Difference between revisions of "Part:BBa K5115041"
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<partinfo>BBa_K5115041 short</partinfo> | <partinfo>BBa_K5115041 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_K5115033 BBa_K5115033(ribozyme+RBS+RcnR_C35L+stem-loop)], [https://parts.igem.org/Part:BBa_K5115036 BBa_K5115036(ribozyme+RBS+ | + | This composite part combines [https://parts.igem.org/Part:BBa_K5115033 BBa_K5115033(ribozyme+RBS+RcnR_C35L+stem-loop)], [https://parts.igem.org/Part:BBa_K5115036 BBa_K5115036(ribozyme+RBS+Hpn+stem-loop)] and [https://parts.igem.org/Part:BBa_K5115035 BBa_K5115035(ribozyme+RBS+MTA+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''. |
| − | MTA is a protein that can bind with nickel ions to reduce its | + | 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. |
| − | sequester metals that accumulate internally to reduce nickel's | + | |
| + | ===Usage and Biology=== | ||
| + | To ascertain which order the three proteins should be assembled for the best results, we create six different parts containing all the sequential possibilities. We finally confirm that the order of the three parts don't affect much on their effect. We choose to use [https://parts.igem.org/Part:BBa_K5115038 ribozyme connected: MTA, hpn, RcnR_C35L] at last. | ||
===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=== | ||
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Latest revision as of 11:29, 2 October 2024
ribozyme connected: RcnR_C35L, Hpn, MTA
Introduction
This composite part combines BBa_K5115033(ribozyme+RBS+RcnR_C35L+stem-loop), BBa_K5115036(ribozyme+RBS+Hpn+stem-loop) and BBa_K5115035(ribozyme+RBS+MTA+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
To ascertain which order the three proteins should be assembled for the best results, we create six different parts containing all the sequential possibilities. We finally confirm that the order of the three parts don't affect much on their effect. We choose to use ribozyme connected: MTA, hpn, RcnR_C35L at last.
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 303
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 875
- 1000COMPATIBLE WITH RFC[1000]