Lecture 19 - Recombinant DNA technology

Question 1

Diabetes

Answer 1

Type I (childhood onset) and Type II (15% lifelong risk)

Goal of diabetes treatment is to return normal levels of insulin

Question 2

Insulin timeline

Answer 2

Previously in the 1960s an individual would be treated with purified pig and cattle insulin. Insulin is purified from the pancreas of cattle or pigs. Human amino acid sequence is similar but not identical. Human insulin is formed by 2 chains; an alpha and a beta chain which are held together by disulphide bonds. Two disulphide bonds between the cysteine in the alpha and beta chain and one intramolecular disulphide bond between two cysteine in alpha chain. Cow insulin has two amino acid residues that are different in the alpha chain and another amino acid change in the beta chain, pig insulin has only one change from thr to ala however these differences especially for type I diabetics that hack an autoimmune deficient can potentially be lethal so giving pig or cow insulin can do anything from mild irritation at injection to full blown anaphylactic shock. Also it is not always 100% pure.

Human sources have also been used - issues around safety (pathogen transmission), yield and source of protein (tissue availability)

Recombinant DNA technology born in 1973 so can be used when considering insulin as treatment for diabetes

Question 3

What are recombinant DNA technologies?

Answer 3

Joining bits of DNA together (sometimes from different species). These are then inserted into an organism to produce (express) a useful protein.

technology that combines genes from different sources into a single DNA molecule

Question 4

Recombinant DNA

Answer 4

DNA that has been formed artificially by combining constituents from different organisms.

Question 5

Plasmids are critical elements for recombinant DNA technologies

Answer 5

(Usually) circular pieces of double stranded DNA 
Replicate independently of the host’s chromosomal DNA 
Common in bacteria, but also found in eukaryotes. 
Provide a benefit to hosts e.g. antibiotic resistance 

Plasmids are naturally found in bacteria. Found independent of the chromosome so that are extra chromosomal. Uses the machinery of the host cell.

Question 6

Key components of DNA plasmids

Answer 6

Origin of replication (ORI)
• allows initiation of replication using host DNA polymerase

Antibiotic resistance gene
• allows selection of cells containing plasmid.

Promoter
• Drives expression of your favorite gene (e.g. insulin or GFP) in cells with appropriate
transcription factor machinery
•Changes to all expression in prokaryotes, eukaryotes, specific cell types

Take advantage of the plasmid and make a recombinant DNA plasmid, which allows us to insert this bit of DNA into any cells we want to and express whatever we want in it, most of the DNA is derived from the bacteria or the endogenous plasmid source, we can add several features to this and usually we want to add our favourite gene and we also need to add a promoter which will drive gene expression and your RNA polymerase is to sit on the plasmid end and transcribe the gene and we also put an antibiotic resistance gene in there which allows us to select out the cells that have taken this plasmid up and any that don’t have this will not survive antibiotic treatment and lastly and ORI which allows DNA polymerase to replicate this plasmid

Question 7

Tools used for recombinant DNA technologies

Answer 7

Restriction enzymes
DNA ligases

Restriction enzymes and DNA ligases are used to manipulate DNA and bacteria are used to amplify DNA

Question 8

What are restriction enzymes?

Answer 8

Naturally found in bacteria - defines system to degrade foreign DNA and insert our favourite promoter and transferee into the bacterial plasmid so that we can use it for recombinant DNA technologies

Cut dsDNA at specific sequences - to cut and paste the promoter and the transgender into the plasmid we take advantage of restriction enzymes which are naturally found in bacteria which bacteria use for a defence mechanism. Restriction sites are where you can cut the plasmud, the restriction enzymes don’t cut the bacterial DNA as well because the bacterial DNA is highly methylated and so it isn’t recognised by the bacteria’s own restriction enzymes

Enzyme that cuts DNA at a specific sequence of nucleotides

Question 9

What are DNA ligases?

Answer 9

Complementary base pairing - then we repair the phosphodiester bond to allow the regeneration of a circular piece of DNA
DNA ligase catalyses the formation of phosphodiester bond to repair nick in DNA backbone
Sticky ends that we can now insert our favourite gene into, to insert the gene we first have to make a copy of it through PCR amplification and add restriction sites so that you have complimentary sticky sites to play with

enzyme that chemically links DNA fragments together

Question 10

Amplifying plasmids

Answer 10

Transformation = transfer of plasmids into bacteria 
Transformed bacteria selected by antibiotic resistance contained on plasmid 
Expression of plasmid gene in bacteria (if bacterial promoter). 
Amplification of bacteria and purification of DNA for downstream uses e.g. PCR, cloning, transfection into other cells or organisms

Question 11

The universal genetic code

Answer 11

All organisms ”read” the same codons as the same amino acids. 

AUG = methionine 
UGA = stop codon 

Significance = we can transform a human gene into bacteria and it will still make the same protein.

Question 12

The catch when cloning eukaryotic genes for expression in prokaryotes

Answer 12

Prokaryotic genes do not have introns. Prokaryotes don’t have the machinery to process eukaryotic introns therefore use coding sequence only!

Eukaryotes have introns and exons - exons code for expressed sequences and the introns are the intervening sequences within DNA and DNA within bacteria do not contain introns. Prokaryotes have no splicing mechanisms and it would retain the introns within the mRNA which would give you a protein with the incorrect sequence and to get around this we only need to use the coding sequence when we make recombinant protein within bacteria so our plasmids needs to be a copy of the mRNA rather than a copy of the original DNA - so to do this you take the mRNA of interest and make a copy of it using reverse transcriptase and then take this copy DNA into the bacterial plasmid

Question 13

start and stop codons

Answer 13

Start: AUG
Stop: UAA, UAG, UGA

Question 14

Genetic transformation

Answer 14

Process of inserting DNA from one species into another species to create a transgenic organism.

Question 15

There are three systems that can be used to produce therapeutic proteins…

Answer 15

Prokaryotic cells (bacterial) 
Eukaryotic cells (yeast are single celled eukaryotes then there are mammalian cells also) 
Whole animals/Transgenic animals

All require the use of a cloning vector derived from bacterial plasmids

Question 16

What is a bacterial plasmid?

Answer 16

A plasmid is a small circular double-strand- ed DNA molecule, which can be found naturally within a bacterial cell.

Plasmids are physically seperated from the host cell’s chromosomal DNA and can replicate independently. Independent replication of the plasmid is facilitated by an AT-rich region called the origin or replication (Ori). While the chromosomal DNA contains all the essential information required for living, plasmids contain extra genes that provide a benefit to the host cell.

Plasmid genes often allow the host cell to survive in an environment that would otherwise be lethal or restrictive to growth, e.g. providing antibiotic resistance, resistance to heavy metals or providing a metabolic function allowing the bacterium to survive on a particular nutrient.

Question 17

What is a cloning plasmid?

Answer 17

A vector is a plasmid that has been engineered to contain certain sequences so that it can be used as a tool in molecular cloning, which is essential for the production of therapeutic proteins.

Modified (or artificial) plasmids are the most commonly used cloning or expression vectors, serving to drive the replication (and/or expression) of recombinant DNA sequences within host organisms. Similarly to natural plasmids, vectors must carry an Ori sequence in order to replicate independently within their host cells. In addition, vectors contain a site into which DNA fragments of interest (e.g. gene sequences) can be inserted. In an expression vecor, this region includes a strong promoter, which will facilitate transcription of any inserted DNA sequence. The resulting mRNA will be translated by the host cell’s machinery to a protein (e.g. a therapeutic protein).

Cells carrying vectors must be identified from all other cells. This is done with the help of a selectable marker, which is usually an antibiotic resistance gene. This gene will code for a protein that will give the bacteria resistance to particular antibiotics.

These plasmids are extracted from bacterial cells and modified in order to serve a specific purpose