Experimental Design and Methodology:

Throughout the course of this project we have developed a thorough, multi-pronged approach to testing the efficacy of the various components of our system.

Purification of MS2 phage-like particles:

Next, the MS2 phage-like particles are purified using a variety of techniques. Each technique is analyzed for its effectiveness in terms of both purity and time/material resource costs:

1) Sucrose Purification

Sucrose purification involves placing samples on a sucrose gradient followed by centrifugation, which will separate particles based on size. Following centrifugation, particles from different bands in the sucrose gradient will be fractionated and further purified. This will allow us to first separate the P22 or MS2 from other proteins.

2) Size-Exclusion Chromatography (SEC)

Size-Exclusion Chromatography involves separating the proteins based on size through a porous gel bead column. The protein will be filtered through the column with an aqueous solution. The pores in the gel allow the sample to elute based on size. Samples of the elutions will be taken at certain time points and the absorbance of the protein will be measured. A chromatogram will be produced by plotting the absorbance on the y-axis and the elution time will be on the x-axis. The chromatogram will confirm the presence of the purified protein-based on size, and in which fraction, to be further purified.

3) Ion Exchange Chromatography (IEC)

Ion-Exchange Chromatography (IEC) involves separating biological macromolecules based upon the charge of the species in a sample loaded onto a column. The impure sample is loaded onto a column at a certain pH and is passed through the column matrix, which has either positively (in the case of anion-exchange chromatography) or negatively (in the case of cation-exchange chromatography) charged functional groups bound. As molecules are passed through the resin, charged molecules bind to the opposite charged functional groups in the resin. A salt gradient of increasing concentration is then applied to the column, where molecules with few charged groups are eluted first, followed by those with many charged groups. Some important considerations for ion-exchange chromatography are the flow rate of the pump attached to the column determines the resolution of the separation of the charged molecules that are eluted. The charged ions in the elution buffer used should be matched to the charge of the resin within the column.

4) Affinity Chromatography

Some of our parts have polyhistidine-tags (His-tag) that allow us to purity them using Nickel Affinity Chromatography. Using a column with immobilized nickel ions bound to beads in resin, when the sample is added, proteins with His-tags bind to the metal ions, while all other molecules are eluted. Imidazole is used to cleave the His-tag, eluting the capsid proteins.

Confirmation of MS2 phage-like particle assembly:

The assembly of the phage-like particles is confirmed using a variety of orthogonal metrics:

1) Size Exclusion Chromatography (SEC)

To confirm assembly using size-exclusion chromatography we analyze the results of a chromatogram, in tandem with SDS-PAGE gels to determine whether we have capsid proteins of the expected size. This is possible as higher molecular weight species elute from the column before the lower molecular weight species.

2) Analytical Ultracentrifugation (AUC)

Our next approach involves analytical ultracentrifugation (AUC), a first-principle biophysical characterization method for biological macromolecules in the solution phase. Through which hydrodynamic parameters, such as the sedimentation coefficient, diffusion coefficient, partial specific volume, and purity of various analytes can be determined. In order to confirm the presence of the Cas13a-crRNA complex within the MS2 capsid, sedimentation velocity experiments using UV absorbance detection can be used to develop a sedimentation profile of a sample, which can then be analyzed using the UltraScan analysis software. The various analytes within the profile are binned, which allows for sedimentation coefficients across the distribution to be correlated to expected molecular weights. An inference can then be made as to whether capsids are fully loaded, partially loaded, or empty. As fully loaded MS2 capsids, which possess a larger molecular weight, will sediment slower, which corresponds to a larger sedimentation coefficient. In order to validate these results, various rotor speed and temperature replicates can be employed to better characterize the capsids and Cas13a-crRNA complex in solution.

3) Transmission Electron Microscopy (TEM)

Transmission electron microscopy is used as an orthogonal metric to analytical ultracentrifugation (AUC). Although it is not favorable as TEM cannot distinguish partially loaded capsids from fully loaded capsids due to its purely surface-level observational abilities.

4) Dynamic Light Scattering

Using the fluorescent tag on Cas13a to track its activity with cyanobacteria using fluorescent microscopy, confocal microscopy or cytometry techniques. This would also allow us to determine the molecular weight, the diameter of the capsid, and observe whether our sample is pure or not.

Confirmation of the encapsulation of Cas13a and the crRNA by the MS2 capsid:

confirm the encapsulation of Cas13a and the crRNA by the MS2 capsid, a variety of analytical techniques, similar to those employed in confirming the assembly of the MS2 capsid:

1) Size-Exclusion Chromatography (SEC):

To confirm that Cas13a and the crRNA have been encapsulated by the MS2 capsid, size-exclusion chromatography can be used to separate the contents of a mixture based on molecular weight, where the higher molecular weight species elute from the column before the lower molecular weight species. In this case, the loaded capsids should elute from the column first, as they possess a higher molecular weight than the unloaded capsids.

2) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE):

SDS-PAGE can be used to observationally determine the molecular weight of capsids. In this case, the full, partially loaded, and empty capsids all have different molecular weights and would migrate different distances through the polyacrylamide gel.

3) Analytical Ultracentriguation (AUC):

Analytical ultracentrifugation can be used to characterize and compare the molecular weights of the species of a mixture, as well as provide information on the sedimentation and diffusion properties of the species in solution. While the precise shape of the molecules can not be determined, the anisotropy or sphericality of the species can. In tandem, these metrics provide a determination of the relative loading amount of the capsids.

4) Electron Microscopy (TEM):

Transmission electron microscopy can once again be used as an orthogonal metric to analytical ultracentrifugation (AUC). However, TEM cannot distinguish partially loaded capsids from fully loaded capsids due to its purely surface-level observational abilities.

Confirming the activity of Cas13a on the target mRNA:

Next, to confirm the activity of Cas13a on our target mRNA and the intended cell death of cyanobacteria, the following methods are used:

1) Cell viability test

In our cell viability test, Cas13a is incubated with a guide RNA that codes for a fluorescent protein. Therefore, the decrease in fluorescence will determine Cas13a effectivity.

2) polymerase chain reaction (qPCR)

Treated mRNA and untreated mRNA samples will be quantified to determine the presence of the target mRNA. Samples are aliquotted at various time points for analysis using liquid chromatography and mass spectrometry. These techniques yield information about the amount of protein present at a specific cycle.

3)Activity assays

Our crRNA is inserted into E. Coli culture to see if our system will cleave the E. Coli’s RNA. This can also be repeated in a colour assay using fluorescent protein tagging.

4) Quantifying the activity of mlrA in breaking down microcystins following the cleaving action of Cas13a

quantify the activity of mlrA in breaking down microcystins following the cleaving action of Cas13a, the minimum inhibitory concentration the enzyme will be determined through iteratively testing a series of dilutions.

Testing our system in a natural setting:

Finally, when testing our system in a natural setting, we will implement our system in samples of water taken from lakes contaminated with cyanobacteria. We will then measure concentrations of cyanobacteria before and after the implementation of our system to determine the effectiveness of our system in a natural environment. This will involve using a control cyanobacteria sample without our system and a variety of different concentrations of our treatment. The scale of these concentrations will be determined using scaled-up estimates based on the concentrations required at smaller-scales in the lab environment