Microorganisms and Biotechnology Uses

• Biotechnology is technologies that involve the use of living organisms, or their products, to benefit humans

Microorganisms and Biotechnology

• Microorganisms are used to benefit humans in various ways
• Molecular genetic tools are important in influencing and improving the use of microorganisms

Common Use of Microorganisms

• Production of medicines examples: Antibiotics and vitamins
• Production of medicines examples: Synthesis of human insulin in recombinant E. coli
• Food fermentation examples: Cheese, yogurt, vinegar, wine, and beer
• Biological control examples: Control of plant diseases, insect pests, and weeds
• Biological control examples: Symbiotic nitrogen fixation
• Biological control examples: Prevention of frost formation
• Bioremediation includes the cleanup of environmental pollutants such as petroleum hydrocarbons and synthetics that are difficult to degrade

Human Insulin Production in Bacteria

• Insulin is a hormone
• Insulin is made of two polypeptide chains called the A and B chains
• Insulin regulates several physiological processes, particularly the uptake of glucose into fat and muscle cells
• Insulin is produced by the β cells of the pancreas
• People with insulin-dependent diabetes have a defect in their β cells and cannot synthesize enough insulin

Gene Knockouts

• Gene Knockouts (KO) are produced to understand gene function and human disease
• Gene knockouts study how the loss of normal gene function affects the organism
• The genomic DNA of a cell or a model organism is perturbed, permanently preventing expression of a specific gene
• A specific gene is always inactivated

RNA Interference (RNAi)

• RNA interference (RNAi) is when double-stranded RNA (dsRNA) results in sequence-specific gene silencing
• mRNA is targeted for degradation using RNAi
• RNAi allows the process of knocking down the expression of genes post transcriptionally

Gene Knockout vs. Knockdown

• Gene knockout is where there is no gene, mRNA, or protein
• Gene knockdown is where there is a gene present where the mRNA is made but gets degraded

Gene Cloning

• Gene cloning isolates and makes many copies of a gene

Cloning Experiments

• Cloning experiments involve two kinds of DNA molecules
• Chromosomal DNA serves as the source of the DNA segment of interest
• Vector (Plasmid) DNA serves as the carrier for the DNA segment that is to be cloned
• Vector (Plasmid) DNA can replicate independently of the host chromosomal DNA

Cloning Experiments

• First, obtain cellular tissue from the organism of interest
• Second, break open the cells
• Finally, extract and purify DNA

Vector (Plasmid) DNA in Cloning

• The cell that harbors the vector is called the host cell
• When a vector is replicated inside a host cell, the DNA that it carries is also replicated
• The sequence of the origin of replication determines whether a vector can replicate in a particular host cell

Plasmid/Vector Features for Bacteria

• The ori is the bacterial origin of replication, so it can replicate in bacteria
• A bacteria construct contains a gene conferring resistance to an antibiotic such as ampicillin

Plasmid/Vector Features for Yeast

• Plasmids are replicated in yeast by an autonomously replicating sequence (ARSH4)
• Plasmids are maintained in yeast cells with a centromere (CEN6)
• A plasmid is selected for by growing leu2 defective strains containing the plasmid in a medium lacking leucine (-Leu)
• A shuttle vector has the ability to replicate in both bacteria and yeast

Restriction Endonucleases or Restriction Enzymes

• Restriction enzymes are made naturally by many species of bacteria
• Restriction enzymes protect bacterial cells from invasion by foreign DNA (bacteriophages)
• Recognized sequences are typically palindromic
• A sequence is palindromic when it is identical when read in the opposite direction in the complementary strand
• An EcoRI recognition sequence is 5'-GAATTC-3' and 3'-CTTAAG-5'

Gene Cloning

• The action of a restriction enzyme creates recombinant DNA

Restriction Endonucleases in Cloning

• Restriction endonucleases are used in cloning
• The endonucleases generate blunt or sticky ends

Gene Cloning Steps

• 1. Human chromosomal DNA is cut to generate thousands of fragments
• 2. Only one fragment inserts into a vector
• 3. This becomes a recombinant vector

Competent Cells & Transformation of gene

• Competent cells are cells that are able to take up DNA
• Transformation includes uptake of plasmid vectors by a bacterial cell
• Antibiotic resistance to the host cell
• Identifies cells that have taken up the vector

Recombinant Plasmids

• Re-circularized vectors form blue colonies (not useful)
• Hybrid vectors form white colonies (contains insert)
• lacZ encodes β-galactosidase
• Bacteria that have picked up the cloned gene can be identified

Identifying Recombinant Plasmids

• β-galactosidase is an enzyme that hydrolyzes lactose
• β-galactosidase is encoded by the LacZ gene
• Active β-galactosidase cleaves X-gal to form a bright blue insoluble pigment

Cloning Amplifies Sequence Copies

• The net result of gene cloning is to produce numerous copies of a gene
• A single bacterial cell usually takes up a single copy of a vector
• The vector gets replicated by the host cell many times and there are 25-50 plasmids in each cell
• The bacterial cell divides approximately every 20 minutes, generating a population of many millions of cells overnight

Polymerase Chain Reaction

• Polymerase chain reaction (PCR) copies a specific sequence of DNA
• PCR can copy DNA without the aid of vectors and host cells
• Enough must be known about the gene of interest to have the sequence of 2 short primers
• Repeated cycles will produce thousands of copies of the region of interest, even from a large background of other sequences

PCR Cycle

• The steps of a PCR cycle are:
• Denaturation separates DNA strands with high temperature
• Primer annealing is done with a lower temperature, which allows primers to bind to template DNA.
• Primer extension is done by incubating at a slightly higher temperature, encouraging DNA synthesis

Material Needed for PCR

• Template DNA contains the region that needs to be amplified
• Oligonucleotide primers are complementary to sequences at the ends of the DNA fragment to be amplified and are synthetic and about 15-20 nucleotides long
• Deoxynucleoside triphosphates (dNTPs) provide the precursors for DNA synthesis
• DNA polymerase is the Taq Polymerase: DNA polymerase isolated from the bacterium Thermus aquaticus
• The thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases

Repeated Cycles in PCR

• PCR is carried out in a thermocycler, and all the ingredients are placed in one tube
• The sequential process of denaturing-annealing-synthesis is repeated for many cycles
• There are usually 20 to 30 cycles of replication
• After 20 cycles, a target DNA sequence will increase 220-fold (~1 million-fold)
• After 30 cycles, a target DNA sequence will increase 230-fold (~ 1 billion-fold)

PCR Uses

• PCR allows a specific DNA segment can be amplified from a complex mixture of other sequences
• PCR works for one gene out of an entire genome
• Requires prior knowledge about the sequence of the template DNA
• It's required to construct the synthetic primers

Site-Directed Mutagenesis

• Analysis of mutations can provide important information about normal genetic processes
• Site-directed mutagenesis alters the sequence of cloned genes
• The DNA sequence is altered in a specific way
• The site-directed mutant can then be introduced into a living organism
• This will allow the researchers to see how the mutation affects expression of a gene, function of a protein, and phenotype of an organism
• The vector and insert are denatured into single-stranded DNA prior to the experiment
• A mismatch is created and the mutant can be identified by DNA sequencing and used for further studies

Gel Electrophoresis

• Gel Electrophoresis separates DNA fragments based on size
• Needed: a gel, an electrical field and a power supply
• Anode (negative end) and cathode (a positive end)
• A buffer conducts electricity (contains ions) and completely covers the gel
• The Green buffer makes the sample visible.
• DNA has to be loaded with a pipette into the wells.
• Negatively charged DNA, moves from the negative end to the positive end
• DNA can be separated based on topology (shape)
• Linearized DNA is between supercoiled and nicked circles in terms of speed
• Fastest is Supercoiled while Slowest is Nicked circles
• Ethidium Bromide allows visualisation of DNA

Electrophoretic Mobility Shift Assay (EMSA)

• The binding of a protein to a labeled fragment of DNA slows movement through a gel.
• Left lane: DNA without protein bound
• Right lane: DNA with protein bound (at a higher molecular mass)

Western blotting

• Primary antibody is added that binds to the protein of interest
• Secondary antibody is added that binds to the primary antibody
• Secondary antibody is also attached to an enzyme called alkaline phosphatase
• When the colorless compound XP (5-bromo-4-chloro-3- indolyl phosphate) is added, alkaline phosphatase converts it to a dark purple dye
• Lane 1: β-globin polypeptide present
• Lane 2: proteins from brain cells
• Lane 3: proteins from intestinal cells
• Brain and intestinal cells do not synthesize β globin

Northern Blotting

• Northern Blotting Is Used to Detect a Specific RNA
• mRNA is isolated from a sample of cells.
• mRNA is separated by gel electrophoresis
• The separated bands are blotted to a nylon membrane and then placed in a solution containing a labeled DNA probe
• The DNA probe is complementary to tropomyosin mRNA
• mRNA molecules that are complementary to the probe appear as labeled bands (shown in purple)