Reporter Journal

Week 1: May 1 - May 3

This week we attended the appropriate safety courses required by our University to work in the lab. Besides that, the undergraduate supervisors presented about the principles of Genetics and Synthetic Biology to the new team members.

Week 2: May 6 - May 10

This week we participated in a general molecular biology workshop to refresh our memory of techniques used in molecular biology.

Week 3: May 13 - May 17

We continued the molecular biology workshop. This week we also divided up into our respective groups for the project and decided research priorities.

Week 4: May 20 - May 24

We examined three problems related to ferritin. Firstly, we investigated protein isolation of ferritin. We are considering the protocols created by Santambrogio et al. (1993), as they have been used by multiple other authors as well. This paper uses precipitation followed by column chromatography, and verification with spectroscopy, PAGE and SDS-PAGE (Levi 1994). Alternatively, we may consider using his-tags for Ni-NTA column purification.

Secondly, we examined the feasibility of using magnetism to manipulate ferritin. Commercial horse spleen ferritin was tested on a magnetic Dynabead rack, and as expected, it did not move, as the core should not be magnetic. We must thus synthesize magnetoferritin wherein the native ferrihydrite core is converted to magnetite--a paramagnetic material which can be attracted to magnetic fields (Jordan et al. 2010, Wong et al. 1998). We were able to attain some general knowledge of magnetism in nano-scale material, including a basic understanding of blocking temperatures, superparamagnetism and magnetic moments, all magnetism-related measurements. But we are ill-equipped to model this system theoretically in terms of calculated forces. We also explore the possibility of alternative magnetic particles, in the case ferritin is too small to exhibit magnetic forces significant enough to move proteins.

Magnetosomes were found to be much larger, but unviable due to the complexity of their creation. The CCMV virus may also be an option, although there is currently little known about it and therefore further research into this topic is required. (Lohsse et al. 2011).

Because we also intend to scaffold proteins to ferritin as in our TALE DNA-binding proteins, we looked into fusions to both C- and N-terminals of ferritin. The N- and C- termini of ferritin subunits are present on the outside and inside of the nanoparticle respectively, and therefore N-terminal fusions are an obvious choice to ensure TALEs are present on the exterior of the nanoparticles (Dorner et al. 1985).

Week 5: May 27 - May 31

While we are unsure of exactly how ferritin will be used in our project, any capacity in which we use it requires isolating recombinant ferritin from E. coli. We investigated which terminus to locate purification tags (eg, His tags) on ferritin subunits. Ingrassia et al. (2006) found that small amino acid additions to the C-terminus of either heavy or light chain ferritin are not deleterious to its function. Thus, we are contemplating fusing proteins to the N-terminal of ferritin as discussed last week, and using the C-terminus for tags to isolate recombinant subunits.

We also contemplated methods for controlling expression in terms of proportions of H and L subunits in completed ferritin. This could influence iron dynamics of completed ferritin. Lee et al. (2002) overcame this issue by fusing the heavy and light chains together to ensure that completed nanoparticles have a 1:1 ratio of each subunit. We are trying to determine pitfalls of this expression strategy. Finally, we investigated methods for validating nanoparticle formation. Namely, we considered fluorescence spectroscopy as tested by Parker et al. (2008), where 410nm output was indicative of iron incorporation as per assembled ferritins.

Apart from using magnetism to manipulate magnetoferritin to isolate pathogenic DNA, we developed a backup approach. Watt et al. (2012) constructed a nanoscale battery where ferritins with two different cores were oxidized and reduced on gold electrodes. We are contemplating using a split TALE capture system to bring ferritin into close association of an electrode. Current could then flow from ferritin into an electrode to indicate binding of pathogenic DNA. Finally, we considered how we might construct a magnetic device to isolate ferritin. Bushart et al. (2006) discussed method for isolating metal chelating magnetoferritin for metal purification. However, the necessary magnetic field strengths, mesh density, or mass of isolated compounds in these systems were not explicit. Thus, we are still uncertain about the feasibility of using magnetism to isolate proteins attached to magnetoferritin.

Week 6: June 3 - June 7

The application of ferritin in our project has changed substantially. Due to uncertainties of using magnetism to isolate proteins attached to ferritin, our team proposes instead to use it as a nanoscale protein scaffold. Our team was inspired by the work of REFERENCE et al. (2011) who used gold nanoparticles bound to ssDNA to detect DNA in a lateral flow strip assay.

We have decided to modify the aforementioned system. We’ll replace gold nanoparticle with ferritin and ssDNA with TALEs targeted to pathogenic E. coli markers. These nanoparticles will bind pathogen DNA via TALEs, be drawn up through a strip via capillary action, and become immobilized with complementary TALEs affixed to other regions on the strip. A reporter marker will also be fused to the recombinant ferritins to allow visualization of the pathogenic DNA.

This system is unique since double stranded sample DNA could be detected without having to first melt the DNA. Cheap prototypes for such a system could be constructed and would be suitable for cost constraints in the beef industry. Moreover, this system is amenable to scale-up for the identification of multiple pathogenic markers on a single lateral flow strip. We are, however, unsure as to what to use as a reporter enzyme.

We also met with two U of C professors in nanoscience to discuss the feasibility of our magnetism idea. Dr. Max Anikovskiy, an instructor from the nanoscience program, allowed us to use a 50 lb. pull force rare earth magnet to manipulate native horse spleen ferritin. However, there were no changes over a three hour period. We also spoke to Dr. Simon Trudel from the nanomagnetism research group. He seemed to think our idea was feasible, but might require optimizing with respect to the solvent system in which the particles are dissolved. From both these meetings, it emerged that modelling the fluid dynamics is beyond our capabilities. Our best bet is to test these ideas experimentally, one step at a time, by first synthesizing and testing magnetoferritin.

Week 7: June 10 - June 14

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Week 8: June 17 - June 21

An issue we addressed this week was the assembly of gene constructs to test ferritin in our system. A tentative strategy plan has been proposed. Coordinating this plan with the TALE and linker teams is necessary to ensure compatibility among the many components of this project. Upon receiving commercially-synthesized genes, we will order primers to use the Golden Gate assembly method.

To test whether the synthesis of magnetoferritin is successful, we must first synthesize it using the method proposed in week 4, where the native ferrihydrite core is converted to apoferritin then magnetite. Using a magnet with a relatively strong pull force (about 50 lbs.), we hope to see the magnetoferritin respond in solution.

We must also confirm the applicability of using ferritin as the Prussian blue reporter system. After generating ferritin with a Prussian blue ferrihydrite core, we will manipulate the core type, pH, coloured substrate, and temperature to test the feasibility of this method. If these tests are futile, we may need to develop an enzymatic reporter that will function in parallel with our system. The necessary materials have been obtained for these experiments, and results will follow shortly.

Week 9: June 24 - June 28

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Week 10: July 1 - July 5

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Week 11: July 8 - July 12

Our team reviewed primers design for our Golden Gate Assembly with emphasis on ensuring proper ligation of our products. This involved matching melting temperatures, ensuring there were no palindromic sequences, and considering the other requirements involved. Primer design is integral to our overall project since we must successfully use Golden Gate Assembly to synthesize our parts. Much of our time has been spent towards this goal.

Also, Ferritin gene sequences have arrived. We have transformed, MiniPrepped and ready as a template for the Golden Gate primers.

Week 12: July 15 - July 19

The primers for golden gate assembly arrived this week and we conducted the PCRs required for our first attempt at making our constructs. The final ligation product was run on a gel and appears promising, showing a 1500bp band, most likely representing our construct. Despite this, transformations have failed and therefore we have to look back at our primers and experimental design.

We now have a more set idea of how we’re going to synthesize magnetoferritin. We will most likely be using a flask with a rubber stopper that will allow inflow and outflow of the nitrogen gas, which will be bubbled into the solution with an air stone. Nitrogen is free to exit a small port in the stopper and reagents can easily be added via injection through the rubber stopper. Safe handling of the thioglycollic acid is paramount, therefore equipment has been ordered (elbow-length gloves, respirator, apron, and something else), and attempts at synthesis will be conducted as soon as we get the equipment.

Week 13: July 22 - July 26

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Week 14: July 29 - August 2

Realizing the time constraints we have for the submission of our parts, we reviewed our Golden Gate assembly experiment to ensure we can submit the parts to the registry. In case this method fails, we also began development of Gibson assembly primers to see if this assembly assembly method cooperates with us.

We began characterization of prussian blue ferritin through kinetics experiments. Our first goal was the construction Michaelis-Menten plot to determine the activity of our peroxidase-like catalyst with the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) substrate which ended up looking like this: INSRERT GAPHHHH These experiments then continued on to optimization experiments wherein we determined the optimal pH, temperature, and concentrations of ABTS and H2O2. We intend to repeat these experiments 3,3',5,5'-tetramethylbenzidine so that we could compare the two substrates and determine the most viable one for our final system.

Week 15: August 5 - August 9

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Week 16: August 12 - August 16

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Week 17: August 19 - August 23

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Week 18: August 26 - August 30

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Week 19: September 2 - September 6

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Week 20: September 9 - September 13

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Week 21: September 16 - September 20

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Week 22: September 23 - September 27

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Week 23: September 30 - October 4

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