Part:BBa_K5136202
J23104-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 J23104-RiboJ-B0034-LMT-linker-sfGFP-B0010 was constructed. 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 constructed BBa_K5136202 with J23104 promotor, RiboJ, B0034 RBS, LMT-linker-sfgfp, and B0010 terminator, the transformed cells were selected by colony PCR. The experiment result was shown in Figure 1.
Characterization of Signal Peptides
In circuit design, promoter strength is a crucial factor that decides the strength of the circuit output. Meanwhile, the metabolic burden of different promoters also needs to be considered, since the growth rate of the engineered bacteria will affect the circuit performance. Hence, when choosing promoters, both their metabolic burden value and promoter strength have to be counted. However, for the Anderson promoter, not all promoters with higher promoter strength have higher metabolic burden, which means the relationship between the two effects is not linear. Here, based on the metabolic burden value and promoter strength of Anderson promoters measured by 2019 Austin_UTexas, followed by model analysis, we categorized the Anderson promoters into four classes. Each of the classes represents a different interaction between the metabolic burden and promoter strength. Depending on this, users of the Anderson promoters can choose the appropriate promoter, considering the combined effect of metabolic burden and promoter strength, more directly. To further review the effect of different classes of promoters in real applications, please refer to our Model page (https://2024.igem.wiki/XMU-China/model).
In our project, to characterize the secretion efficiency of the LMT-mediated system controlled by consistutive promoters, we constructed circuits driven by promoters of different strength (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 strength and thus evaluate its secretion capability (Figure 3).
Based on the experimental results (Figure 3), we selected the promoter J23104 in our final application due to its optimal secretion efficiency. Although weak promoters result in less protein production, all the proteins will be translocated, hence have a higher secretion efficiency. On the other hand, although strong promoters drive high protein production, they cause 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, please refer to our Contribution page (https://2024.igem.wiki/XMU-China/contribution) for further information.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 277
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