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.

This 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]

Usage and Biology

The Hpn can reduce the toxicity of nickel to the E.coli cell.

Get details in BBa_K5115063.

Characterization

Growth curve of E.coli

Before we test the nickel absorption ability of the E.coli, we must make sure that the Ecoli can survive in the waste water. Hpn being the major detoxifying part, we launched experiments on E. coli expressing Hpn compared to E. coli without Hpn expression in mediums containing different concentration of nickel ions.

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. 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. 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. 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.

Ni absorption experiment

In order to accurately measure the concentration of nickel ions, we adopted the spectrophotometry and used dimethylglyoxime as a color developer, which can specifically bind with Ni. For more details about its protocol, please visit our experiments wiki.

Figure 4. Close-up view of the spectrophotometer we used.

Figure 5. Every centrifuge tube contained a medium waiting to be made spectrophotometric.

Ni absorption with a single part introduced

Ni absorption with two parts combined

Ni absorption with three parts combined

Ni absorption with four parts combined

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.