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.