Difference between revisions of "Part:BBa K2921350"

Revision as of 09:03, 21 October 2019

Strong Promoter + RBS + 6xHIS + BacFerr + GS-linker + mRFP + Double Terminator

This construct constitutively expresses a colored metal-binding fusion protein: bacterioferritin linked with mRFP (Basic part: BBa_E1010). According to iGEM14_Berlin’s basic part page of BBa_K1438001, Bacterioferritin is a protein that contains bacterial ferritin and can store iron. The mRFP serves as a functional color reporter, allowing for the convenient visible or UV detection of the location of bacterioferritin.

Construct design

This construct was created to constitutively express BacFerr-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 BacFerr (Basic part: BBa_K1438001), 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 BacFerr 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 K2921350 using the primers VF2 and VR, which resulted in the expected size of around 1.6 kb.

Characterization

We used SDS-PAGE to check for BacFerr-GS-mRFP expression in E. coli carrying our construct. Bacterial cultures expressing BacFerr-GS-mRFP were grown overnight at 37°C, lysed and run on SDS-PAGE gels. BacFerr-GS-mRFP is approximately 46 kDa, and we observed a strong signal at that size in the BacFerr-GS-mRFP lysate sample, suggesting that BacFerr-GS-mRFP is being expressed in the transformed E. coli.

Functional Assay with Iron

Our construct produces intracellular BacFerr-GS-mRFP 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, our experimental group was cells expressing the BacFerr-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 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 bacterioferritin 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 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 protein-cell 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-GS-mRFP fusion protein, as compared to the RFP only negative control. There is a percent difference of -70.7% between the mean absorbance values of the experimental and control group, suggesting a decrease in extracellular iron concentration in the presence of BacFerr-GS-mRFP. This shows that proteins are capable of binding to iron ions, thus increasing the cell’s ability to retain metal ions from their environment.

Bacterioferritin and Bacterioferritin-RFP increase cellular retention of iron ions. After two hours of shaking incubation with 15 mM iron 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-GS-mRFP. Cells expressing mRFP only were used as a negative control. Error bars represent standard error. There is a -70.7% 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