Recombinant Bacillus phage phi15 Early protein GP17 (17)
Description
DNA Replication Modulation
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In phage phi29, protein p17 (homolog of GP17) is essential for replication initiation under low multiplicity of infection (MOI). It recruits limiting replication factors to viral DNA origins, enhancing binding of origin-binding protein p6 .
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Proposed mechanism: Oligomerization of GP17 stabilizes protein-DNA complexes, ensuring efficient replication initiation .
Expression and Engineering
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Cloning: GP17 from Bacillus phage PZA (UniProt ID: P08389) has been recombinantly expressed in yeast, yielding a 174-amino-acid protein with a His-tag for purification .
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Storage: Lyophilized form stable for 12 months at -80°C; reconstitution in sterile water with 50% glycerol recommended .
Biotechnological Utility
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Growth Decoupling: In phi15-engineered P. putida systems, phage proteins like gp16 (not GP17) inhibit host RNA polymerase, diverting resources to recombinant protein production .
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Biofilm Degradation: Phi15’s depolymerase (unrelated to GP17) reduces P. putida biofilms by 20–40% within 8 hours, highlighting the potential of phage proteins in biofilm control .
Research Gaps and Future Directions
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Functional Validation: Direct studies on phi15 GP17 are scarce. Homology modeling and knockout experiments are needed to confirm its role in replication or structure.
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SynBio Applications: Phi15’s transcriptional machinery (e.g., RNA polymerase) has been optimized for high-yield expression in P. putida . Integrating GP17 into such systems could enhance phage-based biotechnology.
Comparative Analysis of Phage GP17 Homologs
Product Specs
Target Background
Q&A
Experimental Design for Studying GP17 Function
Q: How can researchers design experiments to study the function of GP17 in the context of phage phi15 replication? A: To study GP17, researchers can employ a combination of genetic and biochemical approaches. This includes constructing recombinant plasmids expressing GP17, analyzing its interaction with other phage proteins, and assessing its role in DNA replication using in vitro assays. Additionally, comparing GP17 sequences across related phages like PZA and phi29 can provide insights into conserved regions essential for function .
Data Contradiction Analysis in GP17 Studies
Q: How can researchers resolve contradictions in data regarding the functional importance of different regions of GP17? A: Contradictions can arise from differences in experimental conditions or methodologies. To resolve these, researchers should conduct comparative analyses using standardized protocols across different strains. For instance, comparing the effects of deletions in the central and 3'-end-proximal parts of GP17 on phage replication efficiency can help identify critical regions .
Advanced Research Questions: GP17 Evolution
Q: What advanced techniques can be used to study the evolutionary reorganization of GP17 in the phi15 phage family? A: Advanced techniques such as phylogenetic analysis and comparative genomics can be employed to study the evolutionary dynamics of GP17. This involves aligning GP17 sequences from various phages, constructing phylogenetic trees, and analyzing gene rearrangements over time. Additionally, experimental evolution studies can provide insights into how GP17 adapts under selective pressure .
Methodological Considerations for GP17 Expression Systems
Q: How can researchers optimize expression systems for GP17 in non-native hosts like Pseudomonas putida? A: Optimizing GP17 expression in Pseudomonas putida involves using orthogonal promoters and fine-tuning expression levels. This can be achieved by employing systems like the phi15 RNAP, which offers a stringent and tunable expression platform. Additionally, using growth-decoupling strategies with proteins like phi15 gp16 can enhance protein yield without affecting cell viability .
Bioinformatics Tools for GP17 Analysis
Q: What bioinformatics tools are available for annotating and analyzing GP17 sequences? A: Researchers can use tools like Phamerator for comparative genomics and genome annotation. This software helps in identifying putative genes, understanding genomic arrangements, and predicting protein functions. Additionally, databases like PhagesDB provide a repository for phage genomes and annotations .
Comparative Genomics of GP17 Across Phage Families
Q: How can researchers compare GP17 across different phage families to identify conserved motifs? A: Comparative genomics involves aligning GP17 sequences from various phages and identifying conserved regions. This can be done using software like MUSCLE or ClustalW for sequence alignment. Conserved motifs can indicate functional importance and provide targets for mutagenesis studies .
Challenges in GP17 Expression and Purification
Q: What challenges might researchers face in expressing and purifying GP17, and how can these be overcome? A: Challenges include low expression levels and protein instability. These can be addressed by optimizing expression vectors, using chaperones to enhance protein folding, and employing affinity purification methods to improve yield and purity. Additionally, using host strains with reduced protease activity can help minimize protein degradation .
Integrating GP17 into Synthetic Biology Applications
Q: How can GP17 be integrated into synthetic biology applications, such as novel expression systems? A: GP17 can be integrated into synthetic biology by designing modular expression systems that utilize its replication functions. For example, using GP17 in combination with other phage components like phi15 RNAP can create robust and tunable expression platforms for various hosts .
Phenotypic Characterization of Phages with GP17 Variants
Q: How can researchers characterize the phenotypic effects of GP17 variants on phage behavior? A: Phenotypic characterization involves assessing parameters like host range, plaque morphology, and one-step growth curves. Variants of GP17 can be engineered and tested in these assays to understand how changes in GP17 affect phage replication and infection dynamics .
Genomic Integration of GP17 for Stable Expression
Q: What strategies can be used to integrate GP17 into host genomes for stable expression? A: Stable genomic integration can be achieved by using CRISPR-Cas systems or homologous recombination techniques. This ensures consistent expression levels and reduces the risk of plasmid loss. Additionally, using weak promoters and optimized ribosome binding sites can help control expression levels .
Example Data Table: GP17 Sequence Conservation
| Phage | GP17 Sequence Length | Conserved Region |
|---|---|---|
| phi15 | 450 bp | 5'-end-proximal |
| PZA | 420 bp | Similar to phi15 |
| phi29 | 480 bp | Highly conserved |
