Educational Cell Free Mini Kit (ECFK)

1. Introduction

This kit is made to give students the opportunity to perform experiments to illustrate what can happen in a cell in a simple and easily accessible way. Hence the kit is based on the « cell-free » technique.

Several experiments are prepared to show four elements:

• The presence, the functioning and the capacity of enzymes to produce a reaction in cells.
• How are proteins synthetized in cells?
• What do cells really need to synthetize these proteins?
• How is the production of proteins regulated?

2. Theoretical context

The cell is a structural and functional unit made of several compartments and is confined by the plasma membrane, which separates the interior to the outside environment. “Cell-free” is a working method that consists of working without the plasma membrane and thus without a living cell. This technique makes transport and storage of reactants simpler, improves biosafety and reduces cost of experiments.

DNA (deoxyribonucleic acid) is a molecule present in every cell. It contains all genetic information of an organism, like a book telling a story. It consists of a double strand that is composed of a string of nucleotides (A, T, G and C) making up a genetic sequence. This genetic sequence is organized into genes allowing, when expressed, the expression synthesis of proteins.

Proteins are macromolecules made up of a sequence of amino acids. Each amino acid is encoded by 3 consecutive nucleotides present in a RNA. Proteins are necessary for the proper functioning of all biological systems. They are among other things responsible for the structure of muscles, the colour of your hair or eyes as well as the ability for a chemical reaction to happen – these particular proteins are called enzymes.

In order for protein production to happen (experiment 2), you need certain conditions:

• The presence of RNA – it’s the code for the protein.
• The presence of ribosomes – a little machine that allows translation of the 3 consecutive bases in the RNA into amino acids.
• The ribosome binding site so that the ribosome can bind to the RNA, scan it and produce the amino acid sequence (experiment 5).
• Amino acids – they are usually present in the cell.
• Energy in the form of ATP that the ribosome can use to advance on the RNA and to link the different amino acids that will make up the protein.
• A stop codon present in the RNA that will signal the ribosome that all necessary amino acids have been linked and that translation has come to an end.

Enzymes are proteins that help to speed up a chemical reaction by providing the reactants with an environment that is favorable for the reaction. Without enzymes, a big number of reactions would never happen (experiment 2).

B-galactosidase, for example, is an enzyme that can cut sugars such as lactose but also a special sugar, chlorophenol red, that changes its colour from yellow to purple when cut. The enzyme ß-galactosidase (also called ß-gal) is thus often used in laboratories as a reporter meaning that the colour changes it produces indicates a reaction taking place. B-galactosidase is made out of structural subunits α and Ω. If one is absent, the enzyme is not working. This mechanism of complementation is also very popular in laboratories to check for the presence of two different components. B-galactosidase is encoded by a gene called lacZ, lacZα and lacZΩ respectively for the subunits.

The principal component of the “cell-free” technique is the lysate, a reactant that allows for protein production for example. Lysate is produced out of a bacterial cell culture. The cells in this culture are first collected at the bottom of a tube, were rid of any contaminants and debris and then suspended. In a final step of sonication, the plasma membranes are disrupted by ultrasound. As a result, the lysate contains all the inner workings of a cell except the membrane and the genome.

This increases biosafety by a lot because no living entities can be found in the solution and all DNA initially contained in the cell has been destroyed. The disadvantage of working without living cells is that the energy generating metabolism can no longer take place. This means that you need to add energy sources to the lysate in order to provide the protein expression machinery with sufficient fuel. Additionally, the storage of lysate is a delicate history, they need to be kept at -80°C. This problem can be avoided by lyophilizing of the lysate, a process that removes all water.

3. Materials

Lyophilised kit

Non lyophilised kit

4. Experiences

To prevent contamination of the experiments and to protect yourself form the substances you are working with, wearing a lab coat and gloves is highly recommended. While you are doing experiments, you should avoid touching your face and doorknobs with your gloves. Furthermore, you should never leave the laboratory with your lab coat and gloves on. Once done with all the experiments, take the gloves off by turning them inside out but try not to touch the parts that came into contact with parts of the experiment with your bare hands.

Normally, tubes containing reactants are labelled according to their content. Here, they are labelled with a letter to simplify the experiments and to avoid situations of confusion about which tube to use.

4.1 Presence of the enzyme ß-galactosidase in a cell

4.1.1. Goal

To test for the presence of the enzyme ß-galactosidase in a lysate made of a cell culture.

4.1.2. Description of the experiment

There are 2 tubes containing lysate, energy solution, a buffer (to provide the appropriate reaction environment) and the substrate of ß-galactosidase (chlorophenol red) that when cuted on will produce a colour change.

Two lysates made from two different cell lines are provided: one of the cultures contains a full version of ß-galactosidase. The other has a non-functional version of the enzyme as the cells cannot produce one of its subunits as they contain a mutation in the gene and are thus missing the information to code the full enzyme.

4.1.3. Protocol

Lyophilised kit

• Rehydrate the lyophilised solutions of BL21 and Dh5α by adding 35 µl of nuclease-free water.

Non lyophilised kit

• Make a mastermix containing:
  • 10 µl of energy solution (label ES)
  • 10 µl of buffer A (label buffer)
  • 4 µl of nuclease free water (label NFW)
  • 4 µl of substrate (label sub)
• Equally separate the mastermix in two tubes and add in one tube 5 µl of solution 1.1 and in the other tube 5 µl of solution 1.2, don’t forget to label the tubes !

For both kits

• Let incubate until the colour change becomes obvious (immediately).
• Which lysate changed colour ?

4.1.4. Expected Results

In one of the two tubes should be able to observe a change in colour. The change from yellow to purple should be produced in the tube containing the lysate produced from the cells that have an intact copy of ß-galactosidase that cuts the substrate chlorophenol red. The other tube stays yellow because it contains an incomplete version of the enzyme and can thus not act on the substrate.

• Can you guess which culturesolution 1.1 or 1.2 contains the full version of the ß-galactosidase ?
• Knowing that the lysate Dh5 has a mutation for the lacZ, guess which tubes 1.1 or 1.2 contain the lysate Dh5 ?
• Does BL21 contain functional ß-galactosidase ? Why ?

4.2. Expression of β-galactosidase

4.2.1. Goal

Cell-free in vitro expression of ß-galactosidase.

4.2.2. Description of the experiment

There are two tubes: one containing a lysate without ß-galactosidase, an energy solution, a buffer, the substrate chlorophenol red and DNA LacZ that codes for ß-gal, the other one everything listed before except the DNA. If the DNA is present in a tube, the full version of ß-gal will be produced when the system is rehydrated and thus functioning again. It is up to you to determine which tube contains the DNA coding for ß-galactosidase.

4.2.3. Protocol

Lyophilised kit

• Rehydrate both tubes 2.1 and 2.2 by adding 35 µl of nuclease free water.

Non lyophilised kit

• Make a mastermix containing:
  • 10 µl of energy solution (label ES)
  • 10 µl of buffer A (label buffer)
  • 10 µl of M15-T7 lysate (label 2.1)
  • 4 µl of substrate (label sub)
• Equally separate the mastermix in two tubes and add in one tube the 4 µl of solution 2.2 and in the other one, add 4 µl of the solution 2.3, don’t forget to label the tubes !

For both kits

• Let the tubes incubate until you see a change of colour (approx. 30 minutes).
• Which tube contains the coding sequence for ß-gal ?

4.2.4. Expected Results

One of the tubes will produce ß-galactosidase due to the presence of LacZ DNA. This shows in a colour change. The other tube is a negative control without DNA that allows the conclusion that the colour change really is due to the presence of DNA.

• What would happen if we add BL21 lysate in a protein expression system containing LacZ DNA ?
• Would it be possible to produce other ß-galactosidase ?
• What would happened if we forgot the energy solution ? Response in the next experiment !

4.3. The necessity of the energy solution in the lysate

4.3.1. Goal

Definition of the importance of energy solution to produce proteins.

4.3.2. Description of the experiment

Energy solution is essential for the production of proteins as will be demonstrated in this experiment. The cells in the lysate are “dead” and can no longer produce all the required material for protein production by themselves. We thus have to provide the lysate will all the essential components for protein synthesis. The energy solution contains a mix of the 20 essential amino acids that make up proteins, ATP (a cell’s form of energy), different salts and some DNAs and RNAs that are needed and have been degraded in the process of making the lysate.

4.3.3. Protocol

Lyophilised kit

• Rehydrate the tube 3.1 by adding 35 µl of nuclease free water.

Non lyophilised kit

• Make solution containing:
  • 5 µl of buffer A (label buffer)
  • 5 µl of M15-T7 lysate (label 3.1)
  • 2 µl of substrate (label sub)
  • 2 µl of DNA coding for ß-galactosidase (label 3.2)

For both kits

• Let it incubate (approx. 5 minutes).
• Witness the constant yellow colour.

4.3.4. Expected Results

The colour of the solution in the tube should stay yellow. Because the energy solution has not been added to the solution, the machinery is not able to produce the enzyme ß-gal and as a matter of fact, no other protein either. The energy solution is an important aspect disadvantage in the field of “cell-free” as it is long and difficult to produce and can only sustain a lysate for so long.

• Why the solution stayed yellow ?
• Try to add BL21 lysat, what happen ? Why ?

4.4. The Polymerase

The polymerase is the cellular machinery that allows transcription of DNA to RNA, which can subsequently be translated to proteins. In order for the polymerase to be able to work properly, it needs a binding site, the promoter. Each different polymerase has its own specific promoter. If your lysate contains a certain type of DNA containing a promoter for another polymerase, almost nothing will be expressed.

In this subsequent experiment, two polymerases are studied: the E. Coli RNA polymerase who need the E. Coli promoter and the T7 RNA polymerase who need T7 promoter for transcription of DNA to RNA.

4.4.1. Goal

Test the principle of the polymerase and its corresponding promoter.

4.4.2. Description of the experiment

The two tubes contain a different lysate each, energy solution, substrate and LacZ DNA with a promoter called T7. To have protein synthesis you thus need T7 RNA polymerase in your lysate. The two lysates used in this experiment are M15-T7 and DH5α. Can you guess which lysate contains the appropriate polymerase?

4.4.3. Protocol

Lyophilised kit

• Rehydrate the two tubes 4.1 and 4.2 by adding 35 µl of nuclease free water.

Non lyophilised kit

• Make a mastermix containing:
  • 10 µl of energy solution (label ES)
  • 10 µl of buffer A (label buffer)
  • 4 µl of substrate (label sub)
  • 4 µl of lacZ DNA (label 4.1)
  • 2 µl of nuclease free water (label NFW)
• Equally separate the mastermix in two tubes and add in one tube the 5 µl of solution 4.2 and in the other one, add 5 µl of the solution 4.3, don’t forget to label the tubes !

For both kits

• Incubate until you see a colour change (approx. 30 minutes).
• Observe the colour change.

4.4.4. Expected Results

One of the tubes should change colour due to the presence of T7 polymerase and the resulting expression of ß-galactosidase based on the added LacZ DNA. The other tube is not able to produce the enzyme due to the missing polymerase. What is the interest of using ß-galactosidase to identify the transcription and subsequent translation of a certain DNA? When you add the coding sequence for ß-gal after your gene of interest you can verify the transcription of the gene of interest. Whenever this process happens, the gene for ß-gal is also transcribed and a colour change is thus produced, indicating that our protein of interest is produced.

• By observing the change of color, which one of the two tubes 4.2 or 4.3 contain lysate from cells that express T7 RNA polymerase ?
• Why one solution stayed yellow ? Why the other one turned purple ?

4.5 The Ribosome Binding Site (RBS) - a laboratory technique allowing the regulation of protein expression

4.5.1. Goal

Identification of the presence of a particular trigger RNA – here a small part of the Zika virus RNA (We do not have the complete RNA of the virus present so no potential danger present!).

4.5.2. Description of the experiment

There are many techniques to regulate protein expression. The one presented here has been specifically developed to be used in laboratories. A toehold switch is a piece of RNA in the form of a hairpin. This hairpin has as a result that both the ribosome binding site and the start codon are hidden for the ribosome and thus the encoded protein isn’t expressed. When a certain trigger RNA is present in the solution of the toehold switch, the latter will unfold and release the RBS and the start codon. The trigger RNA has a sequence that is complementary to the stem of the hairpin and when it binds, it opens up the structure. A ribosome can then bind to the RNA and produce proteins. This system is used to report the presence of a particular DNA, viral for example, that will trigger the toehold and thus protein expression.

The experiment will be done with a toehold that is followed by the LacZα gene, the coding sequence of the α subunit of ß-gal. When the trigger sequence is present in the solution, the toehold will unfold and as a result the  subunit is produced and then assembled with the  subunit to produce a functional version of ß-galactosidase. This will induce a colour change. The lysate contains an incomplete version of ß-galactosidase, the Ω subunit. When the  subunit assembles with the  subunit they create a fully working version of the enzyme. This is a very useful mechanism for studying transcription and translation: you add the coding sequence of LacZα after your gene of interest. When your gene of interest is being transcribed and translated, LacZα is so too. If you add this construct into a lysate containing only the  subunit, the two can assemble.

4.5.3. Protocol

Lyophilised kit

• Rehydrate the two tubes 5.1 and 5.2 by adding 35 µl of nuclease free water.

Non lyophilised kit

• Make a mastermix containing:
  • 10 µl of energy solution (label ES)
  • 10 µl of buffer A (label buffer)
  • 10 µl of M15-T7 lysate (label 5.1)
  • 4 µl of substrate (label sub)
  • 2 µl of toehold (label 5.2)
• Equally separate the mastermix in two tubes and add in one tube the 2 µl of solution 5.3 and in the other one, add 2 µl of the solution 5.4, don’t forget to label the tubes !

For both kits

• Incubate until you see a change of colour (approx. 30 minutes).
• Which tube contains the trigger RNA of the Zika virus ?

4.5.4. Expected results

One of the tubes should change colour as the trigger RNA is present in the solution and thus unfolds the toehold switch. This is inducing the production of the  subunit of ß-galactosidase and subsequent assembly of a functioning enzyme. The other tube does not contain this trigger and thus also no functioning ß-gal. The first tube would correspond to an infected individual if the sample would originate from blood, while the second one corresponds to a healthy person.

• Which tube contain the trigger RNA ? Why ?
• Does this diagnostic system would be possible if using BL21 lysate for ß-galactosidase expression ?
• If we added a part of a RNA from the influenza, what would happen ? What is the interest ?

5. References

Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., ... & Daringer, N. M. (2016). Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell, 165(5), 1255-1266.

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Jackson, R. (2014). Campbell biology (p. 135). Boston: Pearson.

6. PDF versions

ECFK PDF english version

ECFK PDF french version

Biosafety evaluation ECFK