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''.
+
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 [https://parts.igem.org/Part:BBa_K5115042 ribozyme connected: MTA, RcnR_C35L, Hpn] works the best, please check it for experiment results.
−
+
−
===Characterization===
+
−
====Growth curve of ''E.coli''====
+
−
{|
+
−
| <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>
+
−
|-
+
−
| '''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, 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.
+
−
+
−
'''
+
−
+
−
|}
+
−
+
−
{|
+
−
| <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>
+
−
|-
+
−
| '''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, 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.
+
−
+
−
'''
+
−
+
−
|}
+
−
+
−
{|
+
−
| <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>
+
−
|-
+
−
| '''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, 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.
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 ribozyme connected: MTA, RcnR_C35L, Hpn works the best, please check it for experiment results.
