Overview

Our 2025 iGEM team contributed new forward and reverse primers targeting the mraY gene of Staphylococcus aureus NCTC 8325 to the iGEM Registry. These primers were designed as part of our CRISPR-Cas9 project to disrupt mraY, an essential gene in peptidoglycan biosynthesis. By registering these primers, we aim to provide future iGEM teams with molecular biology tools that can be reused in projects related to antibiotic resistance, bacterial cell wall synthesis, or CRISPR-based genetic engineering.

This year, our team added 8 new parts and 2 new collections to the iGEM Parts Registry. These include the mraY NCTC 8325 inhibition and dnaG primase NCTC 8325 inhibition collections, each containing two gRNAs and one forward and reverse primer.

mraY NCTC 8325 Inhibition Part Collection

Based on the mraY gene sequence from KEGG.

mraY Forward Primer (BBa_25XWBMAC)

GGCAAAAACAAATCGCAGGC

Anneals to the mraY gene and initiates PCR amplification. Useful for guide RNA validation, cloning, or sequencing in CRISPR-Cas9 experiments.

mraY Reverse Primer (BBa_256XT95A)

TGGTAATCTAGATGCGCCGA

Complements the forward primer for PCR amplification of the mraY locus.

mraY Forward gRNA (BBa_25RN887L)

GGUCAAAGUAUUCGAGAAGA

Guides Cas9 to the mraY gene for site-specific cleavage, enabling gene disruption or knockout.

mraY Reverse gRNA (BBa_25R466M4)

ACAGGUAGCUUAGCUUUAGG

Targets the mraY gene for precise CRISPR-Cas9 editing on the reverse strand.

dnaG Primase NCTC 8325 Inhibition Part Collection

Based on the dnaG gene sequence from NCBI.

dnaG Forward Primer (BBa_252Y9EZH)

CAGCAATACCGCATTCGCAT

Anneals to the dnaG gene for PCR amplification and validation of CRISPR edits.

dnaG Reverse Primer (BBa_25KO6KFW)

AGGACCCATTACTGGTGAACA

Complements the forward primer for PCR amplification of the dnaG locus.

dnaG Forward gRNA (BBa_25PHP0ET)

ATAAATTAAGGGAAGCTACA

Guides Cas9 to the forward strand of dnaG, facilitating targeted cleavage near the primase coding region.

dnaG Reverse gRNA (BBa_25NDSX9X)

TAAAACCGACATTTTAGACT

Targets the reverse strand of dnaG for precise CRISPR-Cas9-mediated gene disruption.

Potential Applications

  • CRISPR-Cas9 mediated mraY knock-out in S. aureus
  • PCR verification of mraY and dnaG gene edits
  • Studying bacterial cell wall biosynthesis
  • Projects addressing antibiotic resistance mechanisms

Conclusion

By contributing these primers and gRNAs, our 2025 iGEM team has provided ready-to-use molecular biology tools for targeting essential genes in Staphylococcus aureus. These contributions aim to accelerate CRISPR-based genome editing, antimicrobial discovery, and synthetic biology education for future iGEM teams. We hope that our collections facilitate further research into antibiotic resistance, bacterial cell wall synthesis, and the development of innovative solutions in synthetic biology.