How to Make Chemically Competent E. coli

Summary

In this video on synthetic biology, the process of preparing competent E. coli cells is demonstrated. The steps include preparing two calcium chloride solutions, inoculating and growing E. coli, and diluting the culture for optimal competency. After centrifugation, cells are resuspended in calcium chloride and glycerol solutions and kept cold to maintain competency. The cells are then aliquoted into smaller tubes for immediate use or storage. Careful temperature control is emphasized throughout the procedure to ensure cell viability and efficiency during transformation.

Takeaways

• 🧪 Preparing competent cells involves making two solutions: 100 mM calcium chloride and 100 mM calcium chloride with 15% glycerol.
• 🔬 Accurate measurement is not critical for these solutions; 'close is good enough' as they are forgiving.
• ❄️ Solutions should be stored on ice to maintain a temperature of 4 degrees Celsius to prevent cell lysis.
• 🌡️ The process starts with a single colony of E.coli to ensure genetic homogeneity and lack of contamination.
• 🚫 It's important to work with log phase E.coli for competent cell preparation as they are healthier and more competent.
• 🌡️ The culture is grown at 37 degrees Celsius with shaking until it reaches an OD of 0.5 or 0.6, indicating mid-log phase.
• 🧊 After centrifugation, cells are resuspended in calcium chloride solution and kept on ice to maintain low temperatures.
• 🕒 The cells are left on ice for at least four hours, or even overnight, to become competent.
• 🔄 A second round of centrifugation is performed, and cells are resuspended in calcium chloride with glycerol for transformation readiness.
• 🏗️ The final step involves aliquoting the cells into smaller tubes for convenient use in transformations, with each tube containing enough for multiple uses.

Q & A

What are the two solutions needed to prepare competent cells in synthetic biology?

The two solutions needed are a 100 millimolar solution of calcium chloride and another solution of 100 millimolar calcium chloride with 15% glycerol.

How much calcium chloride is required to prepare the solutions?

One and a half grams of calcium chloride is required for both solutions.

Why is it important to label the solutions near the top and what is the risk if not done so?

The solutions should be labeled near the top because they will be stored on ice, and if the labels get wet, they can fall off.

What is the purpose of adding water to the calcium chloride solution before the full 100 milliliters?

Adding water before the full volume allows room for the glycerol to be added later without exceeding the total volume.

Why is it recommended to be careful when pipetting glycerol?

Glycerol can be difficult to pipette and can coat the entire pipette if not done carefully, which can affect the accuracy of the measurement.

What is the significance of storing the prepared solutions on ice?

Storing the solutions on ice is crucial to maintain the cells at four degrees Celsius, preventing them from lysing if the temperature rises.

What type of E. coli strain is used to start the culture for making competent cells?

A wild type strain of E. coli, specifically mg 1655, is used, but other cloning strains like nab turbo or dh5 alpha could also be used.

Why is it important to start the culture from a single colony of E. coli?

Starting from a single colony ensures that the cells are genetically homogeneous and not contaminated.

What is the purpose of diluting the saturated E. coli culture into a fresh 50 ml culture?

Diluting the culture helps to use freshly growing log phase E. coli, which are the healthiest and most competent for transformation.

At what optical density (OD) should the E. coli cells be to proceed with the competent cell protocol?

The cells should be at an OD of 0.5 or 0.6, indicating they are in the mid-log phase.

How are the cells prepared for the second round of centrifugation in the competent cell protocol?

After the first round of centrifugation, the cells are resuspended in calcium chloride solution and left on ice for at least four hours. For the second round, they are resuspended in calcium chloride and glycerol solution.

Outlines

00:00

🔬 Preparing Solutions for Competent Cells

The video begins with the preparation of two solutions: a 100 mM calcium chloride solution and a 100 mM calcium chloride solution with 15% glycerol. The process involves weighing out 1.5 grams of calcium chloride and adding water to make up the volume to 100 milliliters for the first solution. For the second solution, water is added to 50 milliliters, and then 15 milliliters of 100% glycerol is carefully pipetted in. The solutions are mixed and stored on ice to maintain a temperature of 4 degrees Celsius, which is crucial to prevent cell lysis. Proper labeling of the solutions is emphasized, and the importance of keeping the cells at a low temperature throughout the process is highlighted.

05:02

🌡 Culturing E.coli for Competent Cells

The second paragraph details the process of culturing E.coli for making competent cells. It starts with inoculating a single colony of E.coli into 5 milliliters of LB media in a series of 14 Falcon tubes. The culture is then grown at 37 degrees Celsius with shaking overnight for 8 to 12 hours. The next day, the saturated E.coli culture is diluted 200-fold into a fresh 50 milliliter culture of LB media. The culture is grown further until it reaches an optical density (OD) of 0.5 or 0.6, indicating that the cells are in the mid-log phase and ready to be made competent. The cells are then collected by centrifugation to form a pellet, which is a precursor to the next step in the competent cell protocol.

10:19

🧊 Transforming Cells into Competent State

The final paragraph describes the transformation of pelleted E.coli cells into competent cells. After pouring off the supernatant from the centrifugation, the cells are immediately placed on ice to maintain a temperature of 4 degrees Celsius. The cells are resuspended in 15 milliliters of calcium chloride solution and gently pipetted to ensure a uniform suspension. The cells are then left on ice for at least four hours or overnight. In the subsequent round of centrifugation, the cells are resuspended in 4 milliliters of the calcium chloride and glycerol solution. The competent cells are then aliquoted into smaller Eppendorf tubes for convenience in transformation, with each tube containing 200 microliters, sufficient for multiple transformations. The competent cells are ready for immediate use or can be stored at -80 degrees Celsius indefinitely.

Keywords

💡Competent cells

Competent cells are bacterial cells that have been treated to allow the uptake of DNA from their surroundings, a process known as transformation. In the context of the video, the preparation of competent cells is the main focus, as it is a critical step in genetic engineering. The script describes the process of making E. coli cells competent, which involves resuspending them in a calcium chloride solution and then in a calcium chloride-glycerol solution, ultimately preparing them for DNA uptake.

💡Calcium chloride

Calcium chloride is a chemical compound used in the preparation of competent cells. It plays a crucial role in the process by helping to make the cell membrane more permeable to DNA. In the video, the script mentions preparing a 100 millimolar solution of calcium chloride, which is one of the solutions used in the transformation protocol. The script also describes the careful measurement and addition of calcium chloride to the cells to facilitate the competent state.

💡Glycerol

Glycerol, also known as glycerin, is a simple polyol compound that is used in the second solution prepared in the video. It is added to the calcium chloride solution to help protect the cells during the transformation process. The script specifies the addition of 15% glycerol to the calcium chloride solution, which is then used to resuspend the cells after the first centrifugation step, indicating its importance in preserving cell viability.

💡LB media

LB media, or Luria-Bertani medium, is a commonly used growth medium for bacterial cells. It provides the necessary nutrients for the bacteria to grow and proliferate. In the video, LB media is used to culture E. coli, starting from a single colony to a larger culture that will be used to create competent cells. The script mentions the use of LB media to start a 5 mL culture of E. coli, which is a standard practice in microbiology labs.

💡E. coli

Escherichia coli, commonly known as E. coli, is a species of bacteria commonly used in molecular cloning and genetic engineering. The script describes starting a culture of E. coli from a single colony on an agar plate, which is a standard procedure to ensure a genetically homogeneous population. E. coli is chosen for its ease of transformation and rapid growth, making it a popular host for genetic manipulation.

💡Transformation

Transformation refers to the process by which cells take up DNA from their surroundings. In the context of the video, the preparation of competent cells is aimed at facilitating transformation, allowing scientists to introduce foreign DNA into the bacteria. The script outlines the steps to make E. coli competent, which is a prerequisite for successful DNA uptake and subsequent genetic manipulation.

💡Centrifugation

Centrifugation is a technique used to separate particles from a solution by spinning at high speeds, causing the particles to settle out due to the force of gravity. In the video, centrifugation is used to pellet the E. coli cells after they have been grown in culture. The script describes spinning the cells down to form a pellet, which is then resuspended in the calcium chloride solution to make them competent.

💡OD (Optical Density)

Optical Density, often abbreviated as OD, is a measure of how much light is absorbed by a solution, and it is used to estimate the concentration of cells in a culture. In the script, the OD of the E. coli culture is checked to determine when the cells have reached the mid-log phase, which is the optimal growth stage for making competent cells. The script mentions an OD of 0.5 or 0.6 as the target, indicating the cells are ready for the next step in the protocol.

💡Incubation

Incubation is the process of maintaining a controlled environment, such as temperature and humidity, to support the growth of cells or the development of biological samples. In the video, E. coli cultures are incubated at 37 degrees Celsius with shaking to ensure optimal growth conditions. The script describes incubating the initial 5 mL culture and the subsequent 50 mL culture to allow the E. coli to grow to the point where they can be made competent.

💡Aliquoting

Aliquoting is the process of dividing a larger volume of liquid into smaller, equal portions. In the context of the video, aliquoting is used to distribute the competent cells into smaller tubes for ease of use and storage. The script describes aliquoting the cells into 1.5 mL Eppendorf tubes, allowing for multiple transformations from a single preparation, which is a common practice in molecular biology labs to conserve resources and ensure reproducibility.

Highlights

Introduction to preparing competent cells in synthetic biology.

Preparation of 100 millimolar calcium chloride solution.

Preparation of 100 millimolar calcium chloride with 15% glycerol solution.

Labeling of solutions with care to avoid damage from ice.

Adding water to the calcium chloride solution for proper volume.

Precautions while pipetting glycerol due to its stickiness.

Mixing and storing solutions on ice for later use.

Importance of maintaining a cold temperature to prevent cell lysis.

Starting a culture of E.coli for competent cell preparation.

Use of a single colony for genetic homogeneity.

Incubation of E.coli culture at 37 degrees for optimal growth.

Dilution of saturated E.coli culture into fresh media.

Growth of E.coli in a larger flask for better aeration.

Centrifugation to pellet cells for competent cell preparation.

Resuspension of cells in calcium chloride solution.

Centrifugation and resuspension in calcium chloride with glycerol.

Aliquoting competent cells into smaller tubes for convenience.

Storage of competent cells at -80 degrees for long-term use.

Transcripts

[Music]

hi welcome back to synthetic biology 1

today we're going to be preparing

competent cells so how I like to start

is by preparing my solutions so we have

two solutions that we need to prepare

one is a hundred millimolar solution of

calcium chloride and another one is 100

millimolar calcium chloride with 15%

glycerol so we'll need one and a half

grams of calcium chloride two times

[Music]

all right and let's not forget to label

our solutions so this guy is gonna be

our 100 millimolar calcium chloride and

over here 100 millimolar calcium

chloride with 15% glycerol ok so label

these and I'm being careful to label

these bottles near the top because we're

gonna put them on ice and if the labels

get wet they can fall off ok so now I'm

gonna take my water and I'll go ahead

and add it to the calcium chloride

solution bringing the total volume of

the solution up to 100 milliliters just

pour that in top it off at 100

milliliters I'm also going to add some

water to the calcium chloride solution

but I'm not gonna add the full 100

milliliters yet I'm just going to take

it up to 50 mils so that there's room

left over for adding the glycerol okay

so now let's pipette the glycerol I need

15 mils of glycerol and for this I'm

using a hundred percent glycerol

solution and it can be really a pain to

pipette so I'll have to be extra careful

one tip here is just put the tip of the

pipette into the glycerol because

otherwise the entire pipette will be

coated with glycerol to be a little

patient to get exactly the right level

let the excess glycerol drip off

and now this is why I added some water

to the solution because when I when I

add the glycerol I want to pipette up

and down just to wash the excess

glycerol from the pipette tip

okay cool and now we'll take the water

and just top off the solution bringing

the total volume up to 100 mils you can

you can tell I'm not being too careful

with my measurements for these solutions

and that's okay I don't need to be there

they're quite forgiving and close is

good enough okay

there's my two two solutions prepared

mix them up and I'll store these on ice

so you want to store them on ice so the

most important thing about preparing

competent cells is that once we've

resuspended the cells in the calcium

chloride solution we want to keep them

always ever after at four degrees

Celsius because if we allow the

temperature to come back up the cells

might start to lyse okay so that's our

solutions now we're going to go ahead

and start the culture of cells that

we're going to actually be making

competent so for this I have a plate of

mg 165 5 you might want to use nab turbo

or dh5 alpha or some other cloning

strain this is just a wild type strain

of e.coli and it's it's what I happen to

have ready around the lab okay so we're

going to start out five mill culture of

e.coli in LB media

so these are 14 male Falcon tubes don't

forget to label

see there's my five mils and now I am

going to take an inoculation loop and

pick just a single colony of e.coli from

my plate to start my culture with it's

important when you make competent cells

to always start from a single colony of

e.coli because that way you know that

the cells are genetically homogeneous

and not contaminated okay so now I'm

going to pop this in the incubator and

grow it at 37 degrees with shaking

overnight eight to twelve hours

okay so it's eight to twelve hours later

and my culture of e.coli is nicely

saturated so the next morning when

you're ready to prepare you're competent

cells when you come in what you do is

you'll take these cells and we're going

to dilute them by a factor of 200 into a

fresh 50 milk culture so we always want

to make competent cells using freshly

growing log phase e.coli because they're

the healthiest and they'll be the most

competent so for my my 50 ml culture I'm

using a 250 mil flask it's important to

use a much larger flask than the volume

of cells that you're growing so that the

cells are nicely mixed and well aerated

so they can grow quickly

now to my 50ml of lb media I'm adding

250 microliters of my saturated e.coli

solution

okay

I mix that around so we'll grow that in

the incubator at 37 degrees for two or

three hours until the OD reaches 0.5 or

0.6 which indicates that the cells are

in the mid log phase and ready to become

competent

it's two or three hours later and I've

collected my cells from the incubator

and they're at an OD of exactly 0.5 and

so they are ready to go for the next

step of the competent cell protocol so

I'm going to take my my culture of

e.coli and my freshly log phase e coli

and I'll add it to a 50 ml Falcon tube

like this

and then put it in the centrifuge and

spin it down at a maximum speed for ten

minutes to produce the pellet of cells

for the next step in the protocol

okay so now we've collected our pelleted

cells let's turn them into competent

cells by adding calcium chloride

solution so the first thing that I'm

going to do is very quickly pour off the

supernatant from the centrifugation

store these cells immediately on ice so

from now on everything that touches

these cells should be at four degrees

Celsius or colder now we're going to

resuspend these cells in our calcium

chloride solution here 15 mils

okay so i'll resuspend the cells by

pipetting up and down the pellet should

come apart pretty easily after a few

rounds of pipetting giving us a nice

uniform resuspension of cells we don't

want to spend too long doing this or the

cells will start to warm up okay now I'm

going to leave these cells on ice for at

least four hours or it could be longer

even overnight okay so after the second

round of centrifugation we will treat

the cells the same as before except now

we are going to resuspend them in four

mils of the calcium chloride and

glycerol solution so I'll go ahead and

pour off the wastes

and resuspend in four mils of calcium

chloride glycerol

okay so now these cells are ready to use

they're ready to transform or to store

however you want for the final step I'm

going to aliquot this large culture of

cells into smaller eppendorf tubes that

will be more convenient to transform so

for that I am going to need even more

ice and I'm using this aluminum rack to

cool the cells so this will transmit the

heat very rapidly from the tubes to the

ice and keep everything nice and cold so

I like to aliquot into these 1.5 mil

eppendorf tubes and I usually do 200

microlitre a loquats so a typical

transformation will use 20 microliters

of cells so 200 microlitre a loquats is

sufficient for 10 transformations from

one tube

okay so these competent cells are

finished they are ready to use

immediately or they can be stored at

minus eighty indefinitely

[Music]