DNA

Part:BBa_K5487107:Design

Designed by: Xuerui Tao   Group: iGEM24_UESTC-China   (2024-09-03)


yahK-HA1/2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

1. Length of Homology Arms: Optimal Length: The lengths of the left (HA1) and right (HA2) homology arms were carefully selected to balance recombination efficiency and specificity. Typically, each homology arm was designed to be between 500 to 1000 base pairs. This length is sufficient to ensure high recombination rates while minimizing the risk of non-specific recombination events. Avoiding Repetitive Sequences: The arms were designed to avoid repetitive sequences, which could reduce recombination efficiency or increase the likelihood of non-specific recombination elsewhere in the genome. 2. Sequence Fidelity and Specificity: Exact Sequence Match: The homology arms were designed to have a high sequence identity to the target regions flanking the yahK gene in the E. coli genome. This was critical to ensure specific binding and alignment of the homology arms during the homologous recombination process. Avoiding Off-Target Effects: The selected sequences for HA1 and HA2 were carefully checked against the E. coli genome to avoid regions with high homology to other non-target genomic locations, reducing the risk of off-target recombination. 3. GC Content and Melting Temperature: Balanced GC Content: The GC content of the homology arms was optimized to be in a moderate range (typically 40-60%) to ensure stable annealing during homologous recombination without forming secondary structures or causing difficulties in PCR amplification or cloning steps. Melting Temperature (Tm) Considerations: The melting temperatures of the homology arms were balanced to ensure that they were suitable for the cloning and recombination processes, avoiding excessively high or low Tm values that could affect annealing and recombination efficiency. 4. Restriction Sites and Cloning: Absence of Restriction Sites: The design process ensured that the homology arms did not contain any restriction sites that would interfere with the cloning strategy or subsequent molecular manipulations. This was crucial to avoid unwanted cleavage of the homology arms during vector construction. Compatibility with Cloning Vectors: The sequences were also checked to ensure compatibility with standard cloning vectors used in E. coli, and additional sequences were added as necessary for seamless cloning and vector integration. 5. Codon Optimization and Compatibility: Codon Usage Optimization: Although the homology arms themselves do not typically encode proteins, the regions adjacent to the targeted editing site (especially if new sequences were being inserted) were optimized for codon usage in E. coli to ensure efficient translation if a gene insertion was being conducted. Avoidance of Cryptic Splice Sites: The sequence design avoided cryptic splice sites or secondary structures that could inadvertently affect downstream expression or recombination. 6. Functional Context and Downstream Effects: Consideration of Neighboring Genes: The design took into account the location of neighboring genes and regulatory elements to avoid unintended disruption of gene function or regulation. This was particularly important if the target site was near a promoter or regulatory sequence that could affect gene expression. Orientation and Read-Through Effects: The orientation of the homology arms was designed to ensure correct recombination without causing read-through transcription that could affect adjacent genes or regulatory regions. 7. Verification and Validation Steps: In Silico Verification: Before synthesis, the designed homology arms underwent extensive in silico verification using software tools to predict secondary structures, potential off-target effects, and overall recombination efficiency. Pilot Testing and Optimization: Initial pilot experiments were planned to validate the efficiency and specificity of the homology arms, with optimization steps in place to adjust arm length or sequence if the initial recombination efficiency was suboptimal.


Source

E.coli K12 genome

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