Difference between revisions of "Part:BBa K5115036"

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ribozyme+RBS+Hpn+stem-loop

Introduction

This composite part is composed of hpn coding sequence (CDS), wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. By inserting BBa_K4765020 before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA.^[1] To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS.^[2] In 2023, we extensively tested various stem-loops using BBa_K4765129. For parts we made this year, this strong protective stem-loop sequence was used.

As for the ribosome binding sequence (RBS) after the ribozyme and before the CDS, we used T7 RBS, from bacteriophage T7 gene 10.^[3] It is an intermediate strength RBS according to our 2022 results, which allows us to change it to a weaker J6 RBS or a stronger B0 RBS if needed, enabling flexible protein expression levels between various ribozyme connected parts.

The His-rich putative nickel storage protein plays a crucial role in nickel detoxification. hpn may sequester metals that accumulate internally via a passive equilibrium mechanism (from a high external metals environment).^[4]

Introduction

This composite part is composed of hpn and improved ribozyme-assisted polycistronic co-expression system: pRAP. The hpn is a protein which can bind with nickel ions, get details in BBa_K1151001(Histidine-rich metal-binding protein)

Usage and Biology

The ribozyme-assisted polycistronic co-expression system can ensure that each cistron can initiate translation with comparable efficiency. For more information, please check part wiki of 2022 Fudan iGEM.

Characterization

Growth curve of E.coli

Figure 1: Comparison of E. coli Growth curve with and without hpn in 20 mg/L Ni²⁺ The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing hpn compared to E. coli without hpn expression in a medium containing 20 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5, and equal volumes of the suspension were added to each tube. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing Hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without hpn expression under the same conditions.

Figure 2: Comparison of E. coli Growth curve with and without hpn in 50 mg/L Ni²⁺ The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing hpn compared to E. coli without hpn expression in a medium containing 50 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5, and equal volumes of the suspension were added to each tube. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing Hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without hpn expression under the same conditions.

Figure 3: Comparison of E. coli Growth curve with and without hpn in 100 mg/L Ni²⁺ The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing hpn compared to E. coli without hpn expression in a medium containing 100 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5, and equal volumes of the suspension were added to each tube. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without hpn expression under the same conditions.

Sequence and Features

References

1. ↑ Eiler, D., Wang, J., & Steitz, T. A. (2014). Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme. Proceedings of the National Academy of Sciences, 111(36), 13028–13033.
2. ↑ Liu, Y., Wu, Z., Wu, D., Gao, N., & Lin, J. (2022). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 12(1), 136–143.
3. ↑ The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Olins PO, Devine CS, Rangwala SH, Kavka KS. Gene, 1988 Dec 15;73(1):227-35.
4. ↑ Maier, R. J., Benoit, S. L., & Seshadri, S. (2007). Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 20(3–4), 655–664.