Part:BBa_K5487110:Design
PHCY-yahK-KO
- 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]
Design Notes
1. Target Specificity and Efficiency of sgRNA: Sequence Selection: The sgRNA sequence targeting the yahK gene was carefully designed to ensure maximum specificity and minimal off-target effects. Bioinformatics tools were used to analyze potential target sites within the yahK gene to select a sequence that would likely result in highly effective gene editing with the least possible impact on other genomic regions. sgRNA Scaffold Compatibility: The sgRNA scaffold was designed to ensure optimal interaction with the Cas9 protein, maintaining the correct tertiary structure for effective binding and cleavage. 2. Homology Arm Design: Length and Sequence Accuracy: Homology arms (HA1 and HA2) were designed to be sufficiently long (typically 500-1000 base pairs) to promote effective homologous recombination. The sequences were matched exactly to the genomic regions flanking the target site to facilitate precise and efficient repair mechanisms. Optimization for Recombination Efficiency: The sequences were checked for repetitive elements and secondary structures that could impede recombination processes, ensuring that the homology arms would integrate smoothly without structural issues. 3. Replication and Stability: Origin of Replication (pSC101 ori): The choice of a low-copy number origin like pSC101 ori was intentional to reduce the metabolic burden on host cells, which can help maintain cell viability and stability during cloning and expression phases. Plasmid Maintenance Proteins (e.g., Rep101): Inclusion of replication maintenance proteins ensured that the plasmid could be stably maintained in the host cells across multiple generations, crucial for long-term studies. 4. Antibiotic Selection Marker: Effectiveness and Safety: The ampicillin resistance gene (AmpR) was included with its native promoter to ensure effective selection in antibiotic media. The design also considered the potential impact of antibiotic resistance on host cell metabolism and the ecological implications of resistance gene dissemination. 5. Regulatory Elements for Controlled Expression: Inducible Promoters: Elements like the lac promoter, araBAD promoter, and associated regulatory sites (CAP binding site, lac operator) were incorporated to allow precise control over the expression of editing components. This enables researchers to activate or suppress gene editing mechanisms as needed, depending on the experimental conditions. Termination Sequences: The inclusion of reliable termination sequences like the rrnB T1 terminator ensured that transcription stops appropriately, preventing read-through that could affect downstream genes or plasmid stability. 6. Verification and Cloning Sites: Multiple Cloning Site (MCS): An MCS with diverse restriction sites was included to facilitate the easy insertion or removal of sequences, allowing for rapid customization of the plasmid for various needs. Sequencing Primer Sites (M13 fwd, M13 rev): These sites were strategically placed to assist in sequencing the plasmid, verifying inserted sequences, and confirming the integrity of the constructed plasmid. 7. Safety and Ethical Considerations: Biosafety: The design included measures to ensure that the plasmid and its components would not contribute to horizontal gene transfer or pose other risks in laboratory or environmental settings. Regulatory Compliance: Considerations were also made to comply with regional and international guidelines on genetic modification and antibiotic resistance management. 8. Codon Optimization: For any parts of the plasmid encoding proteins (like Cas9 from another plasmid), codon optimization for E. coli was considered to enhance expression efficiency and functionality within the bacterial host.
Source
yahK-sgRNA and sgRNA Scaffold: The sgRNA targeting the yahK gene is custom-designed based on the genomic sequence of yahK in E. coli. It is synthesized to include a region complementary to a specific sequence within the yahK gene to ensure targeted binding and cleavage by Cas9. The sgRNA scaffold, which supports the proper formation and function of the sgRNA-Cas9 complex, is typically derived from a standard scaffold used in CRISPR technology, often based on the original Streptococcus pyogenes Cas9 system. Homology Arms (HA1 and HA2): These are synthesized based on the genomic sequence immediately upstream and downstream of the yahK gene in E. coli. The sequences are selected to promote efficient homologous recombination, allowing precise deletion or insertion at the target site. Rep101 and pSC101 ori: Rep101 is a protein involved in plasmid replication, often derived from the corresponding replication systems found in naturally occurring plasmids. pSC101 ori, the origin of replication, originates from the pSC101 plasmid, a well-characterized low-copy number plasmid used in molecular biology for maintaining plasmids in bacterial cells. Antibiotic Resistance Gene (AmpR) and AmpR Promoter: The ampicillin resistance gene (AmpR) and its promoter are commonly sourced from beta-lactamase genes found in various bacterial plasmids, which confer resistance to the antibiotic ampicillin. Regulatory Elements (lac operator, lac promoter, CAP binding site, araBAD promoter): These elements are typically derived from the well-studied lac and ara operons in E. coli, which are used widely in genetic engineering for regulated expression of genes in response to environmental signals (such as the presence of IPTG or arabinose). MCS (Multiple Cloning Site), M13 primer sites, rrnB T1 terminator: The MCS is designed to include common restriction sites used in molecular cloning, facilitating the insertion or removal of DNA segments. M13 primer sites are universal primer sequences used in sequencing and are derived from the M13 bacteriophage. The rrnB T1 terminator is sourced from the rrnB operon in E. coli, ensuring efficient termination of transcription.
Application in Projects: In gene editing projects, researchers can use the PHCY-yahK-KO plasmid to specifically knock out the yahK gene in E. coli. The plasmid components, sourced from both synthetic constructs and naturally occurring genetic elements, provide a robust tool for precise genetic modifications. This system is especially useful in functional genomics, metabolic engineering, and synthetic biology applications where precise gene editing and controlled gene expression are critical.
References
Huang, C., Guo, L., Wang, J. et al. Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering. Appl Microbiol Biotechnol 104, 7943–7956 (2020). https://doi.org/10.1007/s00253-020-10819-1