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.