. Contribution .

Overview

As we embarked on our project, we read about the research and contributions of our predecessors. Their insights and experiences sparked our creativity and guided our design, significantly advancing our work. We aim to enrich the knowledge base for future iGEM participants and hope our contributions inspire other teams, fostering a vibrant and diverse iGEM community.

Contribution From XMU-China 2024

The LMT sequence has been identified as a signal peptide capable of directing recombinant proteins out of bacterial cells. This part (BBa_K3739010) was first registered in 2021 (2021 XMU-China) and used to construct the expression system and help prove the function of some signal peptides.

For the contribution of this part, we test the effect of different promoters on the function of LMT signal peptides, to achieve the most appropriate extracellular secretion performance in practical use.

1. Construction of Gene Circuit

We constructed circuits driven by promoters of different strengths (J23100, J23103, J23104, J23106, J23110, J23114), each containing RiboJ-B0034-LMT-linker-sfgfp-B0010. The composite part J23104-RiboJ-B0034-LMT-linker-sfgfp-B0010 (BBa_K5136202) was used as an example, and the rest results of colony PCR are detailed in BBa_K5136200, BBa_K5136201, BBa_K5136203, BBa_K5136204, BBa_K5136205

Figure 1 DNA gel electrophoresis of the colony PCR products of BBa_ K5136202_pSB1C3.

2. Characterization of Secretion Performance

By measuring the fluorescence intensity in the supernatant of each circuit, we can quantitatively analyze the secretion of the LMT signal peptide under different promoter strengths and thus evaluate its performance (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.

We find that this follows exponential kinetics, which is consistent with the T7-mediated secretion. As shown in Figure 3A, secretion efficiency is affected by many factors, but not positively related to promoter strength. The R square (R²) indicated that the fitting effect was better, and J23104 (0.9786) was the highest (Figure 3B).

Figure 3 (A) Fitted curve of fluorescence intensity in the supernatant of circuits driven by BBa_K5136200, BBa_K5136201, BBa_K5136202, BBa_K5136203, BBa_K5136204, BBa_K5136205. (B) R square of fit of BBa_K5136200, BBa_K5136201, BBa_K5136202, BBa_K5136203, BBa_K5136204, BBa_K5136205.

To ensure that these results reflect actual secretion rather than differences in cell growth, we measured fluorescence intensity data in 30 h of culture (Figure 4A) and supernatant (Figure 4B), and calculated the secretion efficiency (supernatant/culture) (Figure 4C).

Figure 4 (A) Fluorescence intensity (30 h) of culture driven by BBa_K5136200, BBa_K5136201, BBa_K5136202, BBa_K5136203, BBa_K5136204, BBa_K5136205. (B) Fluorescence intensity (30 h) of culture driven by BBa_K5136200, BBa_K5136201, BBa_K5136202, BBa_K5136203, BBa_K5136204, BBa_K5136205. (C) Secretion Efficiency (30 h) driven by BBa_K5136200, BBa_K5136201, BBa_K5136202, BBa_K5136203, BBa_K5136204, BBa_K5136205.

Interestingly, when we compared the secretion efficiency at 30 h, we found that high secretion efficiency does not mean high protein content in supernatant and culture. Although the secretion efficiency of the weak promoter and the strong promoter were both higher, the weak promoter could fully realize extracellular secretion due to its low efficiency and less protein production by the exosystem, but the amount of protein in supernatant was less. For strong promoters, although the amount of protein secreted is large, the total fluorescence is low, indicating that the metabolic burden is heavy, which affects the survival rate. If the detection continues, very few bacteria will survive. Also we use multi-scale model to deconstruct the dynamic secretion process of signal peptides accurately. The experimental results aligned well with the model's predictions (see model for more details). By trading off, BBa_K5136202 with promoter J23104 has the most appropriate secretion efficiency.