pEC3111 (pSpy1C-Plac(3)_ffluc)
(Plasmid #218778)

• Purpose: replicative E. coli-S. pyogenes shuttle plasmid for IPTG-inducible expression of the Firefly luciferase in a strain expressing lacI repressor
• Depositing Lab: Emmanuelle Charpentier
• Publication: Lautenschlager et al Front Bioeng Biotechnol. 2024 Jun 7;12:1395659. doi: 10.3389/fbioe.2024.1395659. eCollection 2024.

Backbone

• Vector backbone: pSpy1C
• Total vector size (bp): 5585
• Modifications to backbone: The Plac_mrfp cassette was replaced with the Plac(3) promoter derived and modified from pJWV102-PL-dCas9 as well as the ffluc reporter gene taken from pLZ12Km2-P23R:TA:ffluc.
• Vector type: Bacterial Expression, Luciferase

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For E. coli, use 20 µg/mL chloramphenicol in LB medium. For S. pyogenes, use 20 µg/mL chloramphenicol in THY medium. The IPTG-inducible promoter is only functional in strains carrying the lacI repressor and is inducible with e.g. 1mM IPTG.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ffluc
• Species: Photinus pyralis
• Insert Size (bp): 1653
• Mutation: deletion of mrfp cassette, mutagenesis of the RBS and spacer downstream of Plac (Plac(3))
• GenBank ID: XM_031473197
• Promoter: Plac(3)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: GTAAAACGACGGCCAGTC
• 3′ sequencing primer: CAGGAAACAGCTATGAC

Resource Information

• Supplemental Documents:
  • pSpy1C_Plac(3)_ffluc.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This high-copy replicative shuttle plasmid features the IPTG-inducible promoter Plac(3) driving expression of the Firefly luciferase in the presence of IPTG (e.g. 1mM IPTG). This plasmid is to be used in S. pyogenes strains expressing the lacI repressor (e.g. integrated with p7INT-Pveg_lacI). Luminescence can be detected by adding luciferin to the culture. See associated publication for more details.

Resource Information: The Plac(3) promoter was taken and modified from the PL promoter on the plasmid pJWV102-PL-dCas9 (Plasmid #85588) described in the following publication:
High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Liu X, Gallay C, Kjos M, Domenech A, Slager J, van Kessel SP, Knoops K, Sorg RA, Zhang JR, Veening JW. Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449. PubMed 28490437
The ffluc reporter gene was taken from the original pLZ12Km2-P23R:TA:ffluc plasmid (Plasmid #88900).

Please download the detailed plasmid map using the file linked below