Applications of Serine Integrases in Synthetic Biology over the Past Decade

Ba, Fang; Zhang, Yufei; Wang, Luyao; Liu, Wan-Qiu; Li, Jian

doi:10.3390/synbio1020012

Cited by 5 publications

(2 citation statements)

References 134 publications

(243 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Integrated DNA is more stable in the host and reduces the need for antibiotic-based selection. Serine integrase and recombinase-based systems can remove host range limitations of a replicating plasmid and the efficiency of native homologous recombination to evaluate heterologous genes and pathways (Ba et al., 2023 ; Elmore et al., 2023 ).…”

Section: Discussionmentioning

confidence: 99%

Application of functional genomics for domestication of novel non-model microbes

Bales,

Vergara,

Eckert

2024

Journal of Industrial Microbiology and Biotechnology

View full text Add to dashboard Cite

With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. One-Sentence Summary The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

show abstract

Section: Discussionmentioning

confidence: 99%

Application of functional genomics for domestication of novel non-model microbes

Bales,

Vergara,

Eckert

2024

Journal of Industrial Microbiology and Biotechnology

View full text Add to dashboard Cite

show abstract

“…Analyses of the evolutionary position of this phage have shown that it may have originated from a P2-type temperate phage and nonproliferative homologous fragments of this phage exist in several A. hydrophila strains, as phiO18P is also a temperate phage and has a ORF of phage major capsid protein of the P2 family in its genome ( 67 ). Integrase drives recombination between the phage and specific attachment sites on the host chromosome known as attP and attB during the integration process ( 68 ), while holin can accumulate in the bacterial cytosol and eventually penetrate the peptidoglycan layer, and can achieve control of the lytic cycle through self-regulation of its concentration ( 69–71 ). This result suggests that the genome of phiA051 is characterized by both temperate phage and virulent phage.…”

Section: Discussionmentioning

confidence: 99%

Biologic and genomic characterization of a novel virulent Aeromonas hydrophila phage phiA051, with high homology to prophages

Wang,

Tong,

Jiang

et al. 2024

Front. Vet. Sci.

View full text Add to dashboard Cite

IntroductionAeromonas hydrophila is particularly harmful to freshwater aquaculture, and the search for phage is an effective biological control method, but reports of possible temperate phages and their mutants are rare in this field. In this study, a virulent phage highly homologous to prophage in the genomes of A. hydrophila was collected and preliminary biological characterization was carried out to understand its nature.Materials and methodsWater samples taken from eel ponds in Fujian, China were combined with the strain. Spot test method and double-layer agar plate assay was used for confirmation and purification. Phage virions were observed using transmission electron microscope. A total of 68 strains of Aeromonas spp. were used to determine the host range. MOI groups of 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 were prepared to detect the optimal MOI. The conditions of thermal stability assay were set as 30, 40, 50, 60, 70 and 80°C for 1 h, respectively, and conditions of acid and alkali stability assay were set as 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 of pH. MOI of 0.01 and 0.1, respectively, are set to determine the inhibitory capacity of phage.ResultsA novel virulent A. hydrophila phage designated phiA051 has been isolated from aquaculture water. Electron microscopic observation showed that the phage phiA051 was composed of an icosahedral capsid. The phage phiA051 possesses an optimal multiplicity of infection (MOI) of 0.01, and its burst size was 108 PFU/cell. The phage maintained a high viability at temperatures of 30–50°C or pH 6.0–10.0 for 1 h. Phage phiA051 has certain potentials in rapidly inhibiting the spread of pathogen early in the outbreak, and it has a linear dsDNA with GC content of 60.55% and a total length of 32,212 bp, including 46 ORFs.DiscussionThe phage phiA051 behaved as a virulent phage. However, the BLASTN result showed that 23 of the top 25 hits were genomes of Aeromonas strains. It was suggested that phiA051 was probably derived from some prophage in the chromosome of Aeromonas. Further investigation of the mechanism how phage phiA051 transforms from a temperate phage to a virulent phage will provide a unique perspective and idea to explore the potential of prophages.

show abstract

Orthogonal Serine Integrases Enable Scalable Gene Storage Cascades in Bacterial Genome

Zhang,

Ba,

Huang

et al. 2024

ACS Synth. Biol.

View full text Add to dashboard Cite

Genome integration enables host organisms to stably carry heterologous DNA messages, introducing new genotypes and phenotypes for expanded applications. While several genome integration approaches have been reported, a scalable tool for DNA message storage within site-specific genome landing pads is still lacking. Here, we introduce an iterative genome integration method utilizing orthogonal serine integrases, enabling the stable storage of multiple heterologous genes in the chromosome of Escherichia coli MG1655. By leveraging serine integrases TP901-1, Bxb1, and PhiC31, along with engineered integration vectors, we demonstrate high-efficiency, marker-free integration of DNA fragments up to 13 kb in length. To further simplify the procedure, we then develop a streamlined integration method and showcase the system's versatility by constructing an engineered E. coli strain capable of storing and expressing multiple genes from diverse species. Additionally, we illustrate the potential utility of these engineered strains for synthetic biology applications, including in vivo and in vitro protein expression. Our work extends the application scope of serine integrases for scalable gene integration cascades, with implications for genome manipulation and gene storage applications in synthetic biology.

show abstract

Applications of Serine Integrases in Synthetic Biology over the Past Decade

Cited by 5 publications

References 134 publications

Application of functional genomics for domestication of novel non-model microbes

Application of functional genomics for domestication of novel non-model microbes

Biologic and genomic characterization of a novel virulent Aeromonas hydrophila phage phiA051, with high homology to prophages

Orthogonal Serine Integrases Enable Scalable Gene Storage Cascades in Bacterial Genome

Contact Info

Product

Resources

About