Difference between revisions of "Part:BBa K5115038"
 
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<partinfo>BBa_K5115038 short</partinfo>
 
<partinfo>BBa_K5115038 short</partinfo>
  
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<html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/5115/czh/mineral-logo.svg" alt="contributed by Fudan iGEM 2023"></html>
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<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===
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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''.
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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''.
  
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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.
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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===
 
===Usage and Biology===
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===Characterization===
 
===Characterization===
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====Growth curve of ''E.coli''====
 
 
{|
 
{|
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/20-mg-l-hpn.png" alt="contributed by Fudan iGEM 2024"></html>
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/3-composites.png" alt="contributed by Fudan iGEM 2024"></html>
 
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| '''Figure 1: Comparison of ''E. coli'' Growth curve with and without hpn in 20 mg/L Ni²⁺
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| '''Figure 1. Comparison of Ni²⁺ Uptake Efficiency by Different ''E. coli'' in 50 mg/L Ni²⁺. 
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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.
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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.
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'''
 
'''
  
 
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{|
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===Sequence and Features===
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/50-mg-l-hpn.png" alt="contributed by Fudan iGEM 2024"></html>
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| '''Figure 2: Comparison of ''E. coli'' Growth curve with and without hpn in 50 mg/L Ni²⁺ 
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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.
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'''
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{|
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/100-mg-l-hpn.png" alt="contributed by Fudan iGEM 2024"></html>
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| '''Figure 3: Comparison of ''E. coli'' Growth curve with and without hpn in 100 mg/L Ni²⁺ 
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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.
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'''
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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

contributed by Fudan iGEM 2024

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

contributed by Fudan iGEM 2024
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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 935
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 198
  • 1000
    COMPATIBLE WITH RFC[1000]


References