Part:BBa_K5136205

J23114-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 J23114-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_K5136205 with J23114 promotor, RiboJ, B0034 RBS, LMT-linker-sfgfp, and B0010 terminator, the transformed cells are selected by colony PCR. The experiment result is shown in Figure 1.

Figure 1 DNA gel electrophoresis of colony PCR product of J23114-RiboJ-B0034-LMT-linker-sfgfp_pSB1C3.

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 (Figure 2).

Figure 2 Fluorescence intensity in the supernatant of circuits driven by BBa_K5136200, BBa_K5136201, BBa_K5136202, BBa_K5136203, BBa_K5136204, BBa_K5136205.

Based on the experimental results (Figure 3), we selected the promoter J23104 due to its optimal secretion efficiency. Although weak promoters also demonstrated high secretion efficiency, they led to lower overall protein content in the supernatant. On the other hand, strong promoters caused a high metabolic burden on the cells, reducing survival rates and protein production over time. J23104 strikes the best balance between secretion efficiency and protein yield, minimizing metabolic burden while maintaining sufficient extracellular protein production.

Figure 2 Characterization of Anderson Promoter.

See model (https://2024.igem.wiki/XMU-China/model) for details.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 277