How to Genetically Engineer E. Coli with Tap Water, a Microwave, and Jellyfish Genes

Making bacteria glow.

Selin Filiz

Published in

Visionary Hub

11 min readMay 13, 2022

Okay, I lied.

You need a few extra things besides tap water, a microwave, and jellyfish genes.

This is exactly what the box looked like when I opened it. Not staged.

These past few weeks, I’ve been trying out this experiment from The ODIN to gain a more practical understanding of genetic design. Here’s a breakdown of the project, from start to finish.

*Note: The instructions and materials for this experiment came from The ODIN. I am simply paraphrasing their instructions and explaining the process a little more in-depth.

Skip here to go straight to the procedure.

Pre-Lab

Goal:

In this experiment, we’ll attempt to insert the gene Gfp, which codes for green fluorescent protein (GFP), into E. coli in order to make it fluoresce.

But what’s E. coli?

Escherichia coli is a species of bacteria that lives in the intestines of humans and animals. It is also commonly found in animal feces. Most strains are harmless and are an important part of the human intestinal tract. Some are pathogenic, causing illnesses like diarrhea, urinary tract infections, respiratory illness, and pneumonia when transmitted through contaminated water, food, or direct contact with infected animals and people.

Pathogenic strains are categorized into pathotypes, six of which are associated with diarrhea (these are called diarrheagenic E. coli). These include

Shiga toxin-producing E. coli (STEC) → one of the strains most commonly heard of in the news relating to foodborne outbreaks,
Enterotoxigenic E. coli (ETEC),
Enteropathogenic E. coli (EPEC),
Enteroinvasive E. coli (EAEC),
Enteroaggregative E. coli (EIEC),
and diffusely adherent E. coli (DAEC).

E. coli is also used as a marker for water contamination. E. coli found in drinking water indicates that the water is contaminated, even though the E. coli itself may not be harmful.

The E. coli used in this experiment is non-pathogenic and has been optimized for taking up new genes (see “Bacterial Transformation”).

And Green Fluorescent Protein?

GFP is a protein found in the jellyfish Aequorea victoria that emits a green fluorescence when exposed to light. In Aequorea, GFP interacts with a chemical called aequorin to emit a blue light when calcium ions are added.

The protein itself is made up of 238 amino acids, three of which form the structure that emits visible green fluorescent light. Biologists often use GFP as a marker protein, as it can be attached to other proteins through the use of DNA recombinant technology with the Gfp gene. Once attached, if the cell produces fluorescence, the target gene must also have been expressed. GFP can also be used to label organelles, cells, and tissues.

DNA recombination: Put very simply, two strands of DNA are cleaved at specific sequences to create an ‘overhang’ on each strand that matches base pairs with the other. The two strands are then sealed together with DNA ligase. Check out the “Gene ENGINEERING” section of this article for a more detailed explanation.

This helps scientists see the presence of the molecule within the structure it has been inserted into and is therefore useful in studying gene expression. GFP does not interfere with other biological processes when used in vivo.

For example, you might insert GFP adjacent to a gene for cancerous mice liver cells. When this is introduced to the mouse successfully, the mouse will radiate green fluorescence from the cancerous liver, providing the location of the cells.

The Gfp gene is also heritable, meaning that the offspring of organisms containing the gene will also have it. There are over 150 GFP-like proteins, including blue, yellow, and red variations found in fluorescent coral!

GFP is a photoprotein, a type of protein that gives off light when combined with oxygen, hydrogen peroxide, or other oxidizing agents. Unlike certain chemicals used in bioluminescence, photoproteins require no catalyst.

In Aequorea, aequorin only needs calcium ions to luminesce.

Wait — Is This Bioluminescence? Fluorescence? Luminescence? Phosphorescence? Chemiluminescence?!

Let’s clear this up.

Luminescence is the emission of light by certain materials. It includes chemiluminescence and bioluminescence.
Chemiluminescence is when a chemical reaction leaves a molecule with enough internal energy to produce fluorescence and phosphorescence.
Bioluminescence is a type of chemiluminescence: the production or emission of light by a living organism. It is typically used to evade predators, lure prey, and communicate between members of a species. Most bioluminescent organisms are found in the ocean.
Fluorescence does not involve a chemical reaction. Instead, a stimulating light is absorbed by the medium and re-emitted. This means fluorescing light is only visible in the presence of the stimulating light. One example is highlighter ink.
Phosphorescence is similar to fluorescence but can re-emit light for longer. An example of this would be glow in the dark stickers.

The light from green fluorescent protein is only seen when a stimulating light (in this case, blue light) is shined upon it. Therefore, we’re making the bacteria fluoresce.

Plasmids

So you’ve got your E. coli and GFP. Now what?

For this experiment, the Gfp gene is embedded in a plasmid which will be transferred into the E. coli. Plasmids are small pieces of DNA connected in a circle. They only contain a few genes and are easy to insert into bacteria. Since they are separate from the bacteria’s own chromosomes, they are able to replicate independently and may be passed between different bacterial cells.

In the end, our genetically modified E. coli while be transferred to a plate of LB Kanamycin. Kanamycin is an antibiotic use to treat bacterial infections (AKA, it kills bacteria). Our plasmid also contains a gene for antibiotic resistance, so only the E. coli cells that have been successfully modified with the plasmid (which contains the GFP) will survive on the kanamycin plates. This way we know that there is no unmodified bacteria on the final plate and that the experiment worked!

Plasmids are a lot smaller than the host chromosome and are greater in number, making them easier to isolate in their pure form and therefore convenient for studying DNA in a lab.

Bacterial Transformation: How Does It Work?

Bacterial transformation is simply the process of inserting DNA into bacteria, or enabling bacteria to pick up DNA plasmids from the surrounding environment. For a visual explanation, I recommend you check out this video!

I’ll be explaining each part of this process as it’s carried out in the procedure.

Materials

For this experiment, you will need

LB Agar (powder)
LB Kanamycin Agar (powder)
250 mL Glass Bottle (for pouring plates)
Sterile Water
(lots of) Disposable Transfer Pipettes
7 Petri Plates
Inoculation Loops
Nitrile Gloves
Microcentrifuge Tubes
Microcentrifuge Tubes containing LB Broth
Centrifuge Tube (to measure liquid volume)
Bacterial Transformation Buffer (25mM calcium chloride, 20% PEG 8000)
Orange UV Filter Sheet and Blue Light
(Note: Because the instructions and materials for this experiment came from The ODIN, I will not provide exact measurements.)

Sterility

As with any biology experiment, ensuring sterility to avoid contamination by other bacteria is essential for success. Isopropyl alcohol should be used to sterilize hands, gloves, surfaces, and inoculation loops before performing the experiment.

Preparation

Making Plates

Empty LB Agar Media into the 250 mL glass bottle.
Using the centrifuge tube, measure and add 150 mL of tap water to the glass bottle. Shake vigorously.
Heat the agar in a microwave for a few seconds at a time to dissolve. Do not let the bottle boil over — if it does, let it rest for a minute or two before continuing. DO NOT screw on the cap! Just place it lightly on top.
Keep heating the agar in increments until the agar becomes a translucent clear yellow and there are no visible suspended particles.
Take the bottle out of the microwave and let it cool for 20–30 minutes until it is cool to the touch.
While the bottle is still somewhat warm, one at a time, remove the lid of two petri plates and pour just enough LB Agar from the bottle to cover the bottom half of the plate.
Put the lid of the plates on. The agar media will solidify when it cools.
Let the plates sit out overnight to let the condensation evaporate. Store them upside down (to avoid condensation dripping on the plates) in your fridge at 4 degrees Celsius.
Repeat steps 1–8 with the LB Kanamycin Agar, make five plates.

LB Agar is a type of lysogeny broth containing agar that is typically prepared in petri dishes for the plating of bacterial cultures and the growth of bacterial colonies. It is nutritionally rich, allowing for the growth of most microorganisms. Bacteria cannot digest the agar but can gather nutrition from the LB, meaning that any growth will stay on top of the media where we can easily scrape it off.

A lysogeny broth is simply a nutritionally rich medium used for the growth of bacteria.
Agar is made of polysaccharides from the cell walls of certain species of red algae, namely ogonori and tengusa.

Here, we’re adding liquid to a mix of LB and agar, then ‘autoclaving’ it using the heat of the microwave and, although minimal, the pressure of the expanding hot air underneath the cap. This produces a liquid solution that solidifies over time.

LB Kanamycin Agar is the exact same, except that the mix includes the antibiotic kanamycin, as discussed under “Plasmids.”

Making Competent Bacterial Cells

Add around 100 uL (or about four drops from a disposable pipette) of sterile water to the freeze-dried bacteria to activate.
Shake for one minute to dissolve bacteria.
Pipette all contents onto an LB Agar plate.
Use an inoculation loop to gently spread the bacteria across the plate, drawing three groups of ‘squiggles’ so the bacteria is evenly spread. Allow plate to dry before putting on lid.
Let the plate grow overnight at room temperature for around 2–3 days until white-ish bacteria begin to grow. Placing the plate in a consistent and warm temperature location is preferred.
Use an inoculation loop to gently scrape bacteria off the fresh plate until the loop is filled. Mix the bacteria into the “Bacterial Transformation Mix” until any big clumps have disappeared. The mix should look very cloudy: hazy but not quite opaque. This tube is known as your competent cell mixture.

“Competent” cells are those that are able to intake foreign DNA, which cell walls normally prevent:

Bacteria are negatively charged because of the structure of their lipid bilayer cell membrane. Plasmids are also negatively charged because of the phosphate backbone of the included DNA. These two negative charges repel each other: the cell wall will not allow plasmids to pass through.

To counteract this, calcium chloride and other chemicals and salts (the “bacterial transformation mix”) are mixed with bacterial cells. The positively charged calcium chloride ions bond to the negative phosphate cells on the bacteria’s cell membrane, neutralizing the charge. These competent cells will now be able to take in foreign DNA, as the two charges (one of which is neutral, the other negative) no longer repel each other.

DNA Transformation

Adding the Plasmid

Add 50 uL (about two drops with the disposable pipette) of sterile water to the freeze dried plasmid and shake for one minute to activate.
Add all contents of the plasmid to the competent cell mixture.
Incubate the competent cell mixture in the fridge at 4 degrees Celsius (DO NOT FREEZE) for 30 minutes.
Incubate the competent cell mixture for 30 seconds in 42 degrees Celsius water.

Now that the bacterial cells have been neutralized and are ready to intake foreign DNA, the plasmid is mixed with the cells at 4 degrees Celsius. The temperature is then increased to 42 degrees Celsius (when you incubate the mix in water) and then back to room temperature. This heat shock creates adhesion zones on the bacteria, forming areas of leaky plasma membrane. The cell takes up the plasmid through these adhesion zones.

Adding LB Media

With a pipette, fill a LB powder media microcentrifuge tube almost full of room temperature tap water. Leave room to shake and dissolve the LB media.
With the same pipette, add half of the LB media to your competent cell mixture. Mix gently by flicking the closed tube.
Incubate the tube for four hours at room temperature. If you’re having trouble with the experiment, increase the incubation time to 12 hours for a higher chance of success. This creates your transformation mixture.

Now that the cells have(hopefully) accepted the plasmid, LB powder media is added to the transformation mix and incubated (the LB acts as a source of food during this time). This step allows the bacterial cells to recover before they are replated.

Plating

Take an LB Kanamycin Agar plate out of the fridge and let it warm to room temperature.
With a pipette, mix your transformation mixture and add about four to five drops on top of the LB Kanamycin Agar plate.
With an inoculation loop, spread the bacteria around the plate. Let dry for ten minutes before putting the lid back down. Then, flip the plate upside down to prevent condensation dripping onto your bacteria.
Incubate the plate at room temperature for 36–48 hours.

After the incubation period, the surviving modifying cells reproduce to form colonies. As discussed beforehand, only successfully modified cells will survive: since these bacteria are plated on LB Kanamycin Agar, only those containing the plasmid with the GFP and antibiotic resistance will live on the media.

Congratulations!

You have now presumably genetically modified E. coli with GFP. Any bacteria growing on your LB Kanamycin Agar plate should be successfully modified (unless you’ve introduced contaminants).

In Aequorea, aequorin interacts with calcium ions to create a blue glow. Some of that energy is transferred to GFP, which emits a green glow. To simulate this, shine the blue light on the bacteria through the orange UV filter sheet. The blue light will excite the GFP so it re-emits the energy as green light, and the UV filter will block the blue light so we can see the fluorescence more clearly.

Check out The ODIN to perform your own genetic design experiments.

Sources

Hey! I’m Selin, a 15 y/o looking to accelerate sustainability with synthetic biology and the arts. Enjoyed the article? Your support is appreciated!
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