Difference between revisions of "Part:BBa K5115033"
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<partinfo>BBa_K5115033 short</partinfo> | <partinfo>BBa_K5115033 short</partinfo> | ||
| − | <html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/5115/czh/mineral-logo.svg" alt="contributed by Fudan iGEM | + | <html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/5115/czh/mineral-logo.svg" alt="contributed by Fudan iGEM 2024"></html> |
__TOC__ | __TOC__ | ||
===Introduction=== | ===Introduction=== | ||
| − | This composite part is composed of RcnR_C35L | + | This composite part is composed of RcnR_C35L coding sequence (CDS), wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. By inserting [https://parts.igem.org/Part:BBa_K4765020 BBa_K4765020] before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA <ref>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.</ref>. To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS <ref>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.</ref>. In 2023, we extensively tested various [https://2023.igem.wiki/fudan/part-collection/#ribozyme-assisted-polycistronic-co-expression stem-loops] using [https://parts.igem.org/Part:BBa_K4765129 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 [https://parts.igem.org/Part:BBa_K4162006 T7 RBS], from bacteriophage T7 gene 10 <ref>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.</ref>. It is an intermediate strength RBS according to [https://2022.igem.wiki/fudan/measurement#optimization our 2022 results], which allows us to change it to a weaker [https://parts.igem.org/Part:BBa_J61100 J6 RBS] or a stronger [https://parts.igem.org/Part:BBa_B0030 B0 RBS] if needed, enabling flexible protein expression levels between various ribozyme connected parts. | ||
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| + | The RcnR_C35L can inhibit the induction of rcnA by metal cations, helping us to lock the nickel ion inside our bacteria <ref>Koch, D., Nies, D. H., & Grass, G. (2007). The RcnRA (YohLM) system of Escherichia coli: A connection between nickel, cobalt and iron homeostasis. BioMetals, 20(5), 759–771.</ref>. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | The | + | The RcnR_C35L can endow ''E.coli'' with enhanced ability of absorbing nickel ions. |
| + | |||
| + | Get details in [https://parts.igem.org/Part:BBa_K5115000 BBa_K5115000] | ||
| + | |||
| + | ===Characterization=== | ||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/double-plasmids-rcnr-nik-nix.png" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''Figure 10. Comparison of Ni²⁺ Uptake Efficiency, with and without RcnR_C35L. | ||
| + | The graph shows the percentage of Ni²⁺ absorbed by ''E. coli'' expressing different constructs after 5 hours of growth in a medium containing 20 mg/L Ni²⁺ (''E. coli'' strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 20 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. RcnR_C35L refers to a mutation in which cysteine (C) at position 35 in the RcnR protein was substituted with leucine (L). The results indicate that ''E. coli'' expressing RcnR_C35L consistently has higher Ni²⁺ uptake efficiency compared to ''E. coli'' without RcnR_C35L expression. | ||
| + | ''' | ||
| + | |||
| + | |} | ||
| + | ===Sequence and Features=== | ||
<!-- --> | <!-- --> | ||
Latest revision as of 10:08, 2 October 2024
ribozyme+RBS+RcnR_C35L+stem-loop
Introduction
This composite part is composed of RcnR_C35L 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 RcnR_C35L can inhibit the induction of rcnA by metal cations, helping us to lock the nickel ion inside our bacteria [4].
Usage and Biology
The RcnR_C35L can endow E.coli with enhanced ability of absorbing nickel ions.
Get details in BBa_K5115000
Characterization
|
| Figure 10. Comparison of Ni²⁺ Uptake Efficiency, with and without RcnR_C35L.
The graph shows the percentage of Ni²⁺ absorbed by E. coli expressing different constructs after 5 hours of growth in a medium containing 20 mg/L Ni²⁺ (E. coli strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 20 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. RcnR_C35L refers to a mutation in which cysteine (C) at position 35 in the RcnR protein was substituted with leucine (L). The results indicate that E. coli expressing RcnR_C35L consistently has higher Ni²⁺ uptake efficiency compared to E. coli without RcnR_C35L expression. |
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 303
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ 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.
- ↑ 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.
- ↑ 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.
- ↑ Koch, D., Nies, D. H., & Grass, G. (2007). The RcnRA (YohLM) system of Escherichia coli: A connection between nickel, cobalt and iron homeostasis. BioMetals, 20(5), 759–771.