Part:BBa_K2921650

Promoter + RBS + 6xHis + OprF + GS-linker + mRFP + Double Terminator

This construct constitutively expresses a colored metal-binding fusion protein: OprF linked with mRFP (Basic part: BBa_E1010). According to iGEM14_HUST-China’s basic part page of BBa_K1393001, OprF is an outer membrane porin of Pseudomonas aeruginosa that allows the passage of small hydrophilic molecules such as Cu²⁺ ions. The mRFP serves as a functional color reporter, allowing for the convenient visible or UV detection of the location of OprF.

Construct design

This construct was created to constitutively express OprF-GS-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 OprF (Basic part: BBa_K2921650) 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 OprF ORF for easy protein purification. 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.

This entire construct was synthesized by Twist Bioscience.

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 K2921650 using the primers VF2 and VR, which resulted in the expected size of around 1.8 kb.

Characterization

We used SDS-PAGE to check for OprF-GS-mRFP expression in E. coli carrying our construct. Bacterial cultures expressing either OprF-GS-mRFP or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. OprF-GS-mRFP is approximately 49 kDa, and we observed a strong signal at that size in the OprF-GS-mRFP lysate sample which was not present in the empty vector sample, suggesting that OprF-GS-mRFP is being expressed in the transformed E. coli.

To verify OprF-GS-mRFP expression in E. coli, we subjected OprF-GS-mRFP lysate to SDS-PAGE, expecting a signal at around 49 kDA. On the gel, we saw a signal at around 49 kDA in the OprF-GS-mRFP lane, but not in the empty lane that was used as a control.

Functional Assay with Copper

Our construct produces intracellular OprF-GS-mRFP proteins expected to increase the cells’ capacity to store copper ions. To test the functionality of this protein, we detected the difference in the copper ion storage capacity of construct-expressing cells and negative-control cells. Thus, our experimental group was cells expressing the OprF-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 copper ions over time, to allow the copper ions to diffuse in and out of the cell. Theoretically, for our experimental groups, the copper ions would diffuse into the cell and bind to the active site of the intracellular OprF protein, reducing the amount of copper ions diffusing out of the cell. After 2 hours of incubation, we measured the absorbance of copper 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 copper ions and, thus, a lower absorbance as compared to the negative control. Copper solution was prepared by dissolving CuSO₄ • 5H₂O 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 copper solution. <b> CuSO₄(H₂O)₅ was dissolved in distilled water for a 10 mM Cu 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 copper solution. The cell-copper 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 copper 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 copper solution. The cell-copper 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 copper ions in the supernatant was measured using a spectrophotometer blanked with distilled water.

<b>Experimental setup: measuring extracellular concentrations of cell-metal mixtures. The pelleted bacteria were resuspended in copper solution. After gently shaking the mixture for 2 hours, the absorbance at 800.7 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 lower absorbance values at the peak absorbance of copper, 800.7 nm, for cells expressing the OprF-GS-mRFP fusion protein, as compared to the RFP only negative control. There is a percent difference of -72.2% between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular copper concentration in the presence of OprF-GS-mRFP. This shows that proteins are capable of binding to copper ions, thus increasing the cell’s ability to retain metal ions from their environment.

OprF-GS-mRFP increases cellular retention of copper ions. After two hours of shaking incubation with 10 mM copper (II) ions, all samples were centrifuged to isolate extracellular solution. At 800.7 nm (the absorbance peak of copper ions), lower absorbance was observed in the extracellular solution of cells expressing OprF-GS-mRFP. Cells expressing RFP only were used as a negative control. Error bars represent standard error. There is a -72.2% percent difference between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular copper concentration in the presence of OprF.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 698
  Illegal AgeI site found at 1286
  Illegal AgeI site found at 1398
  
• 1000
  COMPATIBLE WITH RFC[1000]