Part:BBa_K2921400

Strong Promoter + RBS + MBP + Double Terminator

This construct constitutively expresses a colored metal-binding protein MBP. According to iGEM10_Peking’s composite part page of BBa_K346004, MBP is a lead binding peptide that can accumulate lead ions from aquatic environment.

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.

PCR

The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.

We confirmed the size of K2921400 using the primers VF2 and VR, which resulted in the expected size of around 0.8 kb.

Characterization We used SDS-PAGE to check for MBP expression in E. coli carrying our construct. Bacterial cultures expressing MBP were grown overnight at 37°C, lysed and run on SDS-PAGE gels. The expected size of MBP is approximately 18 kDa, but we observed a strong signal at approximately 23 kDa in the MBP lysate sample. This discrepancy in size is likely due to post-translational modifications on the protein such as phosphorylation and glycosylation.

To verify MBP expression in E. coli, we subjected MBP lysate to SDS-PAGE, expecting a signal at around 43 kDa. Instead, we saw a signal at around 23 kDa in the MBP lane. This discrepancy in size is likely due to post-translational modifications on the protein such as phosphorylation and glycosylation.

Functional Assay with Lead

Our construct produces intracellular MBP proteins expected to increase the cells’ capacity to store lead ions. To test the functionality of this protein, we detected the difference in the lead ion storage capacity of construct-expressing cells and negative-control cells. Thus, we had two experimental groups: cells expressing this MBP protein and cells expressing a MBP-mRFP fusion protein (Composite part: K2921420). We had two negative control groups: cells carrying a MBP ORF-only plasmid and cells expressing RFP only. In order to measure cell storage capacity, we incubated cells with the lead ions over time, to allow the lead ions to diffuse in and out of the cell. Theoretically, for our experimental groups, the lead ions would diffuse into the cell and bind to the active site of the intracellular MBP protein, reducing the amount of lead ions diffusing out of the cell. After 2 hours of incubation, we measured the absorbance of lead 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 lead ions and, thus, a lower absorbance as compared to the negative control. Lead solution was prepared by dissolving Pb(NO₃)₂ 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 lead solution. Pb(NO₃)₂ was dissolved in distilled water for a 5mM Pb 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 lead solution. The cell-lead 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 lead 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 lead solution. After gently shaking the mixture for 2 hours, the absorbance at 298.7nm 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 higher absorbance values at the peak absorbance of lead, 298.7 nm, for cells expressing the MBP protein, as compared to the ORF only negative control. This suggests that the cells expressing MBP protein were not capable of binding to the lead ions, as the higher extracellular absorbance suggests a weaker ability to retain lead. This discrepancy in our results can be attributed to the immediate clumping of the cell-lead mixtures that we observed for all of our experimental and control groups. The clumping is most likely a sign of bacteria death in the presence of a high concentration of lead.

MBP increased cellular retention of lead ions. After two hours of shaking incubation with 5 mM lead (II) ions, all samples were centrifuged to isolate extracellular solution. At 298.7nm (the absorbance peak of lead ions), a higher absorbance was observed in the extracellular solution of cells expressing MBP. Cells carrying MBP ORF (BBa_K346004,) only were used as a negative control. This suggests that the cells expressing MBP protein were not capable of binding to the lead ions, as the higher extracellular absorbance suggests a weaker ability to retain lead. This discrepancy in our results can be attributed to the immediate clumping of the cell-lead mixtures that we observed for all of our experimental and control groups. The clumping is most likely a sign of bacteria death in the presence of a high concentration of lead.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]