Part:BBa_K2253001

Constitutive P32 promoter and RBS composite

The P32 constitutive promoter is natively found in the genome of Lactococcus lactis ^[1]. Although the transcriptional efficiency of this promoter has been characterized and tested in Lactococcus lactis and other Gram-positive bacteria, its functionality in Gram-negative species such as E. coli has not been recorded in the literature. We have confirmed that E. coli transformed with a cassette plasmid containing the P32 promoter and E2-Crimson reporter is able to successfully express the E2-Crimson red fluorescent protein.

Usage and Biology

As a PhytoBrick compatible part containing the proper overhangs specified by the iGEM PhytoBrick standard, the P32 promoter can be assembled into a transcriptional unit via Golden Gate assembly to upregulate expression of a gene of interest in E. coli as well as L. lactis. For more information about the PhytoBrick standards set by iGEM, click here.

Experimental Design

To test if our Lactococcus lactis constitutive promoters function well within E. coli, we created a test cassette plasmid containing the E2-Crimson reporter gene inserted downstream of the P32 promoter using BsaI Golden Gate assembly. To create this test cassette, we used the P32 promoter part plasmid, E2-Crimson part plasmid, an M13 terminator part plasmid, connector part plasmids, and the pYTK095 vector as the backbone (Fig. 1). These part plasmids contained a distinct set of overhangs specified by the Lee/Dueber YTK Golden Gate Assembly standard ^[2].

Figure 1. Golden Gate assembly process of the P32 test cassette plasmid.

Top10 electrocompetent E. coli cells were transformed with the P32 cassette assembly and plated on LB + CAM plates. After one day of incubation at 37°C, there was growth of purple-blue colonies, which fluoresced red when placed under UV illumination (Fig. 2). The observed red fluorescence phenotype was likely due to the P32 promoter successfully directing expression of the E2-Crimson reporter gene. From this result, we concluded that the P32 constitutive promoter is indeed functional within E. coli and can thus be used to overexpress genes of interest in E. coli as well as L. lactis.

Figure 2. P32 cassette plasmid transformation plate under normal light (left) and UV illumination (right).

Sequence and Features

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]

References

1. Zhu, D. et al. Isolation of strong constitutive promoters from Lactococcus lactis subsp. Lactis N8. FEMS Microbiol Lett. 363(16): pii: fnv107 (2015).
2. Lee, M. E. et al. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4(9): 975-86 (2015).