Biotechnology and Gene Technologies- F215 Module 2

A clone is a whole organism or cells that carry identical genetic material because they are derived from the same original DNA.

Examples of natural clones: identical twins, plants that are reproduced asexually, bacteria when they asexually reproduce through binary fission.

Asexual Reproduction in English Elm: Healthy elm grows a root sucker so that when the main elm shows symptoms of Dutch elm disease, the main trunk dies leaving the root suckers to grow.

Advantages of Asexual reproduction:

• It is quick, allowing organisms to take advantage of resources in the environment.
• It can be completed if sexual reproduction fails.
• All offspring have the genetic information to survive in that specific environment.

However, there is no genetic variety, so if there is a change in the environment, all offspring are susceptible.

Farmers arificially propagate plants by these two main methods:

• Taking cuttings-section of stem is cut between the nodes (leaf joints). This cutting is treated with plant hormones, then planted.
• Grafting- a shoot section of a plant is joined to an already growing root and stem (a rootstock). 

Micropropagation by tissue culture:

- This is the separation of cells of any tissue type and their growth in a nutrient medium.

1. An explant (small piece of tissue, often from shoot) is taken from the plant.

2. This is placed on a nutrient growth medium. Cells divide and form a mass of cells called a callus.

3. These callus cells are removed and placed on a growing medium to grow into plants.

Cloned animal- one that has been produced using the same genetic information as another animal. They have the same genotype.

There are two ways of artificially cloning animals:

Splitting embryos: Cells from a developing embryo are separated out, and each develops to form a genetically identical 'twin'. 

Nuclear Transfer: A differentiated adult cell's nucleus is placed in an egg cell which has had its own nuclus removed- electrofusion used to reconstruct the cell.

Non-reproductive cloning: This is using cloned cells to generate cells, tissues and organs to replaced damaged ones. 

Advantages: -Being genetically identical tissue, there will not be rejection.

-It avoids the wait for organ donors.

-Cloned cells can be used to generate any type of cell needed.

Biotechnology: Technology based on biology and involves the exploitation of living organisms or biological processes, to improve agriculture, animal husbandry, food science,medicine and industry.

Many biotechnological processes use microorganisms:

• They grow rapidly in favourable conditions, like low temperatures, can be grown anywhere.
• The often produceproteins/chemicals that can be harvested, or can be genetically engineered to do so.
• Can be grown using nutrient materials that would be toxic to humans.

The Growth Curve:

Lag Phase: Organisms adjusting to surroundings; Cells active but not reproducing so population is constant.

Log Phase: Population size doubles each generation as all reproduce.

Stationary Phase: Nutrient levels decrease and waste products build up. Reaches carrying capacity. 

Batch Culture: the microorganism starter population is mixed with a specific quantity of nutriet solution, then allowed to grow for a fixed period with no further nutrient added. At the end the products are removed and the fermentation tank is emptied.

Continuous Culture: nutrients are added to the fermentation tank at regular intervals. 

Asepsis: any measurement taken to ensure contamination does not occur

Any unwanted microorganisms that could cause contamination are called contaminants.

Contaminants cause the following problems:

- Compete with the culture for nutrients and space

- Reduce the yield of useful products from the bacterial culture

- May cause spoilage of the product

- May produce toxic chemicals

- May destroy the culture microorganism and their products

Downstream processing- the extraction of enzymes from the fermentation mixture. Used to purify the product.

Immobilising Enzymes:

Advantages ------------------------------------------Disadvantages

Enzymes are not present with products--------- Immobilisation requires additional time and equip

Enzymes are available for reuse-------------- They can be less active- don't mix freely w/ subtrate

They are more stable- enzymes protected------------------ Contamination is costly

Methods for Immobilisation of Enzymes:

1. Adsorption- mixed with porous immobilising support- binds enzymes non- covalently.

2. Covalent bonding- covalently bonded to an immobilising support by a cross-linking agent.

3. Entrapment- Enzymes trapped in a gel bead/network of fibres. 

4. Membrane Separation- Enzymes physically separated from substrate mixture. 

1. Genomes are mapped- to identify which part of the genome they have come from. 

2. Samples of genome are sheared- they are mechanically broken to form smaller sections of around 100,000 base pairs. These are placed into bacterial artificial chromosomes (BAC) and transferred to E.coli cells, where they grow and replicate- next slide.

3. In order to sequence a BAC section, restriction enzymes are used to cut BAC into smaller fragments.

- These fragments are then separated using electrophoresis which separates them in size order:

A gel plate with agarose is used, covered in buffer solution. Electrodes are attached to each end and a current is passed through the gel. DNA is negatively charged, so moves towards positive end. Shorter lengths move faster as they aren't slowed down as quickly. Nylon then placed over to blot the DNA fragments off and are then analysed further.

- Analysis further could involve DNA probing, using a probe with a complementary DNA section. It ihas a radioactive marker so it can be seen- binding may occur  to strand(annealing).

- Each fragment is sequenced. Computer programmes then compare overlapping sections in order to reassemble the BAC segment.

Polymerase Chain reaction:

This is artificial DNA replication. Samples of DNA can be amplified to generate more material. 

PCR is not identical to DNA replication:

• It can only replicate short strands of DNA
• Primer must be added to trigger the process: short single stranded DNA - bind to DNA sample
• A cycle of heating and cooling is used to separate and bind strands, usually done by helicase.

1. DNA mixed with DNA nucleotides and DNA polymerase. Heated to 95 degrees, splits the complementary strands. 

2. Primers are added and temperature is reduced to 55 degrees. Primers anneal (hydrogen bonding), forming double stranded sections which DNA polymerase can bind to. 

3. Temperature rises to 72 degrees. The enzyme extends the primer section by adding free nucleotides to the DNA strand present to form a complete double strand of DNA. THIS REPEATS

Automated DNA sequencing is very similar to PCR, however, some nucleotides contain a fluorescent marker which stops any more nucleotides joining the chain.This means fragments of many different sizes are created. These are then run through a machine like in electrophoresis and a laser reads the sequence. 

Recombinant DNA- A section of DNA, often in the form of a plasmid (separate DNA to the main bacterial DNA), which is formed by joining two pieces of DNA from different sources.

Stages of genetic engineering:

1. The required gene is obtained- restriction enzymes cut the DNA forming sticky ends.

2. A copy of the gene is placed (packaged and stabilised) in a vector- the sticky ends join to the sticky ends of the vector. These are joined by ligase.

3. The vector carried the gene to the recipient cell, and the recipient expresses the gene through protein synthesis.

To get the gene into the recipient cell, you can use electroporation, microinjection, viral transfer, or use liposomes. 

Replica Plating- the process of growing bacteria on an agar plate, the transferring this to other plates, containing different promoters or inhibitors to see the genetic properties of this bacteria strain.

Somatic Cell Gene Therapy: Ex-vivo therapy (must be treated outside the body)

- Augmentation: Adding genes by engineering a functioning copy of the gene and insert it.

- Kill specific cells: Eliminating certain populations of cells, or making canceruos cels vulnerable to the Immune system.

Disadvantages: It is not permanent.

- When using viruses as a vector repeatedly means the body becomes immune to it.

- Genetic manipulations only assist the patient involved.

Germline Cell Gene Therapy- Germline cells are cells are cells of an early embryo. If you engineer a zygote or a sperm/egg, these will multiply as the embryo grows, so that the new gene is part of the new being.

Advantages: All cells derived from these germline cells will contain the functioning allele. 

- Genetic manipulations can be passed on to the patient's children.

Xenotransplantation: between different species Allotransplantation: between same species