Gene Cloning and Fusion Expression

DNA Cloning

DNA cloning yields multiple copies of a DNA segment or gene

Most methods for cloning pieces of DNA in the laboratory share general features, such as…

The restriction enzymes, plasmids and use of bacteria

Type II Restriction Enzymes (RE)

are enzymes that cut DNA at specific sequences, allowing for the manipulation of genes in cloning and molecular biology.

EcoRI enzyme

A restriction enzyme used to cut DNA at specific sequences, allowing for the manipulation and cloning of genes.

SmaI Enzyme

A restriction enzyme used to cut DNA at specific sites, recognizing the sequence CCCGGG. SmaI is commonly utilized in cloning and molecular biology to facilitate the insertion of new DNA fragments.

Plasmids

Molecules of DNA that are found in bacteria separate from the bacterial chromosome

Essential features for cloning are:

a vector, a selectable marker, and a target gene.

Ampicillin resistance gene (ampR)

Cloning vector

plasmid in gene cloning

Cloning vector definition

A DNA molecule that can carry foreign DNA into a host cell and replicate there

Naturally occurring plasmids have been genetically engineered to

Possess features for use as cloning vectors

Polylinker or multiple cloning site

A synthetic DNA fragment with restriction sequences for several RE can be inserted into a plasmid

Gene Cloning

Using bacteria to producing many identical copies of the same recombinant molecule

Cloning steps

1) Vector + DNA
2) Recombinant DNA
3) Transform Host Cells
4) Select for transformed cells
5) ID/Screen Host cells that contain recombinant molecule

RE and DNA ligase allow

Insertion of DNA fragments into cloning vectors

Experiment regarding RE and DNA ligase

To clone the gene X (350bp) into the Eco RI site of the vector pBR322

Cut ends are…

Cohesive (sticky) and complementary

DNA ligase

Catalyses the formation of the 3'‐5' phosphodiester bond

Recombinant DNA

DNA from two different sources is incorporated in vitro into a single molecule

Bacterial Transformation

Transfer of plasmid/recombinant molecule into bacteria (E.coli)

Phospholipid bilayer of the PM has…

A hydrophilic exterior and a hydrophobic interior

DNA unable to…

Freely pass through the membrane

Induced by laboratory procedures e.g. Calcium chloride transformation

A process used to facilitate the uptake of plasmid DNA by bacteria, typically through chemical treatments that make the bacterial cell membrane more permeable.

CaCl2 treatment of bacterial cells produces…

Competent cells which will uptake DNA after a heat shock step to facilitate transformation.

Colony formation

If a cell is placed on the surface of agar medium, it will start to divide and form a colony

Clone representation

All cells in the colony are descendants of one cell thus they represent a clone

Recombinant molecule

Contains DNA that has been artificially inserted into a vector. This allows for the expression of a desired gene in a host organism.

Identifying the correct clone step 1

Isolate the plasmid DNA from a number of transformants

Isolating the plasmid DNA

Involves extracting plasmid DNA from bacterial cells that have taken up the plasmid during transformation. This step is crucial for analyzing which transformants contain the desired genetic insert.

Identifying the correct clone step 2

Analyse plasmids using restriction enzyme digestion

Agarose Gel Electrophoresis

A technique used to separate nucleic acids or proteins based on size and charge by applying an electric field to a gel matrix.

Restriction Digest

A technique in molecular biology where restriction enzymes (restriction endonucleases) are used to cut DNA at specific sequences, producing DNA fragments useful for cloning, mapping, or analysis.

Agarose Gel Electrophoresis

A laboratory technique used to separate DNA, RNA, or proteins based on their size and charge by applying an electric field to a gel.

To assess if pBR322 vector contains gene X (350bp) using restriction digestion followed by agarose gel electrophoresis steps:

Lane 1. Molecular weight marker
Lane 2. Clone 1 EcoRI digested
Lane 3. Clone 2 EcoRI digested
Lane 4. Molecular weight marker

Gene Cloning and expression is used to

Produce protein drugs

Foreign DNA is inserted into

A plasmid, and the recombinant plasmid is inserted into a bacterial cell

Reproduction in the bacterial cell results in

Cloning of the plasmid including the foreign DNA, which results in the production of multiple copies of a single gene

Cloned genes expressed to

Produce a protein

Examples of proteins produced:

Insulin, growth factors, monoclonal antibodies

Expression vectors

Are plasmids that have been modified for the purpose of expressing high levels of a foreign protein in bacteria. They have been engineered to contain appropriate sequences for transcription, translation and protein purification

The gene encoding the protein of interest is cloned into…

An expression vector that allows high gene expression in the host cell

Production of large amounts of mRNA which can…

Be translated into protein

Produce protein using

The host cells synthetic machinery

Features of an Expression vectors

1. ORI
2. Unique restriction site
3. Small size
4. Selectable marker
5. Promoter
6. RBS
7. Tag protein to aid purification

Fusion Protein

Protein that results from the expression of recombinant DNA

POI

Protein of Interest

Induce large vol of culture for

Optimal induction time

Affinity Chromatography

Purification of the induced protein

Fusion proteins are purified by

Bacterial lysates by passing the lysate through a column containing a resin to which the fusion protein can bind

Other cellular proteins will…

Flow through the column

After washing the fusion protein can be

Eluted or released from the column

Purification of fusion protein

Techniques like affinity chromatography to separate the fusion protein from other cellular components.

Eppendorf experiment

Refers to a molecular cloning experiment where DNA ligase is used to join DNA fragments.

Purpose of DNA ligase in cloning

To covalently join the 3'-OH and 5'-phosphate ends of DNA fragments, forming phosphodiester bonds.

Common components of ligation reaction

Linearized plasmid vector, DNA insert, buffer with ATP/NAD⁺, T4 DNA ligase.

Function of T4 DNA ligase

Catalyzes phosphodiester bond formation between adjacent nucleotides to join DNA strands.

Incubation conditions for ligation

Typically at 16°C overnight or at 20–25°C for 10–30 minutes.

Post-ligation step

The ligation mixture is usually transformed into competent bacteria to propagate recombinant plasmids.

Molecular cloning

A method used to insert DNA fragments into plasmids for replication and expression in host cells