IDT Constructs

DNA-based:

MS2CP_MPA (Ampicillin resistance)

MS2CP_MPA (Kanamycin resistance)

mlrA_NHis_CanTag

mlrA_CanTag

Lbu_Cas13a_NHis_CAnTag

MS2CP_NPolyR_CHis

MPA_NHis

Methodology:

Attempted to transform into competent cells and then plated
Unsuccessful as they were in pUC-IDT cloning vectors, and not expression vectors

Red herring: Attempted to run SDS page of the first attempted transformation to probe for successful insertion of our constructs
Unsuccessful, proteins of the expected size were not found

Attempted to change the constructs from the pUC-IDT cloning vectors to pUC-19 expression vectors
Q5, Q5-HF, and PFU PCR on constructs
Largely successful, occasionally would not work but not because of any specific, overarching issue, just needed repetition.
Q5 gave us the most consistent results
Restriction enzyme digest using Tango
Cleanup using spin column
Ran agarose gel electrophoresis to determine success. The digest was unsuccessful as we did not see bands that correlated with the expected fragment sizes, rather, there was a single band corresponding to the size of the plasmid. Therefore, we hypothesized that we would have more success with a different endonuclease.
Q5 PCR of our constructs; PCR was relatively successful as we saw bands on the agarose gel corresponding to the plasmid size.
Restriction enzyme digest using a class IIM endonuclease, DnpI.
Ran agarose gel electrophoresis to determine success. The digest was unsuccessful as we did not see bands that correlated with the expected fragment sizes, rather, there was a single band corresponding to the size of the plasmid. Therefore, we hypothesized that the Q5 PCR buffer was inhibiting DnpI activity.
Q5 PCR of our constructs; PCR was relatively successful as we saw bands on the agarose gel corresponding to the plasmid size.
Precipitated the DNA using ethanol precipitation
Restriction enzyme digest using a class IIM endonuclease, DnpI. Ran agarose gel electrophoresis to determine success. The digest was unsuccessful as we did not see bands that correlated with the expected fragment sizes, rather, there was a single band corresponding to the size of the plasmid.
T4 Ligation
Transformation into competent cells
Growth on agar plates
Overexpression in LB media

RNA-based:

McyH_crRNA

GFP_crRNA

Methodology:
IVT (In vitro transcription) to replicate RNA
Succesful
RNA Urea PAGE
Succesful

Construct Troubleshooting:

During the course of the lab work this year we were working with two major types of construct: 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.

DNA-based constructs:

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 preformed 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.

After our successful digest, we planned to preform a T4 ligation to insert our genes of interest into a Puc19 expression vector plasmid, which would then be transformed into bacterial cells. Since our constructs (and the associated antibiotic resistance genes) would then be on expression vector plasmids, we would theoretically be able to plate them on media containing the appropriate antibiotic and see growth. After that, we would preform an overexpression in LB media, and confirm the presence of our proteins of interest with an SDS PAGE.

RNA-based constructs:

McyH_crRNA

GFP_crRNA

Our two RNA constructs are used to express the guide-RNAs that would have been used with our CRISPR system. For these constructs, we used in-vitro transcription to express the RNA segments of interests, and used urea PAGEs to confirm successful transcription. Both of these were successful, and did not require troubleshooting in the ways that the DNA constructs did.

Education

Our team taught elementary school and middle school students from École Agnes Davidson School and Senator Joyce Fairbairn Middle School about our project.