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Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin
From 2013.igem.org
Prussian Blue Ferritin
Prussian Blue Ferritin
What is Ferritin?
Ferritin is a ubiquitous iron storage protein found in both prokaryotes and eukaryotes that allows cells to keep iron in a soluble and non-toxic form (Munro, 1990). Ferritins across different species have very similar architecture and function, despite variations at the primary structure level (Harrison and Arosio, 1996). The 450 kDa protein shell consists of 24 subunits that can be composed of both heavy and light chains (Lawson et al., 1991) (Figure 1). Inside this shell is room for an iron core composed of up to 4500 Fe (III) atoms stored as ferrihydrite phosphate (Ford et al., 1984). The goal of our project is to make use of this natural nanoparticle as both a scaffold and a reporter system.
Figure 1. Ribbon visualization of a fully assembled ferritin protein.
How can Ferritin be a Reporter?
Iron is worthy of attention because of its ability to participate in Fenton chemistry. In this reaction, iron acts as a catalyst to cause the disproportionation of hydrogen peroxide into oxygen-radical species (Figure 2). The resulting hydroxyl radical can cause the oxidation of common horseradish peroxidase substrates such as 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 3,3’,5,5’-tetramethylbenzidine (TMB) to produce a colourimetric output. The standard iron core of ferritin, however, is not very suitable for catalyzing this process due to the lack of catalytic ferrous ions. The catalytic activity of ferritin can be increased by synthesizing ferritin with a magnetite core (Zhang et al., 2010). The synthesis of this ”magnetoferritin” however is time consuming and inefficient due to the procedure being prone to oxidation.
Figure 2. Chemical equation for the disproportionation of hydrogen peroxide into oxygen radical species using iron as a catalyst; Fenton chemistry.
An alternative to increasing the catalytic activity of ferritin would be to provide the necessary ferrous ions for the Fenton chemistry via the iron complex Prussian blue. This compound could act as a surface modification agent to give the ferritin core a Prussian blue surface. Prussian blue has previously been shown to have high catalytic activity and is believed to act as a source of negative charge at appropriate pH levels to yield an affinity with positively charged peroxidase substrates such as TMB (Zhang et al., 2010).
Synthesizing Prussian Blue Ferritin
The creation of Prussian blue ferritin is a relatively simple process that involves the surface modification of the iron core of normal ferritin. To accomplish this, purified ferritin is combined with the compound potassium ferrocyanide under acidic conditions to produce the iron complex Prussian blue (Zhang et al., 2013). This chemical reaction is shown below:
Figure 3. Chemical equation for the synthesis of Prussian blue ferritin using potassium ferrocyanide.
The chemical reaction can easily be observed as there is a visible change from the colour of ferritin to the blue colour of Prussian blue ferritin (Figure 4). This reaction takes place overnight and then the Prussian blue ferritin is collected via dialysis. For this process, we made use of commercial horse spleen ferritin purchased from the commercial supplier Sigma-Aldrich. This commercial ferritin is structurally very similar to the human ferritin we will be producing in our bacteria, which we anticipate would display very similar properties to one another, making use of the commercial reagent a good starting point to demonstrate our concept.
Figure 4. Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period.
Kinetic Testing of Prussian Blue Ferritin
The next step in moving forward with using Prussian blue ferritin as a potential reporter was to determine the kinetic characteristics of the catalyst. This analysis is necessary to understand how our reporter will react over time in relation to different substrate concentrations. Previous data (Zhang et al., 2013) had shown, that the catalytic activity can be fit to Michealis-Menten kinetic models, which gives us an opportunity to evaluate the Prussian blue ferritin as a reporter. Our analysis composed of varying both the chromogenic substrate (TMB or ABTS) concentration and the hydrogen peroxide concentration.
Our kinetic analysis also included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 5 and 6). For both of the substrates we can see that normal ferritin has a very low catalytic activity. This is excellent news as it means that our Prussian blue modification is an effective method to create a strong catalyst. It also means that the presence of unmodified ferritin is not active enough to pose a significant risk for producing spurious results in our system.
Figure 5. 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 6. 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.
In order to complete our kinetic analysis we had to determine the catalytic properties of our Prussian blue ferritin according to the Michaelis-Menten kinetic model. For these tests we varied the colourimetric substrate concentrations (ABTS and TMB) (Figures 7 and 8). We also varied the hydrogen peroxide concentration in association with TMB as this the first chemical compound that will react in the system (Figure 8). This test was not repeated for ABTS as we had decided TMB was a more viable substrate at this point in the summer. Based off of the calculated catalytic constants (Table 1) it would appear that TMB is a more effective substrate for our system.
Figure 7. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of ABTS. Absorbance readings were taken at 415 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 6).
Figure 8. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of TMB. Absorbance readings were taken at 650 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 5).
Figure 9. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of hydrogen peroxide. Absorbance readings were taken at 650 nm which measure the breakdown of TMB. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data.
| Catalyst | Enzyme Concentration (M) | Substrate | Km (mM) | Vmax (Ms-1) | Kcat (s-1) | Kcat/Km (M-1s-1) |
| Prussian Blue Ferritin | 1.31 x 10-9 | ABTS | 0.448 | 1.25 x 10-8 | 9.51 | 2.12 x 104 |
| Prussian Blue Ferritin | 1.31 x 10-9 | TMB | 0.0432 | 1.12 x 10-7 | 85.3 | 1.97 x 106 |
| Prussian Blue Ferritin | 1.44 x 10-9 | H2O2 (TMB) | 0.0176 | 1.45 x 10-8 | 10.1 | 5.71 x 105 |
Optimization of Prussian Blue Ferritin Reaction Conditions
The next step of our process was to determine the optimal conditions in which our commercial Prussian blue ferritin reporter could operate. This is a very important step in determining how we will move forward with creating a portable sensor device. The first variable tested was pH. For this variable we can see that the lower the pH the higher the activity of Prussian blue ferritin in the case of the ABTS substrate. As we raise the pH for this substrate we see that the activity drastically drops off (Figures 10 and 11). We did not test below a pH of 2 as the ferritin subunits would dissociate at this point. For the TMB substrate however we see that the activity has a much higher relative activity at a neutral pH compared to the ABTS substrate. This would be beneficial in our system as it means the substrate solution can be a lot less harsh, which will prevent chances of functional disruption to other proteins on the strip.
Figure 10. pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.
Figure 11. pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.
The next variable we tested was the effect of temperature on the commercial Prussian blue ferritin (Figures 12 and 13). For both substrates we can see that the as the temperature increases. Armed with this information we know that if can increase the incubation temperature of our system when we add the substrate solution we can potentially decrease the amount of time it will take our system to produce a signal from the presence of our target DNA.
Figure 12. Temperature optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.
Figure 13. Temperature optimization of commerical Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.
Does Prussian Blue Ferritin Work on a Strip?
After doing the kinetic analysis of the Prussian blue ferritin it was necessary to think in terms of our actual device. There are many factors that we must balance in order to design the most optimal system. How much Prussian blue ferritin will give us a positive result that we can see with the naked eye? Which substrate will give us a result the fastest? What substrate colour is the easiest to see (Figure 14)?
Figure 14. Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.
In order to test the Prussian blue ferritin in a strip environment we blotted varying concentrations of Prussian blue ferritin onto nitrocellulose; the material which will make up our strip system. From the results below it became readily obvious that TMB produced a much more distinct and identifiable colour compared to ABTS (Figures 15 and 16). Further testing also revealed Prussian blue ferritin was able to produce a detectable reaction of TMB when only 2.5 nanograms of the Prussian blue ferritin was present. We anticipate that this detection limit could be improved given more refined methods of applying the protein to the nitrocellulose. We also decided based on the results of this experiment and previous experiments we have decided that TMB is the optimal substrate for our system. Overall TMB produces a colour output that is much easier for the human eye to see as well as that it can be in a less harsh substrate solution which limits the potential of functional disruption of other proteins on our strip. We could potentially explore more sensitive substrates to see if they interact with Prussian blue ferritin differently or are more sensitive with our system. We also tested Prussian blue ferritin in a basic prototype housing. You can see the results of this experiment on the Prototype page.
Figure 15. Blots of Prussian blue ferritin on nitrocellulose (5 µL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.
Figure 16. Blots of Prussian blue ferritin on nitrocellulose (5 µL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.
What did we build this summer?
In order to make use of the data we have gathered from characterizing commercially purchased horse spleen ferritin in our strip system we had to develop constructs that would be functional in our strip system. The first ferritin part we constructed and and purified the protein from this summer is BBa_K1189018 (Figure 17). This part consists of the heavy and light subunits of ferritin fused together. These subunits will still successfully assemble to form the fully assembled ferritin nanoparticle. Also attached is an E coil which will allow this protein to bind with proteins that have a fused K coil. In the case of our system this will allow us to bind BBa_K1189018 via coil interactions as a reporter to BBa_K1189029; one of our DNA binding constructs.
The second construct of interest (BBa_K1189021) is once again composed of our ferritin subunits but this time it has a TALE protein directly fused to the ferritin subunits (Figure 17). This part allows more flexibility in trying out different variants of our strip system as in this case we have the capability to create a reporter that is directly fused to part of our detector system. Both of these constructs are expressed under the promoter PlacI allowing for the expression of these parts as we need the proteins produced and purified for our in vitro system.
Figure 17. The two ferritin constructs used for the testing of Prussian Blue ferritin catalytic activity
So can our Recombinant Ferritin become Prussian Blue Ferritin?
After our proof of concept efforts and once our own constructs of ferritin (BBa_K1189018, BBa_K1189021) were purified it was necessary to see if this same treatment could be applied to bacterially produced human ferritin. The same synthesis reaction to cause the Prussian blue surface modification of this ferritin was performed except that it was scaled down to match the concentrations of our ferritin. Our own Prussian blue ferritin was then exposed to the TMB substrate (Figure 18). From the results we can see that the ferritin with the E coil attached had excellent catalytic activity (BBa_K1189018). The same ferritin with TALEs attached did not display as high of activity (BBa_K1189021). While this result is disappointing it may be remedied with further optimization of the Prussian blue synthesis reaction. It also does not signify a major problem as we have multiple variants of our strip system and ferritin fused directly to TALEs is not a part that is absolutely necessary.
Figure 18. Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 µL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself. Due to limitations on the protein available only one replicate was performed. Zero time points do not have low absorbance as colour change was rapid and began before measurements started.
How does the use of Coils versus Direct Fusions of TALEs Affect our Prussian Blue reporter?
After successfully confirming that we could convert our own ferritin proteins that were produced from the parts we constructed (BBa_K1189018, BBa_K1189021) into Prussian blue ferritin the next step was to evaluate how the design of our parts could potentially affect the reporter activity of our Prussian blue ferritin. Based on the spatial modelling performed by our team it was suggested that assembly of the ferritin nanoparticle with TALE proteins directly fused was highly unlikely. This is because the TALE proteins are significantly larger than the ferritin subunits. Their size would likely result in steric hindrance and prevent the assembly of the full ferritin protein. In order to test the predictions put forward by our modelling we ensured that our protein samples were balanced in order to have the same number ferritin cores in each sample. The catalytic activity of these proteins was then compared. From the data gathered we saw that the Prussian blue ferritin with fused coils (even if TALES are additionally bound to the ferritin via coils) was more effective as a reporter than having the TALE proteins directly fused to the ferritin nanoparticle (Figure 19). The results from this experiment suggest that the predictions made by our model were correct. Using coils however alleviates this issue as these coils are small and would not interfere in the ferritin self-assembly but can be used to attach our TALES to create the FerriTALE.
Figure 19. Samples of our parts that were converted to Prussian Blue ferritin were mole balanced in order to ensure that the same number of effective ferritin cores are present in every sample. Additionally the ferritin-coil fusion was incubated with the TALE-coil fusion part in order to allow their binding for a separate trial. Negative controls include unconverted recombinant ferritin, bovine serum albumin and a substrate only control. Samples were incubated with a TMB substrate solution for 10 minutes at a pH of 5.6. Absorbance readings were taken at the 10 minute time-point at a wavelength of 650 nm. An ANOVA (analysis of variants) was performed upon the values to determine that there was statistical difference in the data gathered (based off of three replicates). A t-test was then performed which determined that the * columns are significantly different from the ** column (p=0.0012). Neither * column is significantly different from each other (p=0.67).
Future Directions
This summer we were able to successfully characterize Prussian blue ferritin as a reporter for our system as well as demonstrate that we could use our constructed parts to produce our own functional Prussian blue ferritin. This characterization included a full kinetic analysis, pH optimization, temperature optimization, and exploring the use of Prussian blue ferritin in a strip assay. In conjunction with our modelling efforts we also were able to evaluate the efficacy of different techniques of constructing our FerriTALE. Moving forward with the data gathered so far on ferritin we would like to further explore its application to our system. This includes fine-tuning the use of ferritin as a reporter for our system based off of the modelling performed. In order to increase the efficacy of the Prussian blue ferritin produced from our parts we would like to also explore different iron loading techniques for ferritin in order to ensure that the nanoparticle is fully saturated with iron before it undergoes the Prussian blue synthesis process. In the context of our prototype we would also like to explore different methods of applying our Prussian blue ferritin to our strips during experiments in order to see if we can reduce the minimum level of detection for our strip assay. We are excited to use ferritin in the context of our full system and to see how future iGEM teams can make use of ferritin.
