Difference between revisions of "Part:BBa K5115038"
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<partinfo>BBa_K5115038 short</partinfo> | <partinfo>BBa_K5115038 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 combines [https://parts.igem.org/Part:BBa_K5115035 BBa_K5115035(ribozyme+RBS+MTA+stem-loop)], BBa_K5115036(ribozyme+RBS+ | + | This composite part combines [https://parts.igem.org/Part:BBa_K5115035 BBa_K5115035(ribozyme+RBS+MTA+stem-loop)], [https://parts.igem.org/Part:BBa_K5115036 BBa_K5115036(ribozyme+RBS+Hpn+stem-loop)]and [https://parts.igem.org/Part:BBa_K5115033 BBa_K5115033(ribozyme+RBS+RcnR_C35L+stem-loop)] . We introduced this ribozyme-assisted polycistronic co-expression system from [https://2022.igem.wiki/fudan/parts 2022]. By inserting [https://parts.igem.org/Part:BBa_K4765020 ribozyme sequences] between CDSs in a polycistron, the RNA sequences of Twister ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons ''in vivo''. |
| − | With this design, we achieve co-expression of [ | + | With this design, we achieve co-expression of [https://parts.igem.org/Part:BBa_K5115050 MTA], [https://parts.igem.org/Part:BBa_K1151001 Hpn], [https://parts.igem.org/Part:BBa_K5115000 RcnR_C35L] at similar level. MTA is a protein that can bind with nickel ions to reduce its toxicity to the ''E.coli''. The Hpn is a protein that can sequester metals that accumulate internally to reduce nickel's toxicity to the ''E.coli''. RcnR_C35L can regulate the nickel ion channel proteins in the cell membrane to tune the nickel ion transport rate. |
| + | |||
| + | ===Usage and Biology=== | ||
| + | This part is eventually chosen as a component of [https://parts.igem.org/Part:BBa_K5115068 mineral nickel module], tuning the nickel ion transport rate and reducing nickel's toxicity to the ''E.coli''. | ||
===Characterization=== | ===Characterization=== | ||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/3-composites.png" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''Figure 1. Comparison of Ni²⁺ Uptake Efficiency by Different ''E. coli'' in 50 mg/L Ni²⁺. | ||
| + | The graph shows the percentage of Ni²⁺ absorbed by ''E. coli'' expressing different constructs after 5 hours of growth in a medium containing 50 mg/L Ni²⁺ (''E. coli'' strain: BL21 DE3, leaky expression, no IPTG induction). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 50 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture at 37°C with a rotating speed at 220 rpm. With out parts for Ni²⁺ uptake, there was no significant difference in the efficiency of nickel absorption between the modified ''E. coli'' and control. | ||
| + | ''' | ||
| + | |} | ||
| + | ===Sequence and Features=== | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Latest revision as of 11:06, 2 October 2024
ribozyme connected: MTA, Hpn, RcnR_C35L
Introduction
This composite part combines BBa_K5115035(ribozyme+RBS+MTA+stem-loop), BBa_K5115036(ribozyme+RBS+Hpn+stem-loop)and BBa_K5115033(ribozyme+RBS+RcnR_C35L+stem-loop) . We introduced this ribozyme-assisted polycistronic co-expression system from 2022. By inserting ribozyme sequences between CDSs in a polycistron, the RNA sequences of Twister ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo.
With this design, we achieve co-expression of MTA, Hpn, RcnR_C35L at similar level. MTA is a protein that can bind with nickel ions to reduce its toxicity to the E.coli. The Hpn is a protein that can sequester metals that accumulate internally to reduce nickel's toxicity to the E.coli. RcnR_C35L can regulate the nickel ion channel proteins in the cell membrane to tune the nickel ion transport rate.
Usage and Biology
This part is eventually chosen as a component of mineral nickel module, tuning the nickel ion transport rate and reducing nickel's toxicity to the E.coli.
Characterization
|
| Figure 1. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 50 mg/L Ni²⁺.
The graph shows the percentage of Ni²⁺ absorbed by E. coli expressing different constructs after 5 hours of growth in a medium containing 50 mg/L Ni²⁺ (E. coli strain: BL21 DE3, leaky expression, no IPTG induction). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 50 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture at 37°C with a rotating speed at 220 rpm. With out parts for Ni²⁺ uptake, there was no significant difference in the efficiency of nickel absorption between the modified E. coli and control. |
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 935
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 198
- 1000COMPATIBLE WITH RFC[1000]