Crispr Cas9, the relatively new kid on the genetic engineering block, has taken off in
popularity among the non-scientific public.
There are even kits you can buy for yourself and experiment with Crispr and other more
conventional genetic modification techniques.
Sold by the company, The Odin, The ‘Bacterial CRISPR and Fluorescent Yeast Combo kit’
is one such kit, with everything you need to get started.
For this Crispr experiment, a non-pathogenic strain of E. Coli is used, and baker’s yeast,
or Saccharomyces cerevisiae is used in the other.
The goal of the yeast experiment is to get the cells to produce a green fluorescent protein,
or GFP for short, so that it ‘glows in the dark’ under blue or uv light.
The goal for the E. coli Crispr experiment is to make a precise edit to the protein rpsL,
or Ribosomal subunit protein.
The modified E. coli will be grown on agar plates that contain streptomycin, an antibiotic,
which normally binds to the rpsL preventing the production of proteins by the cell’s
ribosomes.
Without proteins the E. coli can’t grow and replicate.
This modification now imparts E. Coli with antibiotic resistance by preventing streptomycin
from binding to the rpsL.
The Yeast and E. coli are going to need a medium to grown on and food to eat.
So the first step is making LB Agar plates for the E. coli, and YPD Agar plates for the
Yeast.
We will make LB Strep/Kan Agar plates for the modified E. coli, which contains the antibiotics
streptomycin and kanamycin and YPD Agar with G418, or Geneticin
another antibiotic which will ensure that only the Yeast with the GFP survive.
But first - wear gloves.
Agar is like jello, so is the process of making agar plates.
Dump each tube of powdered Agar into the glass bottle provided.
Some sort of funnel is helpful.
Pour in 150 mL of water, a half turn of the cap, and then microwaved at 30 second intervals
for a couple of minutes so the agar doesn’t boil over.
Once all the agar has dissolved and it’s cooled down a bit, but still warm, it’s
time to make plates.
But don’t let it cool down too much, or you’ll get clumps.
Go slowly, and pour only enough agar to fill the plate.
Each tube is enough for about 7 plates.
You only need one each, but experiments fail, so redundancy is good.
Flip the plates over when complete so the condensation doesn’t drip into the agar.
I waited a bit before doing this to make sure the agar was solid enough.
Before continuing, let the plates cool down for an hour, or at 4 degrees Celsius
you can store them in the fridge for later use.
We will use the LB Agar and YPD Agar plates to grow fresh E. coli and yeast, which increases
the odds for a successful experiment.
I don’t advise doing both experiments at the same time as it can get really confusing,
but for brevity's sake, I’ll demonstrate them together in this video.
Using an inoculation loop, collect the E. coli from the bottle along this faint white
line called an agar stab.
Wildly spearing the Agar is inadvisable and ineffective.
With the loop flat against the agar, gently streak out the bacteria on the LB Agar plate.
Try not to pierce the agar.
Add water to the top of one of the tubes of dried yeast.
I was being cautious and used the pipette for this.
Shake the tube until the yeast has dissolved.
Set the Pipette to 100 microliters and grab a tip.
Pipette out the yeast solution onto a YPD Agar plate and carefully streak out the yeast.
Allow the E. coli to grow overnight for about 12-18 hours, and the yeast for 12-24 hours.
Before any genetic engineering can take place, we need to get all the components that will
do the work inside the cell.
The cell walls of yeast and E. coli, by themselves, don’t usually allow just anything in.
Only by successfully making the cell’s competent are we able to reliably get the modified DNA
and the other components pass the cell walls.
This process, called transformation, requires a few components.
The Yeast experiment uses Polyethylene Glycol, or PEG, Lithium Acetate, and Single Stranded
Carrier DNA derived from Salmon.
The E. coli experiment uses a different concentration of PEG, Dimethyl Sulfoxide and Calcium Chloride.
These components perform several key functions such as shielding the negative charge of both
the DNA and cell wall, DNA would otherwise be repelled, and making the cell wall more
porous.
The single stranded DNA in the Yeast experiment, isn’t performing any modifications, instead
it’s used to overwhelm the cell’s defenses so the nucleases are more likely to digest
them rather than the Plasmid DNA that has the coding for the Green Fluorescent Protein.
For both the E. coli and yeast transformation mixes, pipette out 100 microliters of each
and add it into a new microcentrifuge tube.
One for each.
Discard the tip!
With a seperate inoculation loop for both, gently scrape up some of the yeast and
E. coli.
Mix it into their respective microcentrifuge tubes with the transformation mix.
About two loop fulls of yeast and E. coli should be enough.
With a new tip for each, you can pipette the mixture up and down to help the process along.
Discard the tip!
Mix until the liquid is opaque with no big clumps.
I’ll call these the E. coli or yeast competent cell mixtures.
Set the pipette to 10 microliters and add a new tip.
Grab the tube with the Cas9 Plasmid.
Pipette out 10 microliters and add it into your E. coli competent cell mixture.
Then 10 microliters from the guide RNA tube.
And a final 10 from the Template DNA tube.
Use a new pipette tip for each!
Put the E. coli mix in the fridge for 30 minutes.
Now back to the yeast.
Grab the Yeast GFP Expression Plasmid tube and pipette out 10 microliters into your yeast
competent cell mix.
Discard the tip!
Both of the competent cell mixes will now go through heat shock, which is another part
of the transformation process to make the cell walls more permeable.
The yeast will undergo its heat shock for one hour in 42 degree celsius water.
I improvised my own method with one of the measuring tubes provided and a meat thermometer
to prevent it from cooling down too quickly so I wouldn’t have to leave the water running.
I occasional put this tube into some hotter water when needed when the temperature dipped
too much.
The E. coli only needs 30 seconds in 42 degree water so I just used a bowl.
After the heat shock it’s time to add the food.
Grab a microcentrifuge tube of LB media for the E. coli competent cell mix, and a YPD
one for the yeast.
With room temperature water fill each tube to the top.
I used a pipette for the LB media, but feeling more confident, I used the tap for the YPD
media.
Shake the tubes until all the media is dissolved.
Pipette out 900 microliters of YPD media into your yeast competent cell mix.
Discard the tip!
And 200 microliters of the LB media into the E. coli mix.
This pipette can only do 100 microliters, so you’ll need multiple trips.
And then incubate.
Lacking a proper incubator, I made my own.
I suppose I could have used this for the heat shock, but the instructions said to use water,
and making stuff is fun.
Incubate the E. coli mix for 2 hours at 37 degrees celsius or 4 at room temperature if
you don’t have an incubator.
And 30 degrees for the yeast mix for 4 to 6 hours or overnight at room temperature.
I made multiple competent cell mixtures for each in case of failure.
This allows the cells to recover from the transformation process and let the engineer
processes do its thing.
The Yeast and E. coli experiment use different engineering methods.
E.Coli bacteria are prokaryotes, and Yeast cells are eukaryotes.
ONE of the important differences, is that eukaryotes carry their main genetic information
inside a membrane bound nucleus, while prokaryotes have a nucleoid which is not enclosed in a
separate membrane from the rest of the cell.
Bacterial, as well as some other types of cells, also have Plasmid DNA.
A plasmid is a, “short circular DNA sequence” that “can replicate independently of the
bacterial chromosome.”
Using our own plasmids is key to both of these experiments.
For the Yeast experiment we are going to use a Plasmid that contains the code for a Green
Fluorescent Protein, and if successful, it will be inherited by the daughter cells and
glow green under the blue light when viewed with the yellow filter glasses in the kit.
Put simply, a plasmid has a segment called the Origin of Replication, where the host
cell starts the replication process and another that codes for Antibiotic resistance.
After this round of incubation, the modified yeast will be spread onto a YPD agar plate
with G418, or geneticin, an antibiotic.
If the yeast doesn’t replicate with the GFP Plasmid, it will therefore not have the
antibiotic resistance and won’t be able to grow on the plate.
Lastly, the insert, sandwiched between restriction enzyme sites.
It’s where you can insert the gene you want replicated - the Green Fluorescent Protein
for Yeast, the Cas9 protein and guide RNA for the E.Coli experiment.
CRISPR Cas9 are acronyms for, Clustered Regularly Interspaced Short Palindromic Repeats and,
CRISPR associated protein 9.
The method is derived from the immune defense system of bacteria.
This strain of E.Coli has over 4 million base pairs, and the Crispr Cas9 process will result
in a change to just one of them.
Here’s how CRISPR works in this experiment.
The E. coli, because of the plasmids we introduced, start producing the Cas9 protein and the guide
RNA or sgRNA.
The guide RNA is itself two components, trans-activating CRISPR RNA, or tracrRNA and CRISPR RNA or
crRNA.
Next, the guide RNA binds to the Cas9 protein.
Together, it locates the specific area of interest on the E.coli’s genome to which
the crispr RNA binds to, having the corresponding matching base pairs.
The Cas9 then cuts both strands of the DNA.
The cell would then start to repair its cut DNA.
But the custom DNA that we introduced works as a repair template, so the cell knows how
to repair the cut.
Our template DNA is nowhere near as large as the genome of the E.Coli, but it’s long
enough to trick the E.coli to use it as a repair template, as it has identical base
pairs on either side of the cut.
The only change is one base pair, a Guanine - Cytosine substitution for a Thymine - Adenine
base pair.
That one change will prevent streptomycin from binding to the rpsL protein.
This is a very simplified animation.
There are much better explanations and videos out there.
Check out the links in the card above or in the description for the video by Bozeman Science
and the McGovern Institute video.
Now it's time to see if the yeast and E. coli have been successfully engineered.
On a LB Strep/Kahn plate,
pipette out 100 microliters of the E. coli competent cell mixture
Discard the tip!
And on a YPD G418 plate, pipette out 400 microliters of the yeast mix.
Carefully streak out the E. coli and yeast with an inoculation loop or a plate spreader.
Let the plates dry for 10 minutes then put the lids back on and flip it over.
Incubate the E. coli one last time at 37 degree celsius for 16-24 hours, or 48 hours at room
temperature.
And 30 degrees celsius for the yeast for 1-3 days or 2-4 at room temperature.
Success...for the most part.
The E. coli experiment went well, out of the four plates I incubated only one had no growth.
The yeast experiment had some odd results.
I had yeast growing on all the plates, but I only seemed to have one fluorescent yeast
colony on each, especially compared to the example plate provided by The Odin.
I noticed this while the yeast was incubating for the final time so I ran the experiment
again, using up all the materials, but with the same results.
Those plates were from both.
I figured that the only yeast that should have survived are ones that replicated the
plasmid with the antibiotic resistance.
I decided to keep incubating the plates, and it took a couple of weeks to get some better
results.
There was still just the one dominant colony, but it appeared the others were at least somewhat
fluorescent under the light.
O spoke with a biochemistry professor about this and
she explained that microwaving the agar may have inactivated the antibiotic as it’s
usually added into the agar after it’s heated.
You can find these kits, and others on The Odin website, links in the description.