genetic engineering Flashcards
what is recombinant DNA technology?
It allows genes to be transferred between different organisms
examples of recombinant DNA technology
- insulin for type 1 diabetics
- herbicide resistant crop plants (BASTA)
what is recombinant DNA?
When DNA from 2 different organisms is combined it is called recombinant DNA
What are transgenic or genetically modified organisms?
Organisms containing recombinant DNA
what is genetic engineering?
It allows genes to be transferred between different organisms
Why can one organisms DNA be accepted and transcribed by another organism?
- The genetic code is universal and the same in all living organisms
- An mRNA codon will code for the same amino acid in all organisms
How Can We Make Recombinant DNA and Transgenic Organisms?
- Isolate the DNA gene fragment
- Insert DNA into a vector (can carry DNA between organisms)
- Transfer DNA into host cells (transformation)
- Identify host cells that have successfully taken up the recombinant gene (selection of transgenic organism)
- Grow the transgenic population
- Extract and purify the protein produced
what are the three ways to isolate gene fragments?
- Use reverse transcriptase enzyme
- Use restriction endonucleases
- Use a gene machine
how would you use reverse transcriptase enzymes to isolate gene fragments?
Reverse Transcriptase makes DNA from an RNA template – it does the opposite of transcription
This is how we obtain the gene for human insulin
what is reverse transcriptase?
An enzyme that can produce DNA from mRNA – comes from a group of viruses called retroviruses (e.g. HIV)
DNA produced is called complementary DNA (cDNA)
how would you use reverse restriction endonuclease to isolate gene fragments?
Restriction enzymes are used by bacteria to cut up the viral DNA.
These enzymes cut DNA at specific sites – this property can be useful in gene technology.
what are restriction enzymes (endonuclease)?
Enzymes that recognise specific DNA base sequences and cut DNA at specific sites 4-8bp long(palindromes) to produce blunt ended fragments or sticky ended fragments
where do restriction enzymes originate?
They originate from bacteria that contain restriction enzymes in order to protect themselves from invading viruses.
what are blunt ends in terms of restriction enzymes?
Some restriction enzymes cut DNA straight across both chains forming blunt ends.
what are sticky ends in terms of restriction enzymes?
Most restriction enzymes make a staggered cut in the two chains, forming sticky ends.
sticky end qualities
Sticky ends have a strand of single stranded DNA which are complementary to each other.
They will join with another sticky end but only if it has been cut with the same restriction enzyme.
how would you use a gene machine to isolate gene fragments?
- Use a computer to determine the DNA sequence of a protein. Computer then designs DNA fragments to build the gene
what is a vector?
A vector is a carrier which can carry DNA into a cell e.g. a plasmid
what are plasmids?
Plasmids are small circular DNA that can be replicated in a bacteria and transferred between different bacteria
what is the process of inserting DNA into a vector? (1)
Ensure target gene and plasmid have been cut with the same restriction enzyme so have complementary sticky ends
what is the process of inserting DNA into a vector? (2)
Ligation – use DNA ligase to join the DNA fragment and plasmid together by forming a bond between adjacent nucleotide in the two fragments of DNA
what bond will join the gene together on the plasmid?
phosphodiester
how can we join target genes with blunt ends to plasmids?
If the target gene of interest was cut to produce blunt ended fragments, then chains of nucleotides can be added to the gene using the enzyme terminal transferase. Complementary nucleotides can then be added to the plasmid to allow them to have complementary sticky ends to be joined by DNA ligase
before a gene can be copied/transcribed what must happen?
Before a gene can be copied/transcribed it has to be inserted into a suitable vector so the transgenic organism will be able to read and use the inserted gene to produce the protein we want.
transformation of host cells with recombinant plasmids
Recombinant plasmids need to be reintroduced into bacterial cells
Bacterial cells must be made “competent” (permeable) to allow them to take up the recombinant plasmid
what is transformation?
Recombinant plasmids need to be reintroduced into bacterial cells
how are the recombinant plasmids and bacteria mixed together?
Recombinant plasmids and bacteria are mixed together in the presence of ice cold calcium chloride
after they are mixed together in CaCl that must happen?
- The bacteria are then heated to 42oC for 2 minutes (heat shock)
- Plasmids can then pass through the cell surface membrane into the cytoplasm
how much Bacteria takes up the recombinant vector?
1%
what about the other 99%?
they are useless
other uses of the useless bacteria
- Some plasmids that have been taken up will have closed up without incorporating the DNA fragment
- Sometimes the DNA fragments join together to make a circular DNA molecule but they will not be able to produce functional proteins
How can Scientists identify the bacteria that have taken up the recombinant plasmid
We can identify if a bacteria has taken up a recombinant plasmid by using plasmids that contain two different genes for antibiotic resistance
identification of the transformed bacteria?
What does the Amp antibiotic resistance geneallow?
The Amp antibiotic resistance gene allows bacteria to grow in the presence of the antibiotic ampicillin so only bacteria that have been transformed with plasmids can grow in the presence of ampicillin
limitations to identifying bacteria that have taken up a recombinant plasmid
However some bacteria may have taken up a plasmid that resealed itself without the addition of the target gene
These bacteria will still grow in the presence of antibiotic but do not contain recombinant plasmids and do not contain the gene of interest
We can identify the transformed bacteria which contain the desired recombinant plasmids because they will not grow in the presence of tetracycline but WE WANT TO BE ABLE TO GROW THEM SO WHAT DO WE DO????
use replica plating technique
how does the replica plating technique work?
- Plate is incubated for 24 hours. Any colonies that grow must contain plasmids but we cant tell if they are resealed or recombinant
- A replica plate is made from the ampicillin plate and left to incubate for 24 hours
- The second replica plate contains tetracycline
-Any colonies that grow must have an intact tetracycline gene & therefore must contain resealed plasmids – these are not the bacteria we want - Which means this colony consists of bacteria which contain the recombinant plasmid where the target gene has inserted into the tetracycline gene in the plasmid and stops it from working so it is UNABLE to grow in the presence of tetracycline – THESE ARE THE BACTERIA WE WANT
what causes type 1 diabetes?
b cells in islets of langerhas in pancreas do not produce insulin
when was insulin discovered?
1922
what is the history of insulin research? (1)
1869, a German medical student named Paul Langerhans discovered clusters of cells, nestled in pancreatic tissue, whose function was unknown.
Later, when it was discovered that these clusters produced insulin, they were named the Islets of Langerhans.
what is the history of insulin research? (2)
In 1889, two German researchers, Oskar Minkowski and Joseph von Mering, found that when the pancreas gland was removed from dogs, the animals developed symptoms of diabetes and died soon afterward.
This led to the idea that the pancreas was the site where “pancreatic substances” were produced.
what is the history of insulin research? (3)
In 1910, Sir Edward Albert Sharpey-Shafer suggested only one chemical was missing from the pancreas in people with diabetes.
He decided to call this chemical insulin, which comes for the Latin wordinsula,meaning “island.”
what is the history of insulin research? (4)
In 1921, a young surgeon named Frederick Banting and his assistant Charles Best figured out how to remove insulin from a dog’s pancreas
what is the history of insulin research? (5)
In January 1922, Leonard Thompson, became the first person to receive an injection of insulin purified from cattle.
Within 24 hours, Leonard’s dangerously high blood glucose levels dropped to near-normal levels
In 1923, Banting and Macleod received the Nobel Prize in Medicine
what is the history of insulin research? (6)
Insulin from cattle and pigs was used for many years to treat diabetes and saved millions of lives, but it wasn’t perfect, as it caused allergic reactions in many patients
Some people objected to the use on religious grounds
what is the history of insulin research? (7)
Frederick Sanger discovered the amino acid structure of insulin in 1950s and was awarded a Nobel Prize for Chemistry
what is the history of insulin research? (8)
The first genetically engineered, synthetic “human” insulin was produced in 1978 using E. coli bacteria to produce the insulin
How do we find the Human insulin gene sequences?
- The β-cells in the pancreas make Insulin. Their cytoplasm contains the mRNA that encodes for insulin protein
- The mRNA is purified and incubated with the enzyme reverse transcriptase This enzyme reverses the process of transcription. The mRNA is copied to produce a cDNA strand
- The DNA strand is then copied using DNA polymerase to make a double stranded molecule. This complementary DNA is called cDNA
The cDNA encodes for the Insulin gene
Overview of production of Human Insulin
- Purify mRNA encoding for Insulin create cDNA
- ‘Stick’ the cDNA into a plasmid vector
- Transfer the vector/cDNA plasmid into bacteria
- Select bacteria that express Insulin
- Grow the bacterial cells in fermentors
- Extract and purify Insulin ready for use
reasons to produce genetically modified crops?
- Pest resistance
- Viral disease resistance
- Herbicide resistance
- Drought and frost resistance
- Longer shelf life
- Produce vaccines “pharming”
What is Agrobacterium tumefaciens?
Agrobacterium tumefaciens is a bacteria that infects wounds in plants and causes tumour production in them
what does Agrobacterium tumefaciens contain?
Agrobacterium tumefaciens contain a plasmid called a Ti plasmid which contains T genes
what happens when Agrobacterium tumefaciens infects a plant?
When the bacterium infects a plant the T genes from its Ti plasmid insert into the host plant cell genome and cause tumour formation in plants
what is Basta?
Basta is a herbicide used in many countries to kill weeds
It also kills crop plants so some crop plants have been genetically engineered to be resistant to Basta
How Does Basta Work?
- Plants naturally produce ammonia which is toxic during metabolic reactions
- Plants remove ammonia by converting it to non toxic glutamine using the enzyme glutamine synthetase
what is the active ingredient in Basta?
The active ingredient in Basta is phosphinothricine
equation for Basta + enzyme?
NH3 + GLUTAMATE –> GLUTAMINE
enzyme= glutamine synthetase
what structure does Basta have?
It has a similar structure to glutamate so acts as a competitive inhibitor of glutamine synthetase
How does Basta work?
it stops glutamine from being formed due to it being a competitive inhibitor. It will cause a build up of toxic ammonia and the plant will die
what have crop plants been genetically engineered to be resistant to?
Crop plants have been genetically engineered to be resistant to Basta by inserting a gene to produce an enzyme called phosphinothricine acetyl transferase (PAT) and introducing the gene into crop plant cells in tissue culture using Ti plasmid as the vector
what is Phosphinothricine acetyl transferase (PAT)?
Phosphinothricine acetyl transferase (PAT) is an enzyme produced by soil bacterium that inactivates phosphinothricine found in Basta
what does Phosphinothricine acetyl transferase (PAT) do?
it converts toxic ammonia to non toxic glutamine and GMO plants will survive being sprayed with Basta
advantages of GMO
- insect resistance
- drought resistance
- herbicide tolerance
- disease resistnace
- increased/enhanced nutritional content
why is insect resistance an advantage?
Insect resistance. Provides season-long protection against target pests, reduces the need for pesticide applications, and lowers input costs and protects insect biodiversity
why is drought resistance an advantage?
Drought resistance. GM crops that express drought resistance can grow in much drier areas, conserving water and other environmental resources.
why is herbicide tolerance an advantage?
Herbicide tolerance. Can grow crops in weed infested fields without ploughing to remove weeds first. This helps prevent soil erosion, and reduce carbon emissions.
why is disease resistance an advantage?
Disease resistance. Through genetic modification, the Hawaiian papaya industry was able to recover from the devastating papaya ringspotvirusthat had crippled the industry.
why is increased/enhanced nutritional content an advantage?
Increased/enhanced nutritional content. Currently in development are genetically modified soybeans with an enhanced oil profile, much like olive oil, made to be longer lasting and trans fat free.
Argument Against GMO in Agriculture
- unknown health effects
- transfer of antibiotic resistance to human gut bacteria
- GMO plants may hybridise with other plants to produce “superweeds”
- Pollen from GMO may pollinate plants on nearby “organic certified farms”
- Evolution of insecticide resistant pests
- Only farmers in developing world will be able to afford the “expensive GMO seeds”
What are ethics?
“a set of standards by which a particular group of people agree to regulate their behaviour, distinguishing an acceptable from an unacceptable activity”
