Team:Lethbridge/Wet-Lab/Timeline
Project Timeline
Wet Lab Work
Part 1. Confirm the assembly of our transport system P22 protein nanocompartments (PNCs) and MS2 phage-like-particles (PLPs).
Part 2. Confirm the assembly of the transport system with CRISPR-Cas13a cargo.
Part 3. Confirming the activity of CRISPR-Cas13a on our target mRNA and confirming the cleaving action of CRISPR-Cas13a to our selected CRISPR-RNA (crRNA) with assistance from mlrA; resulting in cell death of cyanobacteria.
The flow chart below outlines the experiments and procedures that will be utilized in these three components.
The experiments that have been conducted thus far involve using P22 PNCs. However, future experiments in the latter part of our second year plan will be conducted using MS2 PLPs.
Part 1 - Confirm the assembly of our transport system (P22 PNCs and MS2 PLPs)
b. Opening of cells
c. Purify our selected carrier/transport system
Sucrose Purification This 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.
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.
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.
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.
d. Dual Plasmid Transformation
- Encapsulation of Cas13a and our target sequence (the sequence we want to be cleaved), the crRNA.
- We will be employing two different backbones, as different origins of replication and antibiotic resistances are needed.
e. Confirm Assembly
To confirm assembly using SEC we view the results of a chromatogram to observe whether or not we have proteins of the correct size.
ii) Analytical Ultracentrifugation (AUC)
Our next approach involves analytical ultracentrifugation (AUC), a first principles 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.
iii) Transmission electron microscopy (TEM)
Can be used as an orthogonal metric to AUC (although TEM cannot distinguish partially loaded capsids from fully loaded capsids).
iv) Dynamic Light Scattering
Use the fluorescent tag on Cas13a and 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.
Part 2 - The confirmation of capsid assembly into our selected carrier:
a. Confirm Encapsulation of Cas13a and the crRNA by MS2
We can compare chromatogram results to the results from the SEC done with the empty capsids MS2 to see if there are any differences in size.
- Examine molecular weight of samples containing encapsulated Cas13a compared to empty capsids.
- Using transmission electron microscopy we will view capsids containing Cas13a.
- Using AUC (see above)
Part 3- Confirming the activity of Cas13a on our target mRNA and activity of mlrA
a. Confirm Cas13a cleaves the crRNA resulting in cell death.
- Test Cas13a with a guide RNA that codes for a fluorescent protein.
- Decrease in fluorescence will determine Cas13a effectivity.
- Treated mRNA and untreated mRNA samples will be quantified to determine the presence of the target mRNA.
- This yields information about the amount of protein present at a specific cycle.
- Insert our crRNA into E. Coli culture and see if our system will cleave the E. Coli’s RNA - Or do a colour assay using fluorescent proteins
b. Confirm that the enzyme mlrA breaks down microcystins following the cleaving action of Cas13a.
- Measure mlrA activity by incubating the enzyme in different concentrations of microcystin extract.
c. Determining minimum inhibitory concentration through testing a series of dilutions.
d. Testing our system in a natural setting.
- Using a control without our system.
- Using different concentrations of our system