Part:BBa_K1189018

Human ferritin di-subunit with E coil w/ LacI promoter

This part was created by fusing the heavy chain and light chains (BBa_K1189024 BBa_K1189025) of human ferritin together. It is expressed under the lacI promoter (BBa_J04500) and has a his-tag for protein purification. An E-coil (BBa_K1189011) is included in order to allow binding of parts containing the respective K-coil (BBa_K1189010). Characterization of this part was done primarily with commercially purchased ferritin. This ferritin is structurally very similar to our recombinant ferritin and does not differ in its chemical properties (Figure 1).

This construct can be used as a reporter through a modification of the iron core to form Prussian Blue (Figure 2). The resulting molecule can then catalyze the formation of radicals from hydrogen peroxide, which can then cause a colour change in substrates such as TMB or ABTS (Figure 3).

Figure 1. Ribbon visualization of a fully assembled ferritin protein.

Figure 14. Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.

Figure 3. Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period.

We performed a kinetic analysis of our Prussian blue ferritin. We included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 4, 5). For both of the substrates we can see that normal ferritin has a very low catalytic activity compared to our modified ferritin.

Figure 4. Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 µL of 10 mg/mL substrate was used in a 242 µL reaction volume.Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data.

Figure 5. Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 µL of 10 mg/mL substrate was used in a 242 µL reaction volume. Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1289