Designing Cell Culture Plasmids

(NEEDS CONTENT)

(Explain what the CRISPR 3.0 Plasmid is and what it should contain)

(Explain/give step on how to design CRISPR 3.0 Plasmid)

- Include link to reference to designing a plasmid

Summary

Plasmids are singular, circular pieces of DNA most commonly found in prokaryotes such as bacteria. Their circular structure is different from the linearized forms of DNA found in eukaryotes, but plasmids are still able to be expressed by eukaryotic cells if the plasmids are able to be introduced into the cells' nuclei. We have the ability to design plasmids virtually (such as through _____ programs) to contain specific genes of interest to us, and we can then order these plasmids to be developed and shipped to our lab. Once we obtain the initial plasmid DNA, we can utilize our transformation and maxi prep protocols to obtain a larger stock of the plasmid. From this stock, we can use our cell culture and transfection techniques to introduce the plasmid into our cultured cells, ideally resulted with our genes of interest being expressed in the cultured cells.

In Our Lab

CRISPR

In terms of our current goals for the CRISPR aspect of the Cell Culture Project, we would need to design a CRISPR 3.0 (prime editing) plasmid that is able to both target and edit a CFTR gene. Key components of this plasmid would need to include a catalytically impaired Cas9, a reverse transcriptase, and a prime editing guide RNA (pegRNA) with a specific sequence that can both target the CFTR gene (near a PAM motif), as well as complex with the nicked DNA to act as a primer for the reverse transcriptase. Finally, the pegRNA would need to have a specific sequence to that would allow the reverse transcriptase to "edit" the CFTR gene and synthesis a desired nucleotide sequence into the gene at the nicked location. More information about the components of the prime editing system can be read about here.

COVID-19

In Our Lab

(talk about the kinds of things that would need to be incorporated into the COVID plasmids, and how to it (i.e. what programs to use))

- also mention why its important to have these components in a plasmid (ex: eGFP and Ruby will be combined with proteins so that the regions these proteins are located in the cell can be observed under fluorescence, as well as their interactions)

- eGFP

- RUby

- Decoy?

- hACE2?

Using Ruby3 for fluorescence

mRuby3 is a fluorescent protein that fluoresces red in response to green light (absorbs 558 nM and emits 592 nm)

Two potential mRuby3 plasmids have been investigated so far, but more optimal plasmids could potentially be identified.

Plasmid Option 1: pKK-mRuby3-TEV

This plasmid contains the mRuby3 sequence, but seems to be mostly designed for the insertion of a protein sequence of interest into the plasmid via Ligation Independent Cloning to allow the protein to be expressed with the Ruby3 tag (although, it says a traditional restriction and ligation approach can still be done). This plasmid would be shipped to us as a TetOn system, but we would likely need to remove the Tet operator. This could be done through q5 PCR to amplify the entire plasmid (except for the tet operator) to have a DNA product that contains the same sequence as the plasmid minus the tet operator. When reading about the plasmid, I (Blake) don't see any reason why we couldn't use this plasmid to express mRuby in a cell (once the Tet operator is removed) without a protein sequence inserted, but this could be further investigated and confirmed.

Plasmid Option 2: mRuby3-C1

This plasmid is designed to be cloned in bacterial colonies with resistance to Kanamycin, but is designed for mammalian expression. So, assuming we get Kanamycin, it seems we could clone this plasmid to collect a plasmid that would express mRuby3, then transfect it into Calu3 cells as needed. It is my understanding we could also insert a protein sequence into this cloning vector as well, although this plasmid doesn't seem to be intended for ligation independent cloning.

Additonally, this expression vector could be used for expressing mRuby3. The mRuby3 sequence would need to be removed from a separate plasmid (i.e. from one of the two plasmids described above) and inserted into this expression vector. This expression vector utilizes a tetOff system, meaning the inserted gene (mRuby3 in this case) will be transcribed unless tetracycline is present. When tetracycline is present, the transcription of the gene will cease.