Part:BBa_K2253000

Constitutive P8 promoter and RBS composite

The P8 constitutive promoter is natively found Lactococcus lactis. 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 P8 promoter and E2-Crimson reporter is able to successfully express the E2-Crimson red fluorescent protein. The P32 promoter and RBS sequence are natively found in the genome of Lactococcus lactis. However, we ordered the promoter and RBS sequence as a gBlock using the sequence information provided by Zhu et al. (2015).

Usage and Biology

As a PhytoBrick compatible part, P8 promoter can be assembled into a transcriptional unit via Golden Gate assembly method to upregulate expression of a gene of interest in E. coli as well as L. lactis. of a gene of interest in E. coli as well as L. lactis.

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 (which encodes a red fluorescent protein) inserted downstream of the P8 promoter using BsaI Golden Gate assembly. To create this test cassette, we used the P8/P32 promoter part plasmids, E2-Crimson part plasmid, an M13 terminator part plasmid, connector part plasmids, and the pYTK095 vector as the backbone.

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File:T--Austin UTexas--p8p32test.jpg

Figure 3. Golden Gate assembly process of the P8 and P32 test cassette plasmids.

Sequence and Features BBa_K2253000 SequenceAndFeatures ===References=== # Zhu, D. et al. Isolation of strong constitutive promoters from Lactococcus lactis subsp. Lactis N8. FEMS Microbiol Lett. 363(16): pii: fnv107 (2015).