Part:BBa_K2921420
Promoter + RBS + MBP + mRFP + Double Terminator
This construct constitutively expresses a colored metal-binding fusion protein MBP linked with mRFP. 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. The mRFP serves as a functional color reporter, allowing for the convenient visible or UV detection of the location of MBP.
Construct design
This construct was created to constitutively express MBP-mRFP. 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 MBP-mRFP (Basic part: BBa_K346004), and a double terminator (BBa_B0015) to end transcription.
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 K2921420 using the primers VF2 and VR, which resulted in the expected size of around 1.5 kb.
Characterization
We used SDS-PAGE to check for MBP-mRFP expression in E. coli carrying our construct. Bacterial cultures expressing MBP-mRFP were grown overnight at 37°C, lysed and run on SDS-PAGE gels. The expected size of MBP-mRFP is approximately 43 kDa, but instead we observed two separate signals at approximately 23 kDa and 26 kDa in the MBP-mRFP lysate sample. The expected size of MBP only is around 18 kDa and the expected size of mRFP is around 25 kDa. Seeing that the MBP and mRFP were expressed separately, we designed a DNA construct (BBa_K2921450) in which we added a GS-linker to connect the two proteins.
To verify MBP-mRFP expression in E. coli, we subjected MBP-mRFP lysate to SDS-PAGE, expecting a signal at around 43 kDa. Instead, we saw separate signals at approximately 23 kDa and 26 kDa in the MBP-mRFP lane. Seeing that the MBP and mRFP were expressed separately, we designed a DNA construct (BBa_K2921450) in which we added a GS-linker to connect the two proteins.
Functional Assay With Lead
Our construct produces MBP-mRFP 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 the MBP-mRFP fusion protein and cells expressing the MBP-only protein. We had two negative control groups: cells expressing RFP-only and cells carrying an MBP ORF-only plasmid.
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 MBPprotein, 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 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 protein-cell mixtures, The pelleted bacteria were resuspended in lead solution. After gently shaking the mixture for 2 hours, the absorbance at 298.7 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 higher absorbance values at the peak absorbance of lead, 298.7 nm, for cells expressing the MBP-mRFP fusion protein as compared to the negative controls. This suggests that the cells expressing MBP-mRFP 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 and MBP-RFP decrease cellular retention of lead ions. After two hours of shaking incubation with 5 mM lead ions, all samples were centrifuged to isolate extracellular solution. At 298.7 nm (the absorbance peak of lead ions), higher absorbance was observed in extracellular solution of cells expressing MBP-RFP. Cells carrying MBP ORF (BBa_K346004) and expressing RFP (BBa_K880005 + BBa_E1010) were used as negative controls. This suggests that the cells expressing MBP-mRFP 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1123
Illegal AgeI site found at 1235 - 1000COMPATIBLE WITH RFC[1000]
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