Prokaryotes + Biotechnology

Prokaryotes

Archaea: single celled prokaryotes that are distinct from bacteria, they are often extremophiles (live in extreme environments)

Bacteria: single celled prokaryotes that are distinct from archaea

Similarities with Eukaryotes

Ribosomes (70s only)

Cytoplasm

Plasma membrane (no cholesterol)

DNA/RNA

Flagella

Cytoskeleton

Cell wall (peptidoglycan)

Glycolysis

Differences to Eukaryotes

No membrane bound organelles

Binary fission

Circular DNA

0.2-2um diameter (roughly speaking)

Nucleoid proteins

Capsule (polysaccharide and or protein)

H+ Pump

ETC occurs in plasma membrane

Pili

Sex pili: allow chromosomes to transfer between bacteria

Requires F-factor to produce sex pili

F-factor: fertility factor

F+ bacteria: can transmit the F-factor via conjugation to F-bacteria, making it F+

Locomotive pili: involved with motion of the bacteria

Fimbriae: involved in adhesion/cohesion and sometimes motion

Gram staining

Gram positive bacteria:

Thick cell wall

Gram stain colour: purple

Gram negative bacteria:

Thin cell wall

Gram stain colour: pink

Outer membrane of lipopolysaccharides (often toxic)

Harder for body to kill, more resistant to antibiotics

Plasmids

circular molecules of extrachromosomaL DNA present in bacteria

Plasmids and Restriction Enzymes

In biotechnology, plasmids are used as vectors (vehicle for transferring genetic information)

Restriction enzymes: are nucleases that can cut DNA at specific sequences, used by bacteria to destroy viruses, used by humans to cut out specific genes

Restriction enzymes recognise palindromic sequences, they cut out single strands with sticky ends that re-anneal to complementary strands

DNA Cloning, Recombinant DNA Technology

Recombinant DNA Technology: brings together DNA from various sources, creating new sequences of DNA

We insert the gene into a vector (plasmid) so that it can be cloned as the cell reproduces

Steps in DNA Cloning:

1. Identify target DNA

2. Cleave out w restriction enzyme (endonuclease)

3. Identify cleavage site on plasmid

4. Restriction enzyme (endonuclease) cleaves site

5. Annealing, attachment of specified genes with ligase

6. Plasmid inserted into host bacteria

7. Bacteria replicates gene

Polymerase Chain Rxn

The aim of PCR is to copy DNA

1. Identify desired gene

2. Denaturation: heat applied to separate the DNA strands

3. Annealing: cooling of strand and addition of primers

4. Synthesis: heat resistant DNA Polymerase (Taq Polymerase) carries out DNA replication

5. Repeat, each repeat doubles the DNA (1 to 2 to 4 to 8 to…)

6. number of copies = 2^n

Ingredients

Target DNA portion

Taq polymerase

DNA nucleotides (A,T,C,G)

Primers

Gel Electrophoresis

Aim is to separate and compare various strands of DNA, which can be done due to DNA having a negatively charged backbone

A gel electrophoresis machine will have a positive terminal where the DNA will want to travel to, but as the DNA must travel through a gel the smaller pieces will get further

Steps:

1. Restriction enzymes are used to cut out DNA for comparison. Different DNA sample types will have different sizes.

2. DNA is placed into electrophoresis machine

3. The machine contains agarose gel and a current

4. One end contains wells where DNA is placed

5. DNA will be sorted, smallest pieces closest to the positive end, larger pieces further away

   Southern Blotting: DNA is separated in gel electrophoresis

   Northern Blotting: RNA is separated in gel electrophoresis (genes that will be expressed are transcribed to RNA)

   Western Blotting: Proteins are separated in gel electrophoresis

Gene Therapy

Medical approach that involves altering genes inside a person’s cells to treat or prevent disease, can be performed in many ways:

1. Replacing a mutated gene that causes disease w a healthy copy of the gene.

2. Inactivating a mutated gene that is functioning improperly

3. Introducing a new gene into the body to help fight a disease

   Two main types of gene therapy:

   1. Somatic gene therapy: most common type, changes cannot be inherited by offspring

   2. Germline gene therapy: type targets reproductive cells, can be passed on to offspring

Gene Therapy Techniques

Needles: very thin needles can insert DNA into host cells

Electroporation: using electricity to increase membrane permeability

Retroviruses: insert their genome into host cells

Retrovirus process for gene therapy

1. Identify target gene, i.e. missing/mutated gene, the correct gene can be prepared for insertion

2. Select appropriate vector e.g. retrovirus

3. Prepare vector, viral genome is removed, RNA version of gene is inserted

4. Vector delivery: retrovirus is inserted into the cells (often stem cells) of patient, in vivo= directly into patient, ex vivo= cells are extracted, treated, cultivated, then returned to patient

5. Integration of gene: retrovirus integrates RNA into cell, patient now has correct gene, the more cells have the gene the more alleviated the patient.