3.8.4.1: Recombinant DNA technology

What is Recombinant DNA technology ?

• This technology is the combining of different organisms’ DNA, which could enable scientists to manipulate and alter genes to improve industrial processes and medical treatment.

Why is transfering DNA fragments possible ?

Transferring DNA fragments between/within species is possible because the genetic code is universal, and transcription and translation work the same in all organisms too.

State the 3 methods of producing DNA fragments

1. Reverse transcriptase

2. Gene machine

3. Restriction endonucleases

Explain how restriction endonucleases cut DNA and the difference between sticky ends and blunt ends.

• Restriction endonucleases are enzymes that cut DNA at specific base sequences known as recognition sequences/ restriction sites

• These enzymes occur naturally in bacteria as a defence mechanism against foreign DNA.

• Each restriction enzyme has an active site complementary to a particular DNA sequence and cuts the DNA at a specific location.

• Some restriction enzymes cut both strands at the same position, producing blunt ends.

• Others cut the strands at different positions, producing staggered ends with overhanging bases.

• These overhanging sections are palindromic and are known as sticky ends

What does it mean by the term palindromic ?

• This means that the base sequence on one strand (or sticky end) is the same sequence as on the other strand but in reverse

Describe how reverse transcriptase is used to produce cDNA from mRNA.

1. This enzyme naturally occurs in viruses, such as HIV, and it makes DNA copies from mRNA.

2. A cell that naturally produces the protein of interest is selected.

3. These cells should have large amounts of mRNA for the protein.

4. The reverse transcriptase enzyme joins DNA nucleotides with complementary bases to the mRNA sequence.

5. Single-stranded DNA is made (cDNA).

6. To make this DNA fragment double-stranded, the enzyme

   DNA polymerase is used.

The cDNA is intron-free because it is based on the mRNA template.

Describe how a gene machine is used to produce a gene from the amino acid sequence of a desired protein

1. The amino acid sequence of this protein is determined

2. From this the mRNA codons are looked up

3. The complementary DNA triplets are worked out and the gene produced

4. The genes are checked using standard sequencing techniques and those with errors are rejected

What are the advantages of using the Gene Machine

• Produces DNA quickly and accurately

• No need for a template DNA or mRNA source

• Produces intron-free DNA, suitable for expression in prokaryotes

• Allows production of genes for proteins that are difficult to obtain naturally

• Can help develop medicines and treatments, such as therapeutic proteins

• Computer checks improve biosafety and biosecurity before synthesis

Define the term in vivo cloning

• In vivo cloning is the process of making multiple copies of a gene inside a living organism (usually a bacterium).

• The gene is inserted into a vector such as a plasmid, which is taken up by a host cell and replicated as the cell divides.

What are the key steps in In vivo cloning ?

1. Preparing the DNA fragment for insertion

2. Insertion: of the DNA fragment into a vector

3. Transformation: This is the transfer of DNA into suitable host cells, using the vector

4. Identification: of the transformed cells that have successfully taken up the gene by the use of marker genes

5. Growth/cloning: of the population of transformed cells

What does it mean to amplify DNA fragments ?

• Once the DNA Fragment has been isolated it needs to be cloned to create large quanitities of it

Describe the preparation of a gene for in-vivo cloning.

• A restriction enzyme is used to cut the gene from the donor DNA at specific restriction sites, producing sticky ends

• The same restriction enzyme cuts the plasmid in the middle of one the marker genes

• The gene and plasmid are mixed in a test tube and they join becuase they were cut with the same restriction enzyme and have the same sticky ends (complementary)

• The fragments are joined by DNA ligase (form phosphodiester bonds) to form a recombinant (hybrid) plasmid - containing a mixture of bacterial and foreign DNA

• Several other products are also formed e.g plasmid will simply re-join with themselves and some DNA fragments will join together to form chains or circles

Describe how recombinant plasmids are introduced into bacterial cells.

• The bacterial cells and recombinant plasmids are mixed together in a medium containing calcium ions

• Given a a rapid temperature change - HEAT SHOCK

• Sudden changes in the temperature of the solution make the bacterial membrane more permeable allowing the plasmids to enter

• Some bacteria take up the recombinant plasmid and are transformed

Why are only a small number of bacterial cells transformed ?

• Not all bacterial cells take up plasmids during transformation

• Cell membranes are not equally permeable, even after calcium ions and heat shock

• Some cells take up no plasmid at all

• Some take up non-recombinant plasmids (without the gene)

• Therefore, only a small proportion of cells contain the recombinant plasmid

Why is it necessary to identify transformed cells in in vivo cloning?

• Not all bacterial cells take up a plasmid

• Some cells do not take up any plasmid

• Some take up non-recombinant plasmids (plasmid re-joins without the gene)

• The DNA fragment may join to itself instead of the plasmid

• Therefore, only a small number of cells contain the recombinant plasmid and must be identified

Why are marker genes used?

• Marker genes on the plasmid can be used to idetify which bacteria successfully took up the recombinant plasmid

What are the 3 different marker genes that can be used ?

1. Antibiotic resistance genes

2. Genes coding for fluorescent proteins

3. Genes coding for enzymes

How are transformed bacteria identified using antibiotic resistance genes (first marker gene) ?

• Plasmids used in genetic engineering are engineered to contain an antibiotic resistance gene (e.g. ampicillin resistance)

• Bacteria are grown on agar containing the antibiotic

• Only bacteria that have taken up a plasmid survive

• Observation: bacterial colonies grow on the agar plate where plasmids have been taken up, but no colonies form where bacteria do not have the plasmid

• This identifies bacteria that have been successfully transformed with a plasmid

How is the second marker gene used to identify recombinant plasmids using replica plating?

• The gene of interest is inserted into a second marker gene (e.g. fluorescent protein or antibiotic resistance gene), disrupting it

• This step occurs after bacteria have been identified as transformed using the first marker gene

• Replica plating is used to copy colonies onto different agar plates using sterile velvet

• One plate contains conditions to test the second marker gene (e.g. different antibiotic or substrate)

• Bacteria with non-recombinant plasmids show the marker gene working (e.g. survive or change colour)

• Bacteria with recombinant plasmids do not show marker activity because the gene has been disrupted

• These colonies are identified and cultured to amplify the DNA

How can the jellyfish GFP gene be used in genetic engineering?

• Jellyfish contain a gene that codes for green fluorescent protein (GFP)

• This GFP gene can be isolated and inserted into a bacterial plasmid

• If the gene is expressed, bacteria produce GFP

• This causes the bacteria to fluoresce under UV light

• GFP can be used as a marker gene to identify transformed bacteria or recombinant plasmids

How are enzyme marker genes used to identify recombinant plasmids?

• A gene coding for an enzyme (e.g. lactase) is used as a marker gene in the plasmid

• The gene of interest is inserted into the middle of this enzyme gene, disrupting it

• Bacteria are grown on agar containing a colourless substrate

• If the enzyme is functional (non-recombinant plasmid), it converts the substrate and causes a colour change (e.g. colourless to blue)

• If the enzyme gene is disrupted (recombinant plasmid), no enzyme is produced and no colour change occurs

• Colonies with no colour change are identified as containing recombinant plasmids

How are recombinant bacteria grown and how is the gene product produced?

• Host cells containing the recombinant plasmid are identified and selected

• These bacteria are grown in large numbers to form a cloned population

• A fermenter is often used to provide optimal conditions for rapid growth

• As the bacteria divide, the recombinant plasmid is replicated, amplifying the DNA fragment

• The inserted gene is expressed to produce the desired protein (e.g. insulin)

• The protein can then be harvested and purified for use