Difference between revisions of "Part:BBa K2921550"

Revision as of 09:03, 17 October 2019

K2921550 Strong Promoter-RBS + 6xHis + Metallothionein(CRS5)-mRFP + Double Terminator. This construct constitutively expresses a colored metal-binding fusion protein: metallothionein (Basic part: BBa_K1460002) linked with mRFP (Basic part: BBa_E1010). 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

We inserted an upstream constitutive strong promoter-RBS combination (Basic part: BBa_K880005) and a downstream strong double terminator (Basic part: BBa_B0015) so as to maximize plasmid and protein expression. To ensure that the binding protein and colored protein were fused, we inserted a flexible glycine-serine linker between the binding protein and the chromoprotein. For protein purification in downstream applications, we tagged a 6-histidine after the RBS.

PCR

PCR check results, using the primers VF2 and VR, and sequencing results by Tri-I Biotech confirm this construct. (insert photo)

Characterization

To verify metallothionein-mRFP expression in E. coli, we ran a SDS-PAGE gel, expecting a protein band at ___kDa. We prepared and lysed overnight liquid cultures of cells expressing metallothionein-mRFP, metallothionein only, and mRFP only. The SDS-PAGE reveals a darker band at ___ kDa as compared to the metallothionein-only and mRFP-only controls. (insert photo)

Functional Assay with Nickel

Our construct produces intracellular metallothionein-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 the metallothionein-mRFP fusion protein and cells expressing the metallothionein-only protein. We had two negative control groups: cells expressing RFP-only and cells carrying a metallothionein 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 metallothionein 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(H2O)6 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 the lowest OD600 across all four groups. 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 393 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, 393 nm, for cells expressing the metallothionein-mRFP fusion protein and metallothionein-only protein, as compared to the negative controls. This shows that proteins are capable of binding to nickel ions, thus increasing the cell’s ability to retain metal ions from their environment.

Metallothionein and Metallothionein-RFP increase 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 393 nm (the absorbance peak of nickel ions), lower absorbance was observed in extracellular solution of cells expressing Metallothionein and Metallothionein-RFP. Cells carrying Met ORF (BBa_K1460002) and expressing RFP (BBa_K880005 + BBa_E1010) were used as negative controls. Error bars represent standard error.