Today I am going to show you how I genetically engineered Escherichia Coli bacteria to allow them to survive in usually non-livable conditions.
The goal of this experiment is to engineer E. Coli so that they can live in environments that they normally can’t, specifically Strep plates. Strep plates contain a molecule called Streptomycin and when it binds to ribosomes of E. Coli, it shuts down the protein synthesis process. In other words, the molecule stops the bacteria from reproducing or doing processes that allow it to live and grow.
The mutation that can be made to allow protein synthesis is very small. A change in the three base pairs is enough to allow the E. Coli to grow and multiply in strep plates.
For this to happen a small mutation is made in the bacteria’s rpsL gene that encodes for the highly conserved rps12 protein that prevents streptomycin from binding to ribosomes, allowing the bacteria to grow and multiply in the strep plates.
The mutation that causes this phenomenon is only a couple of base pairs.
As a review, codons are transcribed from genes, which are translated into amino acid sequences. Chains of various amino acids fold in particular ways creating fictionalized proteins, which contribute to DNA synthesis and carry out an organism’s functions.
The mutation in this scenario is a mutation in the rpsL gene causing a change from a Lysine codon to a Threonine codon, in other words, a change from AAA to ACC in the transcribed mRNA.
Lysine is an amino acid that when in a particular position, shuts down ribosome function in E Coli.
Threonine on the other hand multiplies protein production. By switching the Lysine codon to the Threonine we allow E Coli to survive in Strep plates.
If the experiment is successful, we will have a colony of E Coli growing on the strep plates.
Step 1: Make the plates and grow the E Coli bacteria
The first step is to make the plates, which we will let the bacteria grow on. There are two kinds of plates that we use in this experiment. The first is LB Agar and the second is LB Strep/Kan/Arab Agar plates.
The first acts as a home for the bacteria. It provides a livable environment with the right pH level, limitless food, and everything for the bacteria to grow and multiply. It is the standard media for growing bacteria in labs. These plates will be used as a medium to grow E Coli bacteria.
The second as you know contains streptomycin molecules which inhibit the bacteria from thriving and multiplying. These plates will be used at the end of the experiment to test whether the bacteria has successfully been edited.
Before starting the experiment, I would like to give credit to The Odin for providing me with all the necessary equipment to experiment. You can buy a CRISPR kit from their website at www.the-odin.com
First add 150 mL of water into a glass bottle with LB Agar powder, which came with the kit provided. Shake the bottle vigorously and heat the bottle in the microwave for less than thirty seconds several times until the LB Agar powder is completely dissolved. The liquid should be a pale clear liquid after dissolving. Be careful that the solution does not boil and that the lid is placed on top and not tightly closed. Pour the liquid into the bottom of 7 Petri dishes. Do the same for the LB Strep Agar plates.
Let the plates cool for about 30 minutes before putting the lid back on. Then leave them to dry overnight. A couple of hours works fine also. After that flip, the Petri dishes upside down and then store them in the refrigerator.
Growing a sample of E Coli.
Add 100 microliters of sterile water into the provided DH5a tube. Shake the solution and pipette all 100 microliters of bacteria solution onto an LB Agar plate. Use an inoculation loop to spread the E Coli around on the Agar plate. Let the bacteria grow at room temperature for 1–2 days until there are visible whitish bacteria.
Step 2: Create the CRISPR-Cas 9 system to edit the E Coli genome
To summarize quickly how the basic CRISPR Cas 9 system works, the system is made of three components. A guide RNA, a Cas 9 endonuclease enzyme, and a template DNA. The guide RNA corresponds to the target gene or the RPSL gene. It binds to the Cas 9 enzyme leading it to the target DNA location. The Cas 9 enzyme then makes a double-stranded break in the target DNA, allowing intercellular repair mechanisms to attempt to put the DNA back together. One such mechanism is called Homology Directed Repair, where a nucleic acid containing the mutation is provided to act as a template for intercellular repair mechanisms to make an edit. This is a very simplified way to explain how the system works, if you want an in-depth explanation check out two articles that I have written linked in the description.
These components are stored in plasmid form. Cas 9 and gRNA encoding genes are inserted into plasmids and then inserted into the cells.
Bacteria have a semi-permeable cell wall, not allowing our CRISPR system to pass through. To bypass this barrier, we use a bacterial transformation mixture, containing 20% Polyethylene Glycol 8000 PEG 8000 and Calcium Chloride which shields the negative charge of plasmids, helping nucleic acids enter the cell. It helps because the cell membrane and nucleic acids are negatively charged and they naturally repel each other.
Add 55 microliters of sterile water into the tubes containing Cas 9 plasmids, gRNA plasmids, and template DNA. Gently shake the solutions and put the tubes aside.
To make the competent cell mixture, grab a microcentrifuge tube. Pipette 100 microliters of bacterial transformation mixed into the tube. Then, using an inoculation loop, take some bacteria from the LB Agar plates and add them to the tube. After that, pipette 10 microliters of each of the CRISPR components into the tube. Shake everything together until it is dissolved. Make sure there are no big clips of E.Coli.
Step 3: Bypassing the E Coli Cell Wall
The final step is to allow the CRISPR components to enter the E Coli cells by bypassing the E.Coli cell wall. We will be heat-shocking the E Coli, which will break or make holes in its cell wall, allowing our CRISPR components to enter the cell. Take the competent cell mixture microcentrifuge tube and put it in the fridge for 30 minutes. Instead of that, I put it on ice, and it turned out to work out well. Then take it out of the cold environment and incubate it in very warm water for thirty seconds. By doing this we are changing the environment’s temperature drastically.
The next step is to add 1.5 millilitres of tap water into an LB Media Microcentrifuge tube and shake until the LB powder is dissolved. And then pipette 250 microliters of the solution into the competent cell mixture tube. This will aid the bacteria to grow more quickly.
Incubate the competent cell mixture, giving it time to recover from the heat shock and allow the CRISPR system to take action.
Finally, pipette all of the solutions inside the competent cell mixture onto a strep plate to see if our experiment worked!
If the E.Coli grows on the Strep plates it means that the experiment worked!
Check out the video at the very top to see me do this experiment in real time!
