence of antibiotics, as a whole. Dissecting these community-level effects into separate acting entities requires the identification of organisms that carry functional genes for the observed feature. However, unculturable microbes abound in various environments, hence making the identification challenging. Here we present a cultivation-free technique that can be utilized to link functional genes with carrying bacterial species at single-cell resolution. The developed protocol is relatively simple to use, utilizes commercially available droplet microfluidics devices, does not require toxic reagents (as compared to some previous methods), eliminates invalid signals emerging from extracellular DNA, and allows simultaneous analysis of community diversity via 16S rRNA gene sequencing. The method can be customized for any given genetic trait to accurately identify its hosting subpopulation from a heterogeneous and potentially uncultivable bacterial community.

show abstract

Section: Discussionmentioning

confidence: 99%

Single-cell resolution genetic association analysis of heterogeneous bacterial communities by utilizing droplet digital PCR

Dutra

Jalasvuori

Franz

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…To avoid potential off-targets, a systematic BLAST search was conducted for sgRNA candidates. DNA fragments of sgRNA and homologous arms were either ordered from Twist Bioscience or constructed by one-step fusion PCR . Plasmids were constructed using restriction enzyme digestion of vector DNA followed by ligation, with standard NEB Restriction Endonuclease protocols used for restriction digests.…”

Section: Methodsmentioning

confidence: 99%

Genetic Engineering of Acidithiobacillus ferridurans Using CRISPR Systems To Mitigate Toxic Release in Biomining

Chen,

Liu,

Mahadevan

2023

Environ. Sci. Technol.

Self Cite

View full text Add to dashboard Cite

Biomining processes utilize microorganisms, such as Acidithiobacillus, to extract valuable metals by producing sulfuric acid and ferric ions that dissolve sulfidic minerals. However, excessive production of these compounds can result in metal structure corrosion and groundwater contamination. Synthetic biology offers a promising solution to improve Acidithiobacillus strains for sustainable, eco-friendly, and cost-effective biomining, but genetic engineering of these slow-growing microorganisms is challenging with current inefficient and time-consuming methods.To address this, we established a CRISPR-dCas9 system for gene knockdown in A. ferridurans JAGS, successfully downregulating the transcriptional levels of two genes involved in sulfur oxidation. More importantly, we constructed an all-in-one CRISPR-Cas9 system for fast and efficient genome editing in A. ferridurans JAGS, achieving seamless gene deletion (HdrB3), promoter substitution (Prus to Ptac), and exogenous gene insertion (GFP). Additionally, we created a HdrB-Rus double-edited strain and performed biomining experiments to extract Ni from pyrrhotite tailings. The engineered strain demonstrated a similar Ni recovery rate to wild-type A. ferridurans JAGS but with significantly lower production of iron ions and sulfuric acid in leachate. These high-efficient CRISPR systems provide a powerful tool for studying gene functions and creating useful recombinants for synthetic biology-assisted biomining applications in the future.

show abstract

“…A systematic BLAST search for sgRNA candidates was performed to avoid potential off-targets. DNA fragments of sgRNA and homologous arms used for plasmid construction were ordered from Twist Bioscience or constructed by one-step fusion PCR [32]. Plasmids were constructed by restriction enzyme digestion of vector DNA followed by Ligation.…”

Section: Methodsmentioning

confidence: 99%

“…Bioscience or constructed by one-step fusion PCR [32]. Plasmids were constructed by restriction enzyme digestion of vector DNA followed by Ligation.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Genetic engineering of Acidithiobacillus ferridurans with CRISPR-Cas9/dCas9 systems

chen

Liu

Mahadevan

2022

Preprint

Self Cite

View full text Add to dashboard Cite

Genus Acidithiobacillus includes a group of Gram-negative Fe/S-oxidizing acidophilic chemolithotrophic bacteria that are extensively studied and used for biomining processes. Synthetic biology approaches are key means to study and improve their biomining performance. However, efficient genetic manipulations in Acidithiobacillus are still major bottlenecks. In this study, we report a simple and efficient pAFi system (CRISPR-dCas9) and a scarless pAF system (CRISPR-Cas9) for genetic manipulations in A. ferridurans JAGS. The pAFi system harboring both dCas9 and sgRNA was constructed based on pBBR1MCS-2 to knockdown HdrA and TusA genes, separately, of which the transcription levels were significantly downregulated by 48% and 93%, separately. The pAF system carrying pCas9-sgRNA-homology arms was constructed based on pJRD215 to delete HdrB3 gene and overexpress Rus gene. Our results demonstrated that the pAF system is a fast and efficient genome editing method with an average rate of 15-20% per transconjugant in one recombination event, compared to 10-3 and then 10-2 in two recombination events by traditional markerless engineering strategy. Moreover, with these two systems, we successfully regulated iron and sulfur metabolisms in A. ferridurans JAGS: the deletion of HdrB3 reduced 48% of sulfate production, and substitution overexpression of Rus promoter showed 8.82-fold of mRNA level and enhanced iron oxidation rate. With these high-efficient genetic tools for A. ferridurans, we will be able to study gene functions and create useful recombinants for biomining applications. Moreover, these systems could be extended to other Acidithiobacillus strains and promote the development of synthetic biology-assisted biomining.

show abstract

Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

Cited by 6 publications

References 15 publications

Single-cell resolution genetic association analysis of heterogeneous bacterial communities by utilizing droplet digital PCR

Single-cell resolution genetic association analysis of heterogeneous bacterial communities by utilizing droplet digital PCR

Genetic Engineering of Acidithiobacillus ferridurans Using CRISPR Systems To Mitigate Toxic Release in Biomining

Genetic engineering of Acidithiobacillus ferridurans with CRISPR-Cas9/dCas9 systems

Contact Info

Product

Resources

About