Biotechnology

Biotechnology Definition

• the application of organisms, biological systems or biological processes in the manufacturing and servicing industries

Recombinant DNA

• DNA that has been altered by foreign genes

Transgenic organisms

• one that has recombinant DNA and can express it to produce the protein it codes for

• used to produce more complex proteins than can be produced then in prokaryotes

Genetic Engineering

• recombinant DNA technology

• involves the manipulation and combination of DNA molecules from different organisms to produce - genetically modified organisms

Major Goals of Genetic Engineering

• understand the processes of inheritance and gene expression

• better treatment of genetic diseases

• generate economic benefits

Stages of Genetic Engineering - How is it done

1. Obtain a copy of the required gene from a donor organism - isolate the required gene

2. Constructing Recombinant DNA

3. Inserting Recombinant DNA into a host organism

4. Selecting and Screening

Stage 1 - obtaining a copy of the required gene from a donor organism

1. Direct Synthesis - when the sequence of bases is known, it can be artificially synthesized in a lab - this is only suitable for short DNA sequences

2. cDNA - mRNA is extracted and reverse transcriptase converts it into a complimentary DNA strand. DNA polymerase converts c.DNA into two complimentary DNA strands

   1. only these two can be used to isolate genes to insert into bacteria - as this will remove introns. Bacteria dont have enzymes to remove introns, hence will make a useless protein

3. Shotgun Approach - using restriction enzymes which cut specific DNA sequences known as restriction sites - cut palindromic sections of DNA. some produce Blunt ends (wont be attracted to each other as they are cut at the same base pair) or STICKY ENDS (which will attract each other as they are cut at complimentary base pairs)

   1. this produces DNA fragments which can be separated through the use of gel electrophoresis and southern blotting

Finding the DNA fragment with the desired gene - Gel Electrophoresis

1. Each DNA nucleotide carries a negative charge when ionized - doff lengths carry diff total charges

2. mixture of DNA fragments is places in wells of a porous gel (made of agarose)

3. placed in electrophoresis chamber which has a negative electrode at one end and a positive electrode at the other end

4. conducting buffer solution

5. electric field is applied acorss the chamber - hence the gel

6. DNA fragments move through the field towards the (+) charged electrode, smallest dna move the fastest - travel the furthest

7. dna marker consisting of DNA fragments of known lengths is run next to the experimental mixtures to provide a size reference

8. dna is colored - flouresces under UV light

What is a Probe and what is hybridization

• probe is a short, single-stranded piece of DNA (or RNA) that is designed to match a specific DNA sequence.

  It is labeled with something detectable (radioactive tag, fluorescent dye, etc.) so scientists can see where it binds.

• Hybridization is what happens when:

  👉 The probe binds to its complementary DNA sequence.

Southern Blotting Technique - Hybridization with a suitable probe

• pH of electrophoresis gel is made basic - this breaks the hydrogen bonds, hence separating the two DNA strands

• a thin sheet of nitrocellulose is placed on the gel and covered with absorbent paper - absorbent paper draws up the buffer through gel and nitrocellulose

• some DNA fragments are also drawn up via capillary action without changing their relative position

• dna fragments are immobilized on the sheet via heating

• a radioactive probe which is complimentary to the DNA sequence required is poured over the sheet

• unbound probe molecules are washed away

• spots of radioactivity revealed by autoradiography

• gel region containing DNA fragment can be cut out and separated from gel

Define and give an example of a restriction endonuclease

1. an enzyme which cut DNA at specific nucleotide suequences called restriction sides, and most cut palindromic sequences of DNA

2. and example is EcoRI which cuts GAATTC - named after bact, strain and first restriction enzyme to be isolated from this bact

cloning vectors

• carriers for dna

• phages and plasmids

• phages are usually used by larger pieces of DNA

Stage 2 - Inserting foreign gene in a vector using RE and ligases

1. bacterial cells are broken open and plasmids are collected by centrifugation

2. restriction enzyme has been used to isolate donor DNA, the plasmid DNA is treated with the same restriction enzyme

3. the two molecules of DNA are attracted together due to hydrogen bonding

4. DNA ligase synthesizes the sugar-phosphate backbone once more

5. Recombinant DNA is inserted into vector

6. If a phage vector is used, the part of the DNA which causes disease is replaced with the donor DNA

Why is it important that Plasmid or Phage DNA vectors should only have a single recognition site

• this is so it is only cut in one area

• multiple restriction sites can lead to the loss of important genes such as antibiotic resistance etc

Stage 3 - Introducing recombinant DNA into host cells

1. plasmid or phage vector is introduced into a bacterial cell which will allow the vector to multiply

2. E.coli is usually used as it rapidly multiplies and a great deal is known about its genetic makeup

   1. a specific mutant form of e.coli is used which is engineered to only survive in the lab - so if it escapes with with foreign genes - it wont survive to infect humans

3. plasmid is added to a flask of e.coli

4. Ca2+ is used to neutralize the charges on the membrane so it wont repel the plasmid

5. Cells + vector are incubated at 0C to lower the KE of ca2+ and phospholipids to allow the two to bind tg

6. Temperature is rapidly raised to 40C - this causes heat shock causing temporary perforations in the membrane which allows the plasmid to enter - transformation

7. e.coli containing plasmids are then usually transferred and grown onto nutrient agar in petri dishes

8. recombinant phage dna is introduced via infecting the bacteria growing on an agar plate, and recombinant dna integrates itself w host dna and is replicated with the host cell dna and passed down to daughter cell

Stage 4 - Screening and Selection

1. Not all bacteria take up plasmids - using plasmids with an antibiotic resistance gene and growing them on a medium containing that antibiotic, any bacteria that survive must have taken up the plasmid

2. Not all plasmid would have interacted with donor dna - using plasmids which have the gene to produce enzyme b-galactosidase, this enzyme breaks down x-gal into a blue substance. IF foreign dna is inserted at restriction site in the gene - gene will not work. So is bacteria growing on antibiotic are then grown on x-gal medium and produce colorless colonies - took up donor dna and can be isolated for further cloning

   1. these are called marker genes and help the researcher identify which bacterium has taken up donor dna

Replica Plating

• replica plate is made by layering a piece of filter paper over the colonies and some of the bacteria stick to the filter paper

• the filter paper is then treated with a basic solution to break cells open and denature DNA which sticks to filter paper

• dna is fixed through backing/exposure to UV light

• hybridization a radioactive probe containing complimentary bases to the specific gene is added

• film sensitive to radioactive emissions is used to identify where radioactivity is (will appear as dark spots)

• film is aligned w og plates and colony of interest can be found

• bacterial colonies with plasmid with desired gene have been identified and grown in a culture medium producing many copies of the DNA segment

• new gene may be active and used to make useful protein such as human insulin - not normally made in that cell

• biotech firms use fermenters (large tanks which grow many kg of bacteria)

PCR TEST - process

• polymerase chain reaction - fast and cheap way to make more copies of a selected gene

• requires a heat resistant DNA polymerase molecule (Taq polymerase), primers which serve as a starting point for DNA Polymerase and 4 types of dNTPS (dATP, dGTP etc)

1. Denaturation; 90C is used which separates the two DNA strands by breaking h-bonds

2. Annealing; temperatures are lowered to about 55C allowing specific primers to bind to sequence which needs to be replicated

3. Extension; temperatures are raised to 70 allowing DNA polymerase to construct the new DNA segment

4. two strands are once again seperated by denaturation and the cycle repeats over and over

5. this can generate billions of copies of DNA very quickly

Applications of PCR

1. Genetic Testing; several copies of dna are made to analyse for genetic diseases

2. Pathology; test for HIV or Tuberculosis - make enough copies to be detected, if there’s no virus, hence no dna, primer have nothing to bind to and rcn cant take place

3. Forensics - make multiple copies of DNA found at crime scene, which can then be analyzed through DNA fingerprinting techniques

4. Evolutionary Biology - several copies of fossilized DNA can be made so their base sequence is determined and evolutionary relationships among species can be found

Getting new genes into plants - what do we use

• agrobacterium is used as this contains a Ti plasmid which induces tumor growth in plants and causes crown-gall disease

• contains T-DNA which leaves the bacterium and enters the plant cell and binds to plant DNA and brings about this unregulated growth

• replacing the ‘harmful’ part of the T-DNA with a new gene without affecting ability of T-DNA to enter a plant cell and transform it

Process of using Agrobacterium

• agrobacterium cells are cultured

• Ti plasmids are seperated by centrifugation

• Ti plasmids are opened with restriction enzmyes

• mixed with copies of donor gene

• recombinant Ti plasmid is inserted into bacteria via heat shock

• agrobacterium cells which took up the Ti cell - identified using marker genes

• agrobacterium cells are used to infect plant cell

• transformed plant cells are identified and cultured

Using Gene Guns

• required DNA is coated on the surface of a 1micrometer diameter gold/tungsten bead

• these gold beads are shot out at high velocities at target cells or tissues

• a single new plant cell will produce an entire new organism

• giving rise to a transgenic plant

Microinjection

• fertility drug is given to female to stimulate production of extra ova

• fertilization is allowed to occur - fertilized ova are collected

• donor dna is injected directly into one of the pronuclei using a micropipette

• in some not all cases DNA integrates into one or more of the chromosomes

• two pronuclei fuse and egg → zygote

• fertilized ova is trasnferred to one or more foster mothers and offspring are screened for presence of gene

Use of Stem Cells

• stem cells are extracted from an organism created invitro

• using a microinjection a gene is introduced

• the stem cells are screened before putting them back into the embryo

• embryo is then placed into a foster mother

• this will result in a chimera as the organism will have some cells which are normal and those which are transformed

• as a result their gametes may carry the new gene - lead to a fully transgenic next generation

Heat Shock

• ca2+ at 0

• temperature is suddenly raised to 40

• heat shock causes some of the cells to take up the vector

Electroporation

• electric current

• causes small perforations in the cell membrane

• making it more permeable to DNA

Virus Vectors

• this is when the harmful DNA within a vector is replaced preventing them from causing disease

• this is used for somatic cells

Liposomes

• liposomes are small vesicles surrounded with a phospholipid bilayer

• the DNA would be contained within the liposome

• liposome would be able to fuse with the cell membrane of a cell, and hence DNA would enter the cell and bind with nucleus

Production of Human Insulin by Genetically Modified Microorganisms

• insulin used to be obtained from pigs - however due to slight differences in amino acid compositions and impurities some patients were allergic to it and showed damaging effect

• gene for human insulin was inserted into bacterium E.coli and bacterium was grown in fermenters to produce large quantities

• promoter genes- activated, turn a gene on.

• we replace the B-galactosidase gene, and the Lac promoter will turn on the gene in the presence of lactose

• later yeast was used - capable of post-transcriptional and post-translational modification as its a eukaryote

Pharming

• process of producing rare and expensive pharmaceutical proteins for use in medicine using transgenic animals

  • large quantities of proteins can be produced quickly

  • no risk of infection with blood-borne diseases

  • cant be produced by bact bcs they lack post-translational modification machinery

Human Antithrombin in goats - ATryn - WHY DO THEY NEED IT

• antithrombin deficiency - leads to blood clotting and organ failure and death

• so patients take anticoagulatns

• however before a big surgery they must stop taking such medication as this will increase the risk of bleeding complications

• they are instead given atryn intravenously

Production of ATryn

1. human antithrombin gene is placed in goat zygotes via microinjection

2. placed into foster mother

3. transgenic organism produced

4. to ensure that only the mammary glands produce ATryn, a goat specific mammary gland promoter is attached - so that the gene to make antithrombin is only switched on when in the mammary gland

5. protein is collected and purified from the milk

Why will not all offspring produce antithrombin

1. gene wont attach to chromosome - never replicated/expressed

2. may cause an insertion mutation leading to morphological abnormalities in organism - or failure to develop

3. some of the transgenic organism will be male - wont produce milk

Drawbacks to Pharming and ATryn

1. cannot be used if patient is allergic to goat milk proteins

2. very expensive

What is Gene Therapy + 2 Types of Gene Therapy

• replacement of faulty genes with normal genes

1. Germ-Line-Therapy

2. Somatic Cell Therapy

Germ Line Therapy

• microinjection of genes into a fertilized ova with a genetic disorder

• ova are re-implanted into mother

• all offspring are normal because all derived from the corrected cell

• considered unethical because the gene is inheritable and may have unpredictable harmful effects later on

• but some affected parents would like to eradicate their disease from their children and great-grand children

Somatic Cell Therapy

• fault gene is fixed in normal body cells

• only treated person is affected - changes are not inheritable

  • scientists isolate the normal gene and clone it

  • use a vector - either a virus or liposome to introduce gene into nucleus of cell

• vector used are usually viruses which are agents of serious human disease - while the harmful DNA is eradicated there is always an element of doubt

• if gene inserts itself incorrectly - activate a harmful gene (tumor inducing) or deactivate an essential gene

How does somatic Cell therapy work in Sickle Cell Anemia

1. doctors remove some of the patients bone marrow stem cells (MAKE BLOOD CELLS)

2. insert normal haemoglobin gene

3. corrected cells are placed into the body

4. body starts making healthy red blood cells

Problems with Gene therapy

• only available for the very wealthy

• could give rise to designer babies

• over or under expression of gene could have awful consequences - gene must be switched on and off properly

Treatment of X-linked SCID

• Severe Combined Immunodeficiency Disorder

• impaired immune response as they lack B-cell and T-Cell functioning

• men get it more - as it is x-linked

• it involves the interleukin 2 receptor gamma gene IL2RG - located on X-chromosome

• bone marrow transplants do not always work

• GENE THERAPY INVOLVED

  • Isolation of blood stem cells from the bone marrow of each infant

  • insertion of normal gene for interleukin receptor into blood stem cells (blood stem cells r what give rise to immune cells)

  • returning treated cells

  • viral vector caused insertion of DNA next to an oncogene for leukemia, when activated caused childhood leukemia

  • 4/10 ended up developing leukemia - major set back

Applications of Gene Technology in Agriculture

• can add new desired genes directly instead of selectively breeding which is a slower process

• increasing yield

• improve food quality

• resistance to pest, herbicides and disease

• tolerance to env stress

• increase rate of growth

• allows beneficial characteristics to remain - just add the new desired gene - this is more difficult w natural breeding methods

Advantages and Disadvantages of Gene Technology in Agriculture

→ Advantages

• increasing yield - demand for agricultural land will decrease

• resistance to pest - dependence of chemical pesticides will decrease

→ Disadvantages

• if it gets into the wild - it will have a competitive advantage over the other plants and wipe out those other species - massive loss of genetic diversity

  • This concern may be overstated, since traits like disease resistance already exist in wild populations and only provide an advantage under specific environmental conditions.

• relying on one crop plant - if a new strain of fungus or pest comes out it could wipe out a major crop

• big companies will only invest money in genetic engineering technology if they get a patent, however this will devastate farmers as according to the patent the seeds/offspring of a genetically engineered plant is the property of the PATENT HOLDER not the farmer

  • farmer will have to pay royalties to company each time it produces offspring

Production of Pest Resistant Crops - Bt maize

• pests lead to major crop losses

• Bacillus thuringiensis produces a powerful toxin - bt toxin which can kill insect larvae but is relatively harmful to humans

  • damages their gut epithelium - cant absorb nutrients - starve to death

• since toxin is a protein it breaks down rapidly - no env harmful residue

• instead of spraying the bacteria on - which would be very expensive as the bacteria die quickly - regular spraying.

• the gene for Bt toxin is isolated and directly injected into plants giving them permanent protection

• ANY LARVAE WHICH EATS IT WOULD DIE

Environmental Implications of Bt maize

• toxic effects on pollinators and insects which are important

• long term harm to soil ecosystems - bt toxin persists in the soil where it is biologically active harming earthworms and nemotodes

• harmful to aquatic life - if it enters streams etc, it can be toxic to aquatic life

• increased pest resistance - if widespread resistance where to occur to Bt toxin, we would need to apply stronger more toxic pesticides

• increases in other pests of bt maize - Bt maize kills its target pests, which can let other, previously minor pests increase in number

Production of Herbicide Resistant Crops

• weeds reduce crop yield by 10% as they compete with plants for nutrients and water

• herbicides - kill weeds as they attract electrons from the ETC in thylakoid membranes - this disrupts the flow of electrons and thus ATP production

• genes which give plants resistance to herbicides have been introduced

  • either an enzyme to break down herbicide

  • or a production of proteins in etc to have higher electron affinity then herbicide

→ this may spread to the weeds making them resistant

→ may encourage more herbicide use

Genetic Fingerprinting and DNA profiling - How does it work

• method used to identify a person using their DNA

• 95% of DNA is non-coding → a lot of this non-coding DNA contains repeating sequences

• Satellites - large repeating sequences which dont vary from person to person

• Minisatellites - smaller repeating sequences - where the number of repeats vary from person to person

• called VNTRS - variable number tandem repeats

What are minisatellites

• short sequence repeated many times - number of repeats varies from person to person

• variable number tandem repeats

• eg ATGC ATGC ATGC ATGC - same sequence but repeated numerous times. person A would have 5 repeats while person B would have 9 repeats

• by analyzing number of repeats u can match DNA to person

Procedure of DNA profiling

1. DNA is extracted is treated with a restriction enzyme which cuts on both ends of the minisatellite - leaving their variable lengths unaltered

   1. because different people have different numbers of repeats, DNA fragments r different length

2. Agarose gel electrophoresis - seperates fragments according to size

3. Southern Blotting transfers DNA to a nitrocellulose filter

4. radioactive DNA probe with a base sequence which is complementary to part of the minisatellite repeat sequence is then hybridized to the DNA

5. location of probe is found by autoradiography

• PCR can be used to amplify amount of DNA

• pattern for an individual is unique and therefore known as a fingerprint

Types of probes

• Multi-locus probes

  • bind to many minisatellite regions

  • produce many bands

  • pattern looks complex and is very unique

  • works only with good quality DNA - isnt usually available to forensic scientists

• Single-Locus Probe

  • identifies and binds to ONE minisatellite region

  • produces only 2 bands - one from each parents

  • if two probes are used - 4 bands etc

  • used when DNA is degraded or v small amount is available

each minsatellite is ONE band, and you will have 2 bands bcs 1 from mom and one from dad

Applications

• crimes - more to prove innocence rather then guilt bcs two people could theoretically have the same patterns + Degraded DNA may affect results

• settle paternity disputes.