Difference between revisions of "Part:BBa K5136204"

Revision as of 01:41, 1 October 2024

J23110-RiboJ-B0034-LMT-linker-sfgfp-B0010

Biology

RiboJ

RiboJ is a self-cleaving ribozyme that removes the 5' untranslated region, creating a precise mRNA start. This ensures consistent and reliable translation of the downstream LMT-sfGFP fusion, acting as a genetic insulator and enhancing expression predictability(1).

LMT

The LMT signal peptide, derived from Vibrio natriegens, directs the attached sfGFP protein to the periplasm. Once in the periplasm, the LMT sequence is cleaved, leaving the mature sfGFP for study in this compartment.

Superfolder GFP (sfGFP)

This stable, fast-folding version of GFP emits bright green fluorescence, even in harsh environments like the periplasm. It allows real-time tracking of protein expression and localization.

Usage and Design

In order to test the effect of different promoters on the function of LMT signal peptides, this composite part J23110-RiboJ-B0034-LMT-linker-sfgfp-B0010 was constructed, coding for the LMT-sfGFP fusion protein. The LMT signal peptide is responsible for guiding the protein to the periplasm of E. coli, and the success of this targeting can be observed through the fluorescence emitted by superfolder GFP. The inclusion of a flexible linker ensures that both the LMT and sfGFP can fold correctly, maintaining their respective functions. Moreover, the RiboJ ribozyme provides consistent and reliable expression by eliminating variability from upstream sequences, ensuring stable production of the LMT-sfGFP fusion protein for further analysis of protein behavior and periplasmic localization.

Characterization of Signal Peptides

Colony PCR

We construct BBa_K5136204 with J23110 promotor, RiboJ, B0034 RBS, LMT-linker-sfgfp, and B0010 terminator, the transformed cells are selected by colony PCR. The experiment result is showed in Fig. 1.

Characterization of Signal Peptides

To characterize the strength of the signal peptide, we constructed circuits driven by promoters of different strengths (J23100, J23103, J23104, J23106, J23110, J23114), each containing RiboJ-B0034-LMT-linker-sfgfp-B0010. By measuring the fluorescence intensity in the supernatant of each circuit, we can quantitatively analyze the guiding efficiency of the LMT signal peptide under different promoter strengths and thus evaluate its secretion capability.

The figure shows that the fluorescence intensity increases with promoter strength, indicating that the LMT signal peptide more effectively directs the sfGFP to the periplasm and supernatant under stronger promoter conditions. However, differences in secretion efficiency among promoters suggest that the signal peptide's effectiveness is influenced by promoter strength, with possible saturation or efficiency limits.

To ensure that these results reflect actual secretion rather than differences in cell growth, OD₆₀₀ was measured to account for bacterial density. The normalized fluorescence (fluorescence/OD₆₀₀) confirms that the secretion efficiency is not simply a result of higher cell counts. However, variations in secretion efficiency across promoters suggest that the signal peptide's guiding ability is influenced by promoter strength, possibly due to saturation or other limiting factors.

Reference

1. Lou C, Stanton B, Chen Y J, et al. Ribozyme-based insulator parts buffer synthetic circuits from genetic context[J]. Nature biotechnology, 2012, 30(11): 1137-1142.

Sequence and Features