KK11 - recombinant plasmids

Bacteria

• Bacteria replicate their plasmid DNA independently from their circular chromosome

• No. plasmid in each bacterium varies

  • Some can have many or none

  • Vary in length - can be 200kbp

Why transform bacteria?

• Independently replicating plasmids = humans can genetically modify bacteria to synthesise large amounts of protein 

• Involves editing a plasmid to incorporate a target gene of interest

• Recombinant plasmid - a plasmid that is edited to integrate a target gene

• Bacterial transformation occurs

  • Uses of bacterial transformation

• Bacteria can synthesise proteins

Uses of bacterial transformation

• Many uses in the medical and food industries

  • Enables cheaper and more efficient methods of production

• Large-scale production of:

  • insulin to manage diabetes

  • erythropoietin to treat anaemia

  • chymosin for cheese production

  • interferon to treat some cancers

  • growth hormone to manage growth disorders

  • hepatitis B surface antigen for use in the hepatitis B vaccine

  • alpha-amylase for ethanol and high fructose corn syrup production.

Recombinant plasmid

• Cloning vectors

  • Able to self-replicate 

  • Small and can be taken up by bacteria

  • Easy to include antibiotic resistance genres, recognition sites and expression signals

• Need a gene of interest, a plasmid vector, a restriction endonuclease and DNA ligase

Gene of interest

• A sequence of DNA encoding the protein that is wanted to be produced

• DNA sequence of a human protein is isolated and amplified using polymerase chain reaction

  • Inserted into a vector

• Even though the gene of interest comes from another organism, bacteria can use their DNA to synthesise identical protein case genetic code is universal

• Must not have introns before insertion

  • Prokaryotic gene expression doesn't involve RNA processing

  • Bacteria would not know that to do the intron segments

Removal of introns

Can be done in two ways

• Use synthetic DNA

  • Genes are made synthetically in a lab using a DNA synthesiser

  • Introns are not included in the gene when it is made this way

• Use copy DNA (cDNA)

  • Made from an enzyme called reverse transcriptase (RT) - transcribes mRNA backwards into the cDNA

  • Doesn’t contain introns due to the absence of intron in the mRNA being reverse transcribed

Plasmid vector

• Selected into which the gene of interest will be inserted

• Has four important DNA sequences

  • Restriction endonuclease sites – a site on the plasmid that can be recognised and cut by a restriction endonuclease, allowing the gene of interest to be inserted.

  • Antibiotic resistance genes – e.g. amp^R which confers ampicillin resistance or tet^R which confers tetracycline resistance.

  • Origin of replication (ORI) – a sequence that signals the start site for DNA replication in bacteria

  • Reporter gene – genes with an easily identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest.

Plasmid vector other factors

• Must contain two genes that encode for observable traits

  • Like antibiotic-resistant genes or report genes like gfp

  • Encodes for a recognised fluorescent protein

• One of these genes must contain the restriction site of the restriction endonuclease that is to be used

Restriction endonuclease

• Gene of interest and plasmid are cut with the same restriction endonucleases to make identical sticky ends on either end of the DNA sequence

• Overhanging nucleotides of the gene of interest will be complementary to the overhanging nucleotides on the plasmid vector

  • Allows them to form hydrogen booths with each other easily

• Bund end restriction enzymes can be used but are less targeted compared to sticky ends restriction enzymes

  • Blunt ends can bond with any other blunt end

DNA ligase

• Added to join the gene of interest to the plasmid vector

  • Forms phosphodiester bonds in the sugar-phosphate backbone

• Creates a circular piece of DNA called a recombinant plasmid

• Not every plasmid will take up the gene of interest

  • Most plasmids will ligate back with themselves and are called non-recombinant plasmids

• Creates a mixture of both recombinant and non-recombinant plasmids

• Reporter genes are used to distinguish between a recumbent and non-recombinant plasmid

  • Bacteria need to undergo transformation first

Transforming bacteria

• Bacteria will naturally take up free-floating DNA from their environment into their cytosol via transformation

• Take advantage of this process to make bacteria take up recombinant plasmids

Uptake of recombinant plasmids

• Involves the recombinant plasmid being inserted into the cytoplasm of bacteria

  • A process called bacterial transformation

Two methods: heat shock and electroporation

Heat shock

• Requires bacteria and plasmids to be placed in a calcium ion solution on ice

• +ive calcium ions make the plasma membrane more permeable to the -ive charged plasmid DNA

• Solution heated to 37-42℃ for 25-45 sec before returned to the ice

• Sudden changes in temperature make plasma membrane more permeable

• Allows plasmid vectors to cross the phospholipid bilayer and enter the bacteria’s cytoplasm

Electroporation

• An electrical current is passed through a solution containing bacteria and plasmid vectors

• This causes the plasma membrane to become more permeable

  • Allows plasmid vectors to cross the phospholipid bilayer and enter the bacteria's cytoplasm

Antibiotic selection

• To distinguish between transformed and untransformed bacteria the mixture is cultured onto an antibiotic-rich plate

• Transformed bacteria contain the gene necessary for a specific resistant

  • All untransformed bacteria will be killed off when exposed to that antibiotic

• Each colony visible on a plate represents a transformation event where a single bacterium has taken up a plasmid

  • Allows it to survive, multiply and form a colony of identical daughter cells

Separation of transformed bacteria

• Takes up both recombinant and non-recombinant plasmids

  • Needs to be distinguished from one another

• Can be done by one of the two genes that encodes for an observable trait within the plasmid vector

• One of these genes (like a reporter gene) was cut by a restriction enzyme

  • Was the site of the gene of interest insertion

• Can be used to distinguish between recombinant and non-recombinant plasmids

For example

• gfp is a reporter gene that encodes for the green fluorescence protein

  • Glows green under UV light when fully expressed

• In non-recombinant plasmids, this gene is continuous and therefore is expressed 

  • Enables bacteria that have been transformed with non-recombinant plasmids to glow 

• In recombinant plasmids, the reporter gene is split by the gene of interest and is non-continuous

  • Bacteria that have been taken up recombinant plasmids cant glow 

  • Can be distinguished from bacteria with non-recombinant plasmid

Protein production and extraction

• Transformed bacteria are cultured and induced to produce the target protein

• As the bacteria make lots of different proteins, the protein of interest is extracted and purified

Insulin

• Hormone that is responsible for regulating blood glucose levels

• People with diabetes can’t produce or respond to insulin and require it to be administered artificially to the body

• Has a quaternary structure consisting of two polypeptide chains

  • Alpha and beta subunits

• Require two different recombinant plasmids and two different transformed bacteria samples

  • One producing alpha subunit and one producing the beta subunit

  • Two chains must first fold individually then be joined together by a disulphide bridge

Creating the recombinant plasmid

1. Plasmid vector produced with ampR , tetR , and restriction sites

2. Insulin A and B subunit genes (without introns) cut and ligated to form recombinant plasmids

3. Plasmids added to bacteria to create transformed bacteria

4. Bacteria colonies tested to find bacteria that successfully took up recombinant plasmids

5. lacZ gene is inserted into the plasmids

6. Plasmids containing lacZ added to bacteria to create transformed bacteria

7. Bacteria colonies tested to find bacteria that successfully took up recombinant plasmids containing lacZ

8. Insulin subunit genes expressed attached to large β-galactosidase proteins

9. Insulin A and B subunit proteins isolated, purified, and combined to form functional human insulin