Bacterial Transformation and CRISPR Mechanisms

ds-DNA

Only double-stranded DNA (ds-DNA) binds to the recipient cell to initiate the process of transformation.

ss-DNA

Single-stranded DNA (ss-DNA) isolated during transformation cannot be used to re-initiate transformation due to the eclipse phase.

TraJ

TraJ is a transcriptional activator required for the transcription of traY to traX and finO in the F plasmid.

FinP

Translation of traJ mRNA is blocked by hybridization with the antisense RNA FinP, while FinO stabilizes the FinP RNA.

RNAseE

RNAseE can cleave the traJ mRNA, affecting its stability and translation.

plasmid mobilization

Some plasmids are not self-transmissible and depend on a self-transmissible plasmid in the same cell for mobilization.

oriT site

The oriT site is identified by cloning pieces of the plasmid into a nonmobilizable cloning vector, transforming them into cells with a self-transmissible plasmid, and then mixing with potential recipient cells.

generalized transduction

In generalized transduction, a phage infects a bacterium and mistakenly packages bacterial DNA into its heads, which can then be injected into another bacterium during subsequent infections.

immunity phase of transduction

During the immunity phase, the incoming protospacer sequence on the phage DNA is targeted to prevent further infections.

CRISPR arrays

The CRISPR array is transcribed into a single long RNA, which is then cut at the repeat sequences to produce small guide CRISPR RNAs (crRNAs), each corresponding to one of the spacers.

crRNA

When a phage infects a cell, the pairing of a crRNA with the identical protospacer sequence in the incoming phage DNA directs Cas proteins to cut the protospacer DNA, thereby inactivating the phage.

CRISPR interference (CRISPRi)

CRISPR interference (CRISPRi) involves blocking transcription by coupling a small crRNA with a dead mutant of Cas9, which has no nuclease activity, effectively stalling RNA polymerase (RNAP).

new CRISPR spacers

During the adaptive phase, one or more Cas proteins recognize a PAM sequence in incoming phage DNA and integrate the adjacent protospacer as a new spacer into the CRISPR array, causing all other spacers to shift down and the last one to be discarded.

CRISPR array structure

A typical CRISPR array consists of repeated sequences interspersed with spacers of similar length but different sequences, along with a leader region.

PAM sequence

The PAM sequence is crucial for the recognition of incoming phage DNA by Cas proteins, allowing for the integration of the adjacent protospacer into the CRISPR array.

Artificially induced competence

Artificially induced competence in bacteria can be achieved through chemical induction, specifically by treating the bacteria with calcium (CaCl2) and rubidium (RbCl2) ions.

Selectable markers

Selectable markers are used to identify and select cells that have successfully taken up foreign DNA, improving the efficiency of transformation.

Circular DNA

Circular DNA is more efficient and stable during transformation, while linear DNA is more susceptible to degradation by exonucleases.

Transfection

Transfection refers to the introduction of viral DNA or RNA into a host cell.

Electroporation

Electroporation involves exposing cells to a strong electric field, which increases the permeability of the cell membrane, allowing DNA to enter.

Washing cells with 10% glycerol

Washing cells with 10% glycerol helps to remove salts that could interfere with the transformation process.

Protoplasts

Protoplasts are cells that have had their cell walls removed, making them more amenable to DNA uptake.

PEG in transformation

Exposure of DNA in the presence of polyethylene glycol (PEG) helps trap DNA in the cytoplasm, facilitating its uptake by the cell.

Viability of transformed cells

The cell wall must regenerate for the transformed cells to survive and function properly.

Efficiency of transformation

The efficiency of transformation is expressed as the number of colony-forming units (CFU) per microgram of DNA.

Transformation efficiency calculation

The transformation efficiency is 100,000 CFU/µg, calculated as 100 CFU divided by 0.001 µg.

Methods of genetic material transfer

The methods include conjugation, mobilization, transformation, general transduction, and complementation.

Sensitivity of DNA to DNase

DNA inside the cell is insensitive to DNase, allowing it to remain intact while DNA that did not enter the cell is degraded.

Asterisk in DNA uptake experiments

The asterisk indicates that the DNA being referenced is radioactively labeled, which is used for tracking its uptake.