Part:BBa_K2921520
Promoter + Double RBS + Metallothionein + mRFP + Double Terminator
This construct constitutively expresses a colored metal-binding fusion protein: metallothionein (Composite part: BBa_K1460002) linked with mRFP (Basic part: BBa_E1010). According to iGEM14_Cornell’s composite part page of BBa_K1460002, Metallothionein is a metal-binding protein that can tightly chelate heavy metal ions by forming a strong coordination bond. The mRFP serves as a functional color reporter, allowing for the convenient visible or UV detection of the location of metallothionein.
Construct design
This construct was created to constitutively express the Met-mRFP protein. Sequences used for the promoter, RBS, and double terminator came from parts included in the iGEM distribution kit. This construct consists of a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the protein-coding gene Metallothionein (Composite part: BBa_K1460002), the chromoprotein gene mRFP (Basic part: BBa_E1010), and a double terminator (BBa_B0015) to end transcription.
PCR
PCR check results, using the primers VF2 and VR, and sequencing results by Tri-I Biotech confirm this construct.
We confirmed the size of K2921520 using the primers VF2 and VR, which resulted in the expected size of around 2.0 kb.
Characterization
We used SDS-PAGE to check for Met-mRFP expression in E. coli carrying our construct. Bacterial cultures expressing either Met-mRFP or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. The expected size of Met-mRFP is approximately 59 kDa. However, in the Met-mRFP lysate sample, we observed two strong signals at approximately 28 kDa and 48 kDa which were not present in the empty vector sample. Thus, the two separate bands on the SDS-PAGE suggest that the cells expressed Met and mRFP proteins separately, rather than as a single, fused unit. It is likely that the band at 28 kDa is the mRFP protein, which has an expected size of 25.3 kDa, and that the band at 48 kDa is the Met protein, which has an expected size of 34 kDa. This discrepancy in size is likely due to post-translational modifications on the protein such as phosphorylation and glycosylation.
Thus, it is concluded that E. coli with the Met-mRFP plasmid produces Met and mRFP proteins as separate units. Our Met-GS-mRFP (Composite Part: K2921550) or Met-EAAAK-mRFP (Composite Part: K2921570) constructs include a linker between the Met and mRFP genes, ensuring that bacteria express Met and mRFP as a single, fused unit.
To verify Met--mRFP expression in E. coli, we subjected Met-mRFP lysate to SDS-PAGE, expecting a signal at around 59 kDa. Instead, we saw two signals at around 25 kDa and 48 kDa in the Met-mRFP lane, but not in the empty lane that was used as a control. This suggests that the Met and mRFP proteins were expressed as separate units rather than a single, fused unit.
Functional Assay with Nickel
Our construct produces intracellular Met-mRFP proteins expected to increase the cells’ capacity to store nickel ions. To test the functionality of this protein, we detected the difference in the nickel ion storage capacity of construct-expressing cells and negative-control cells. Thus, we had two experimental groups: cells expressing this Met-mRFP fusion protein and cells expressing the Met protein only (Composite part: K2921500). We had two negative control groups: cells expressing RFP only and cells carrying a Met ORF-only plasmid. In order to measure cell storage capacity, we incubated cells with the nickel ions over time, to allow the nickel ions to diffuse in and out of the cell. Theoretically, for our experimental groups, the nickel ions would diffuse into the cell and bind to the active site of the intracellular Met-mRFP protein, reducing the amount of nickel ions diffusing out of the cell. After 2 hours of incubation, we measured the absorbance of nickel ions in the extracellular solution.
By the Beer-lambert law, concentration is directly proportional to absorbance. Thus, for the experimental groups, we expected the extracellular solution to have a lower concentration of nickel ions and, thus, a lower absorbance as compared to the negative control. Nickel solution was prepared by dissolving NiSO4• 6H2O in distilled water. To optimize the absorbance measurements in the downstream experiment, the wavelength at the peak absorbance of metal solutions were first determined using a spectrophotometer.
Experimental setup: measuring the peak absorbance of nickel solution. NiSO4• 6H2O was dissolved in distilled water for a 25mM Ni solution. The solution was measured for its absorbance across the full visible light spectrum using a spectrophotometer.
Overnight bacterial cultures were prepared and standardized to an OD600 of 0.7. Then, the cultures were centrifuged and the pellet was resuspended in nickel solution. The cell-nickel mixtures were gently shaken at room temperature for 2 hours. The cells were then spun down to isolate extracellular solution as the supernatant. The peak absorbance of the nickel ions in the supernatant was measured using a spectrophotometer blanked with distilled water.
Experimental setup: measuring extracellular concentrations of protein-cell mixtures. The pelleted bacteria were resuspended in nickel solution. After gently shaking the mixture for 2 hours, the absorbance at 651.4 nm of the supernatant was measured using a spectrophotometer. It is expected that the extracellular solution of the experimental group has a lower absorbance than the negative control.
Our results indicate that there are lower absorbance values at the peak absorbance of nickel, 651.4 nm, for cells expressing the Met-mRFP fusion protein, as compared to the RFP-only negative control. There is a -40.1% percent difference between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular nickel concentration in the presence of Met-mRFP. This shows that proteins are capable of binding to nickel ions, thus increasing the cell’s ability to retain metal ions from their environment.
Met-mRFP increases cellular retention of nickel ions. After two hours of shaking incubation with 25 mM nickel (II) ions, all samples were centrifuged to isolate extracellular solution. At 651.4 nm (the absorbance peak of nickel ions), lower absorbance was observed in the extracellular solution of cells expressing Met-mRFP. Cells expressing RFP only were used as a negative control. Error bars represent standard error. There is a -40.1% percent difference between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular nickel concentration in the presence of Met-mRFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 789
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1614
Illegal AgeI site found at 1726 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 201
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