Part:BBa_K2921300

Strong Promoter + RBS + Bacterioferritin + Double Terminator

This construct constitutively expresses a colored metal-binding protein bacterioferritin. According to iGEM14_Berlin’s basic part page of BBa_K1438001, Bacterioferritin is a protein that contains bacterial ferritin and can store iron.

Construct design

This construct was created to constitutively express BacFerr 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 BacFerr (Basic part: BBa_K1438001), and a double terminator (BBa_B0015) to end transcription.

PCR

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

We confirmed the size of K2921300 using the primers VF2 and VR, which resulted in the expected size of around 1.0 kb.

Characterization

We used SDS-PAGE to check for BacFerr expression in E. coli carrying our construct. Bacterial cultures expressing either BacFerr or BBa_K880005 (empty vector) were grown overnight at 37°C, lysed and run on SDS-PAGE gels. BacFerr is approximately 19 kDa, and we observed a strong signal at that size in the BacFerr lysate sample which was not present in the empty vector sample, suggesting that BacFerr is being expressed in the transformed E. coli.

To verify BacFerr expression in E. coli, we subjected BacFerr lysate (left gel) to SDS-PAGE, expecting a signal at around 19 kDA. On the left gel, we saw a signal at around 19 kDA in the BacFerr lane, but not in the empty lane that was used as a control.

Functional Assay with Iron

Our construct produces intracellular BacFerr proteins expected to increase the cells’ capacity to store iron ions. To test the functionality of this protein, we detected the difference in the iron ion storage capacity of construct-expressing cells and negative-control cells. Thus, we had two experimental groups: cells expressing this BacFerr protein and cells expressing a BacFerr-mRFP fusion protein (Basic part: BBa_K1438001). We had two negative control groups: cells carrying a BacFerr ORF-only plasmid and cells expressing mRFP only. In order to measure cell storage capacity, we incubated cells with the iron ions over time, to allow the iron ions to diffuse in and out of the cell. Theoretically, for our experimental groups, the iron ions would diffuse into the cell and bind to the active site of the intracellular BacFerr protein, reducing the amount of iron ions diffusing out of the cell. After 2 hours of incubation, we measured the absorbance of iron 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 iron ions and, thus, a lower absorbance as compared to the negative control. Iron solution was prepared by dissolving FeSO₄ • 7H₂O 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 iron solution. FeSO₄(H₂O)₇ was dissolved in distilled water for a 15mM Fe 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 pellets were resuspended in iron solution. The cell-iron 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 iron ions in the supernatant was measured using a spectrophotometer blanked with distilled water.

Experimental setup: measuring extracellular concentrations of cell-BacFerr mixtures. The pelleted bacteria were resuspended in iron solution. After gently shaking the mixture for 2 hours, the absorbance at 776.8 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 iron, 776.8 nm, for cells expressing the BacFerr protein, as compared to the ORF only negative control. There is a -20.9% percent difference between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular iron concentration in the presence of BacFerr. This shows that proteins are capable of binding to iron ions, thus increasing the cell’s ability to intake metal ions from their environment.

BacFerr increased cellular retention of iron ions. After two hours of shaking incubation with 15 mM iron (II) ions, all samples were centrifuged to isolate extracellular solution. At 776.8 nm (the absorbance peak of iron ions), lower absorbance was observed in the extracellular solution of cells expressing BacFerr. Cells carrying BacFerr ORF (BBa_K1460002) only were used as a negative control. Error bars represent standard error. There is a -20.9% percent difference between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular iron concentration in the presence of BacFerr.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 187