iGEM 2022 — Team lethbridge

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### Implementation of Experimental Design:

During this year, most of the work in the lab was focused on the preparation of our constructs. Since this was a two year project, we did still have transformed cells from last year of lab work, which is what we started out using in the lab this year.

### Polymerase Chain Reaction

The first step in the preparation of our constructs was to amplify our construct DNA so that we could use it in future steps. We mainly used a Q5 PCR protocol, though we also used a Q5 high fidelity protocol and a pfu PCR protocol. After PCRs were run, amplification was confirmed with agarose gel electrophoresis, which often yielded negative results, which led us to troubleshoot the protocols in order to successfully amplify our DNA. This involved changing the concentrations of components in order to increase the chances of a successful amplification, as well as the agarose gels themselves.

### Restriction and DNPI Digests

Since all of our constructs are contained within cloning vector plasmids, before we attempted any transformations we had to digest them so our constructs could then be ligated into expression vectors. After digestion, we could then amplify the resulting DNA through a PCR, and confirm the digestion and amplification through an agarose gel. Unfortunately, most of our gels would only show bands corresponding with full plasmids, not digested fragments. We believe that the PCR buffer the constructs were suspended in was potentially inhibiting the enzyme needed for digestion, so cleaning the DNA and resuspending it theoretically would solve this problem.

### T4 Ligation

Once the constructs were cut out of the cloning vector plasmid, we can then use a DNA ligase to insert our constructs into an expression vector, which can then be transformed into bacteria for later expression. Since performing the ligation was dependent on the digestion, there were less ligations performed than many of the other procedures.

### Bacterial Transformation

Multiple bacterial transformations were performed over the course of this year’s lab work, both into BL21 cells, and into DH5a cells. Since all of our constructs include antibiotic resistance genes (either ampicillin or kanamycin) we could then plate them on media that contained appropriate antibiotics and confirm the transformation that way. We transformed our bacterial cultures with both our construct parts ligated into the new expression vectors, and the original cloning vectors.

### In-Vitro Transcription

In-vitro transcriptions were performed on our DNA constructs in order to synthesize RNA. Urea PAGEs were done after the IVTs in order to confirm success. The constructs that were used in the IVTs were Lbu\_Cas13a, McyH\_crRNA, and GFP\_crRNA.

### Agarose Gel Electrophoresis

1% agarose gels were used to confirm the success of our PCRs and digests, mainly at 100V for 45 minutes to an hour.

### Urea PAGE

The urea PAGEs were performed on our IVT products in order to confirm success of RNA transcription. During the course of our lab work, we have changed the concentration of urea used in the PAGE, increasing it in order to break down the RNA samples so they can migrate more easily. We used tRNA as a control for our urea PAGEs. We were able to obtain successful results for the GFP and McyH IVTs, though the Lbu did not migrate as expected for its size, staying at the top of the well although it should have moved below the tRNA.

### SDS PAGE

We only performed one SDS page during the course of our lab work, on transformed bacteria samples in order to confirm the presence of our plasmid of interest within the transformed bacteria. This PAGE was unsuccessful and did not show bands of a size we would expect from our plasmid.

See our notebook!

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/lethbridge](https://gitlab.igem.org/2022/lethbridge).

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## Design and Build:

Our project this year represents the continuation of a two-year project, due to limited lab access throughout the 2021 iGEM season. In 2021, we designed and ordered many constructs for the expression of a Cas13a system and the enzyme mlrA, to be delivered to Microcystis aeruginosa cyanobacteria through the use of a phage-like particle vector. This year, we tested these constructs by working with them in the wetlab.

## Test and Learn:

### Construct Troubleshooting

During the course of the lab work this year we were working with two major types of constructs: DNA based constructs, and RNA based constructs, and as such had to use different processes to prepare and work with our constructs, and different methods to troubleshoot them. Our constructs, and the methods we used to troubleshoot our issues, are listed below.

Our DNA constructs were as follows:

MS2CP\_MPA (Ampicillin resistance)
MS2CP\_MPA (Kanamycin resistance)
mlrA\_NHis\_CanTag
mlrA\_CanTag
Lbu\_Cas13a\_NHis\_CAnTag
MS2CP\_NPolyR\_CHis
MPA\_NHis

Our first step with these constructs was to transform them directly into our competent cells, and then to plate them on media containing the appropriate antibiotic for primary selection. This failed and no growth occurred, and we now know that this was because we were attempting to use the original constructs as delivered to us, which were still in the original pUC-IDT cloning vectors, so the bacteria were not able to express the antibiotic resistance genes included, and could not grow.

After this, we attempted to run an SDS PAGE on our transformed bacterial cells which had been incubating in media containing antibiotics. Because the bacteria were able to survive and grow in the presence of the correct antibiotics, we assumed that meant the transformation was successful. The SDS PAGE showed no bands of the size we would expect, and the conclusion we reached was that the bacteria had developed antibiotic resistance that was unrelated to the one attached to our constructs.

After the unsuccessful initial transformation attempts, we concluded that the reason it was not working was because of the vectors the constructs were in, and attempted to change the constructs from the pUC-IDT cloning vectors to pUC-19 expression vectors.

The first step of any attempts to work with our constructs were PCRs, in order to amplify the amounts of DNA we had to work with. We used Q5, Q5 high fidelity, and PFU PCR protocols, and of these, the Q5 gave us the most consistent results. Though the PCRs were largely successful, we still changed some of the parameters in order to increase our chances of success, such as the volumes of dNTP and water (raising the former, lowering the later).

After our PCRs, we needed to use a restriction digest to cleave our construct from the cloning plasmid. We initially used Tango for this, but we also used DnpI, a class IIM endonuclease. Our digestions often did not work, and when we ran agarose gels on the results we would only find bands corresponding to the full plasmids. After multiple unsuccessful digests, we concluded that the reason it was not working was because our Q5 PCR buffer was inhibiting DnpI activity. The final digests we performed on our constructs after using a spin column to clean them so we could resuspend them in an appropriate buffer were successful, confirming our theory.

## Learn and Design:

For 2022, we decided to improve our project by designing parts that would make it easier to test our proof of concept in the level 1 lab that we have available to us. Additionally, we modified the design of one of our 2021 parts to improve binding affinity and target specificity, and designed a new part to take advantage of a collaboration opportunity with the Lethbridge High School Team and troubleshoot a problem we were having.

The first new parts that we designed this year were for the expression of the mcyH ATP binding cassette (ABC) transporter gene in E.coli. The mcyH gene, originally from Microcystis aeruginosa cyanobacteria, is the target for activating our CRISPR Cas13a system. Since it is unclear whether or not we would be allowed to bring potentially toxic cyanobacteria into our level 1 lab space, we designed a coding region and BioBrick to allow for the expression of this gene in E.coli; once E.coli has been transformed with this construct, it will be able to activate a version of our Cas13a system, which includes a crRNA designed to pair with the version of the mcyH sequence that has been codon-optimized for expression in E.coli.

As well, we improved upon the design of our Lbu crRNA from our 2021 project; this year’s crRNA was designed with the help of RNAfold, which allowed us to locate a target sequence that met the ideal design criteria of being close to the 5' end of the sequence, being a relatively unfolded region in the mRNA secondary structure, and having a high probability of remaining single-stranded.

Additionally, owing to the difficulty we had in expressing our Lbu Cas13a constructs, we collaborated with the Lethbridge High School team to acquire Lwa Cas13a, which they had expressed as a part of their 2019 project. We therefore designed mcyH-targeting crRNA to be compatible with Lwa Cas13a, for both E.coli expressing the mcyH and for cyanobacteria.

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/lethbridge](https://gitlab.igem.org/2022/lethbridge).

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### 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

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The repository used to create this website is available at [gitlab.igem.org/2022/lethbridge](https://gitlab.igem.org/2022/lethbridge).

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### Notable Results

On June 23rd, DNA for the Lbu\_cas13a, GFP\_crRNA, and mcyH\_crRNA were successfully cut
from plasmid vectors through EcoRI restriction digest, dephosphorylated, and amplified by PCR.

On August 3rd, the sequences for the following constructs were successfully amplified via PCR:
6, 7, 8, 9, 10

On September 24th, constructs 1, 2, 6, 7 were digested from plasmid vectors through DPNI
restriction enzyme digestion.

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/lethbridge](https://gitlab.igem.org/2022/lethbridge).