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 of Staphylococcus aureus strain NCTC 8325 and initiates amplification of the target sequence during PCR. It can be used together with a corresponding reverse primer to generate a DNA fragment of the mraY locus for applications such as guide RNA validation, cloning, or sequencing in CRISPR-Cas9 experiments aimed at disrupting mraY.

mraY Reverse Primer (BBa_256XT95A)

TGGTAATCTAGATGCGCCGA

Anneals to the mraY gene of Staphylococcus aureus strain NCTC 8325 and extends in the reverse direction during PCR. When paired with the corresponding forward primer, it enables amplification of the target mraY locus for applications such as guide RNA validation, cloning, or sequencing in CRISPR-Cas9 experiments aimed at disrupting mraY.

mraY Forward gRNA (BBa_25RN887L)

GGUCAAAGUAUUCGAGAAGA

Targets the mraY gene of Staphylococcus aureus strain NCTC 8325 for site-specific cleavage by the Cas9 nuclease. Guides Cas9 to the mraY locus to enable gene disruption, knockout, or other CRISPR-mediated genome editing applications, facilitating studies of cell wall synthesis and essential gene function.

mraY Reverse gRNA (BBa_25R466M4)

ACAGGUAGCUUAGCUUUAGG

Targets the mraY gene of Staphylococcus aureus strain NCTC 8325 for site-specific cleavage by the Cas9 nuclease. Guides Cas9 to the mraY locus to enable gene disruption, knockout, or other CRISPR-mediated genome editing applications, facilitating studies of cell wall synthesis and essential gene function.

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 of Staphylococcus aureus strain NCTC 8325 and extends in the reverse direction during PCR. When paired with the corresponding reverse primer, it enables amplification of the target dnaG locus for applications such as guide RNA validation, cloning, or sequencing in CRISPR-Cas9 experiments aimed at disrupting dnaG.

dnaG Reverse Primer (BBa_25KO6KFW)

AGGACCCATTACTGGTGAACA

Anneals to the dnaG gene of Staphylococcus aureus strain NCTC 8325 and extends in the reverse direction during PCR. When paired with the corresponding forward primer, it enables amplification of the target dnaG locus for applications such as guide RNA validation, cloning, or sequencing in CRISPR-Cas9 experiments aimed at disrupting dnaG.

dnaG Forward gRNA (BBa_25PHP0ET)

ATAAATTAAGGGAAGCTACA

Guides Cas9 to the forward strand of the dnaG gene in Staphylococcus aureus strain NCTC 8325, enabling targeted cleavage near the primase coding region. This gRNA is designed to initiate double-stranded breaks at the specified locus, facilitating CRISPR-Cas9–mediated disruption of dnaG function for studies on DNA replication, bacterial growth, or antibiotic sensitivity.

dnaG Reverse gRNA (BBa_25NDSX9X)

TAAAACCGACATTTTAGACT

Guides Cas9 to the reverse strand of the dnaG gene in Staphylococcus aureus strain NCTC 8325, directing site-specific cleavage at the complementary target site within the primase coding sequence. In combination with the forward gRNA or used independently, this gRNA enables precise editing or functional knockout of dnaG to investigate its essential role in DNA replication and potential as an antimicrobial target.

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.