Difference between revisions of "Part:BBa K2921500"

Revision as of 08:21, 21 October 2019

Promoter + Double RBS + Metallothionein + Terminator

This construct constitutively expresses metal-binding protein Metallothionein. 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.

Construct design

This construct was created to constitutively express the Met 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), 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 K2921500 using the primers VF2 and VR, which resulted in the expected size of around 1.3 kb.

Characterization

We used SDS-PAGE to check for Met expression in E. coli carrying our construct. Bacterial cultures expressing either Met or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. The expected size of Met is approximately 34 kDa, but we observed a strong signal at approximately 37 kDa in the Met lysate sample, which was not present in the empty vector sample. This discrepancy in size is likely due to post-translational modifications on the protein such as phosphorylation and glycosylation.

All the lanes on this figure were run on the same gel. To verify Met expression in E. coli, we subjected Met lysate to SDS-PAGE, expecting a signal at around 34 kDa. Instead, we saw a signal at around 37 kDa in the Met lane, but not in the empty lane that was used as a control. This discrepancy in size is likely due to post-translational modifications on the protein such as phosphorylation and glycosylation.

Functional Assay with Nickel

Our construct produces intracellular Met 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 protein and cells expressing a Met-mRFP fusion protein (Composite part: K2921520). We had two negative control groups: cells carrying a Met ORF-only plasmid and cells expressing RFP only. 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 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• 6H₂in 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• 6H_{2</sub 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 cell-metal 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 protein, as compared to the ORF only negative control. There is a -36.3% 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. This shows that proteins are capable of binding to nickel ions, thus increasing the cell’s ability to intake metal ions from their environment.

Met increased 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. Cells carrying Met ORF (BBa_K1460002) only were used as a negative control. Error bars represent standard error. There is a -36.3% 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.

Sequence and Features