microbiology

• Microorganisms can reproduce through various methods, including binary fission, budding, and spore formation.
• Budding occurs when yeast or fungi produce small outgrowths called buds that eventually detach from the parent cell to form new organisms.
• Binary Fission is the most common method of reproduction among bacteria, where one cell divides into two identical daughter cells.
• Insulin is a protein and it is made up of two amino acids chains held together by disulphide bonds
• A chain: 21 aa

  B chain : 30 aa

  sequence is highly conserved among mammals: biologically active within species
• HB at the c terminus of B chain can lead to the formation of diners and hexamers in the presence of since ions
• insulin is synthesised by B cells produced by the islets of langerhans
• it is involved in regulation of carbohydrates and fat metabolism
• diabetes mellitus is a metabolic disease in which the body cannot control its insulin synthesis
• patients can be treated with injection of external insulin
• natural synthesis of insulin:

  it was initially synthesised as one 86 aa which is preproinsulin
• processed in the cells to make pro insulin
• undergoes subsequent modification to give insulin
• excreted from secretory granules in the cell
• insulin biosynthesis:

  the prep to insulin polypeptide chain is cleaved to form proinsulin in the ER
• 3 parts: A,B,C polypeptide chain

  in the ER, the disulphide bridges form to create a 3D tertiary structure
• the central c part is cleaved to make 3 chains
• A and B are bound by disulphide bridges
• the 3 chains are transported via the golgi body and packaged into the secretory granules which are accumulated in the cytoplasm
• recombinant human insulin was discovered in 1982 to treat type 1 diabetes
• use e coli as it is easier and faster to replicate
• insulin genes clone e.coli to generate recombinant cells that are capable of generating insulin
• e. coli requires very little nutrients to synthesise macromolecules of its cell and support cell division
• this condition can be created easily by simple liquid mediums
• recombinant human insulin was developed by genentech and commercialised by eli lilley

  insulin is produced by recombinant e. coli
• recombinant insulin gene cloning

  plasmid vector and dna fragment cloned
• enzymatically insert dna into the plasmid
• mix e. coli with plasmids with the presence of cacl2 culture on the nutrient agar that contains ampicillin
• transformed e. coli survives
• cells that do not take up plasmid cells will die on ampicillin plate
• individual plasmid replication then cell multiplication then colonies of cells will all have the same amount of recombinant plasmids
• plasmids can be used as

  expression vectors

  cloning vectors
• plasmid vectors are

  naturally occurring pieces of dna found in bacteria

  extrachromosomal

  dna which are covalently closed

  self replicating
• plasmids used in rDNA technilogy are genetically engineered constructs
• selection:

  ampicillin ensures all cells which grow on the plate carry a plasmid
• IPTG is an inducer than prevents the negative regulation of the lacZ gene activating the production of B galactosidase if functional
• X-Gal generates a blue chromogenic product in the presence the enzyme B galactosidase
• functional lacZ- Blue colonies

  cells and plasmids but not DNA insert
• disrupted lacZ- white colonies

  cells and plasmids and dna insert
• recombinant insulin gene expression

  to achieve this:

  there must be

  regulatory gene

  promoter

  ribosome binding site

  translational start codon

  translational terminator

  transcriptional terminator