Part:BBa_K2921550

Metallothionein + EAAAK-linker + mRFP

This construct constitutively expresses a colored metal-binding fusion protein: metallothionein linked with mRFP (Basic part: BBa_E1010). According to iGEM14_Cornell’s composite part: 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 Met-GS-mRFP fusion proteins. 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 (Basic part: BBa_K1460002), the chromoprotein gene mRFP (Basic part: BBa_E1010), and a double terminator (BBa_B0015) to end transcription.

In addition, for downstream applications, we included a hexahistidine tag (6xHis) after the RBS and prior to the Metallothionein ORF for easy protein purification. To ensure that the binding protein and colored protein were fused, we inserted a flexible GS-linker between the binding protein and the chromoprotein.

This entire construct was synthesized by Twist Bioscience.

PCR

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

b>We confirmed the size of K2921550 using the primers VF2 and VR, which resulted in the expected size of around 1.4 kb.</b>

Characterization

We used SDS-PAGE to check for Met-GS-mRFP expression in E. coli carrying our construct. Bacterial cultures expressing either Met-GS-mRFP or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. The expected size of Met-GS-mRFP is approximately 35 kDa, but we observed a strong signal at approximately 36 kDa in the Met-GS-mRFP 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.

To verify Met-GS-mRFP expression in E. coli, we subjected Met-GS-mRFP lysate to SDS-PAGE, expecting a signal at around 34 kDa. Instead, we saw a signal at around 36 kDa in the Met-GS-mRFP 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-GS-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, our experimental group was cells expressing the Met-GS-mRFP fusion protein. Our negative control group was cells expressing mRFP 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 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.

(insert photo)

Experimental setup: measuring the peak absorbance of nickel solution. NiSO₄(H₂O)₆ was dissolved in distilled water for a 25 mM 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. 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.

(insert photo)

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-GS-mRFP fusion protein, as compared to the RFP only negative control. There is a percent difference of -31.7% between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular nickel concentration in the presence of Met-GS-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.

(insert photo)

Met-GS-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-GS-mRFP. Cells expressing RFP only were used as a negative control. Error bars represent standard error. There is a -37.1% 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.

Color visibility

To verify that the mRFP exhibits red even after being linked to Met, we compared the absorbance at 607nm, which is the emission peak of mRFP (Basic part: BBa_E1010), of cells expressing Met-GS-RFP fusion protein to that of cells expressing the Met protein only. First, we standardized overnight bacterial cultures of cells expressing the Met-GS-RFP protein and the Met protein only (K2921500), which should not exhibit color, to the same OD700 to normalize the cell density of the two groups. Then, we diluted the two groups with LB by a ratio of 1:2 to generate a total of four groups for this experiment. After, we used a spectrophotometer to determine the absorbance of the four groups. It is expected that the cells expressing the Met-GS-mRFP protein will have a higher absorbance value at OD607, as it is the peak wavelength of mRFP.

(insert photo)

The mRFP in the Met-GS-mRFP fusion protein does exhibit the color pink. The absorbance at 607nm of the cells expressing the Met-GS-mRFP protein is higher than the cells expressing the Met protein only, which does not have the fluorescent protein..

(insert photo)

The mRFP in the Met-GS-mRFP fusion protein does visibly exhibit the color pink. The overnight liquid culture of cells expressing the Met-GS-mRFP protein is much darker than the cells expressing the Met protein only, which does not express the fluorescent protein..

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 706
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1531
  Illegal AgeI site found at 1643
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 118