Part:BBa_K5487110
PHCY-yahK-KO
The PHCY-yahK-KO plasmid is a specialized tool designed for precise gene editing within Escherichia coli (E. coli). This plasmid, based on the PHCY163 backbone, is a part of a dual-plasmid system used in conjunction with PHCY25A. It is meticulously engineered to target the yahK gene for knockout, utilizing the CRISPR-Cas9 system coupled with sophisticated recombination techniques for gene editing.
Composition of PHCY-yahK-KO Plasmid: yahK-sgRNA: Specifically designed single-guide RNA targeting the yahK gene, integral for directing the Cas9 protein to the desired genomic location to create a double-strand break. This sgRNA is essential for the specificity of the CRISPR intervention. sgRNA-HA1/2: Homology arms flanking the sgRNA sequence, facilitating precise homologous recombination for gene knockout. These sequences enable the E. coli DNA repair machinery to accurately delete or replace the targeted gene segment. Rep101: A protein essential for plasmid replication within bacterial cells, ensuring stable maintenance of the plasmid during cell division. pSC101 ori: A replication origin that allows for the stable, low-copy replication of the plasmid in E. coli, balancing efficacy with minimal metabolic burden on the host. AmpR: Ampicillin resistance gene providing a selection marker for maintaining and identifying successfully transformed cells in antibiotic-containing media. AmpR promoter: Drives the expression of the Ampicillin resistance gene, crucial for effective selection in antibiotic media. M13 fwd and M13 rev: Primer binding sites useful for sequencing and further subcloning efforts, allowing for verification of plasmid integrity and modifications. MCS (Multiple Cloning Site): Contains unique restriction sites facilitating easy insertion or removal of DNA segments, providing flexibility for custom genetic manipulations. lac operator and lac promoter: Elements of the lac operon that regulate the expression of downstream genes in response to environmental conditions, specifically the presence of lactose or IPTG. CAP binding site: Works in conjunction with the lac promoter to enhance gene expression in the presence of cAMP, integral for regulated gene expression. araBAD promoter: An inducible promoter activated in the presence of arabinose, used for precise control over gene expression. sgRNA scaffold: Structural component that supports the correct formation of the sgRNA-Cas9 complex, critical for effective targeting and gene editing. rrnB T1 terminator: Ensures proper termination of transcription, preventing runaway transcripts and maintaining genetic stability.
Functionality and Applications: Gene Knockout: Designed primarily to facilitate the knockout of the yahK gene in E. coli, the plasmid utilizes the CRISPR-Cas9 system to introduce precise genetic alterations. The inclusion of homology arms enables seamless deletion or replacement of genomic sequences through homologous recombination. Functional Genomics and Metabolic Engineering: The plasmid can be used to study gene function, investigate metabolic pathways, or develop bacterial strains with novel properties for industrial, environmental, or pharmaceutical applications. Research and Development: Ideal for academic and commercial research environments where modification of bacterial genomes is required to understand gene function, regulate biochemical pathways, or produce metabolites.
Usage Guidelines: Transformation: Introduce PHCY-yahK-KO into E. coli cells alongside the PHCY25A plasmid, which supplies the Cas9 and RED recombineering system. Use heat shock or electroporation methods followed by selection on ampicillin-containing media. Induction of Editing: Culture the transformed cells under conditions that induce the expression of the araBAD and lac promoters, typically in the presence of arabinose or IPTG, to activate sgRNA and Cas9 expression. Verification: Post-transformation, use PCR and sequencing techniques to confirm the successful incorporation and function of the sgRNA, homology arms, and any edits made to the genome. Application: Employ the modified strains for further experimental analysis, production processes, or as a model for studying bacterial genetics.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4317
Illegal XbaI site found at 4290
Illegal SpeI site found at 1277
Illegal PstI site found at 4278 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4317
Illegal SpeI site found at 1277
Illegal PstI site found at 4278
Illegal NotI site found at 1596 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4317
Illegal BamHI site found at 32
Illegal BamHI site found at 4296
Illegal XhoI site found at 13 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4317
Illegal XbaI site found at 4290
Illegal SpeI site found at 1277
Illegal PstI site found at 4278 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4317
Illegal XbaI site found at 4290
Illegal SpeI site found at 1277
Illegal PstI site found at 4278 - 1000COMPATIBLE WITH RFC[1000]
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