Out of the blue CRISPR kit study guide

lacZ Gene

Codes for an enzyme called beta galactosidase (β-gal). This enzyme metabolizes the sugar lactose

beta-galactosidase

breaks down lactose into glucose and galactose

CRISPR-Cas9

Clustered sequences of viral DNA that have been incorporated into a microbes' genome

Cas9

A bacterial enzyme that cuts both strands of DNA at a very specific location

sgRNA

Single guide RNA

Binds to the Cas-9 enzyme and guides it to a specific place to cut the DNA

PAM

Stands for protospacer adjacent motif. In a nutshell, its where Cas-9 will bind to the DNA

NHEJ

Non-homologous end joining

After the DNA is broken, the cell tries to repair the DNA by inserting random nucleotide. This usually disables the gene

Negatives of using NHEJ

The process may randomly insert or delete one or more bases and can cause mutations that can disrupt gene function or expression

HDR

Homology directed repair

Enzymes patch the break caused by Cas9 using donor template DNA

X-Gal

A sugar like substance that when cut turns a dark blue

E. Coli HB101-pBRKan

The strain of bacteria we are using in this experiment; has a functional lacZ gene and has been modified to express Cas9 enzyme

Arabinose

Sugar used as a biological switch

Turns on the HDR DNA machine repair in E.coli

IPTG

ensures continuous expression of the lacZ gene

Used in the the plates during the lab

PlZ donor

used as the control

Includes a donor template DNA sequence that is used by the HDR machinery to fix double stranded DNA breaks.

Includes an insert sequence which will be inserted into the lacZ gene and impair its function

Does not code for sgRNA

pLZ Donor guide

includes both the donor DNA sequence in pLZ and a sequence that codes for sgRNA

Once transcribed sgRNA direct Cas9 where to cut

IX

Growth medium with the IPTG, X-gal, LB, and arabinose

IX/SPT

Growth medium with the IPTG, X-gal, LB, and spectinomycin

What is the purpose of an arabinose-inducible promoter

when the bacteria is exposed to arabinose, they express the HDR DNA repair machinery

Why are the bacteria on the starter plates blue?

The Cas9 cuts the lacZ gene. this releases B-gal that breaks the x-gal in the bacteria which causes a blue color

If the bacteria on the starter plates did not have a functional lacZ gene, what color would they be expected to be

colorless because the lacZ gene is needed for B-gal to be released to cut the X-gal that causes color expression

How could the IX starter plate influence gene editing in the lab activity

will not be able to edit because it doesnt have the enzymes necessary to carry out the HDR DNA repair system

Repair system is off

How could the IX/ARA starter plate influence gene editing in the lab activity

Has the necessary enzymes needed to carry out the HDR DNA repair system.

Can edit the genes

color of colonies grown from IX bacteria with pLZ donor, Cas9, no DNA repair system, no sgRNA, and a donor template DNA incubated on IPTG, X-gal, spectinomycin media

Why?

Blue colony growth

bacteria starts blue so since under these conditions there is no change to bacteria color since there is nothing to direct the Cas9 where to cut the lacZ gene, they will end blue

color of colonies grown from IX bacteria with pLZDonorGuide, Cas9, no DNA repair system, has sgRNA, and donor template DNA incubated on IPTG, X-gal, spectinomycin media

Why?

No growth, bacteria will die

bacteria dies because the pLZDonorGuide directs Cas9 to a cutting site which it then cuts. Since there is no DNA repair system, the Bacteria will die

color of colonies grown from IX/ARA bacteria with pLZDonor, Cas9, a DNA repair system, no sgRNA, and a donor template DNA incubated on IPTG, X-gal, spectinomycin media

Why?

Blue.

Since there is no cut made in the lacZ gene of the DNA, the color will not change in this bacterial cell

color of colonies grown from IX/ARA bacteria with pLZDonorGuide, Cas9, a DNA repair system, sgRNA, and a donor template DNA incubated on IPTG, X-gal, spectinomycin media

Why?

Colorless

This is because the pLZ donor guide directs the Cas9 where to cut the lacZ gene. After this, the DNA strand is repaired and leads to no blue coloring

Why on some plates will there be multiple colors of colonies

Random mutations during replication of the cells during incubation

Outside bacteria is on the plate that can survive the selectivity

Gene editing was not 100% accurate

Why is SOC nutrient broth used

to encourage bacterial growth

How can colony color be used as evidence of the state of the lacZ gene

If the lacZ gene is functional the color of the colonies will be blue

What are two other experiments that can be used to verify that chromosomal gene editing occurred in the bacteria

DNA sequencing

Gel electrophoresis