Microbio Chpt 12 - EXAM 4

Manipulating DNA → PCR + Nucleic Acid Hybridization

Synthetic biology → used with genetic engineering + biotech in industrial, medical, + agricultural app.

• Genetic Engineering → used in vitro techniques to alter genes in the lab

• Heterologous expression → expressing a gene in a different host

PCR + Polymerases → DNA replication in vitro, multiplying segments of target DNA up to a billionfold during amplification (doubling)

• req. DNA polymerase (naturally copies DNA) + artificial oligonucleotide DNA (instead of RNA) primers

• amplifies stretches of a few kbp target from within a larger DNA molecule template

  1. Denature (seperate strands) template DNA via heating + adding 2 DNA oligonucleotide primers in excess

  2. DNA polymerase extends primers using original DNA template

  3. Heat again w. target in twice original amnt, cool, repeat 20-30x, yielding 10⁶ - to 10⁹ - fold increase

• use of thermostable DNA polymerase (Taq + Pfu) → critical bc of hi temps

  • Pfu polymerase has proofreading activity useful when hi accuracy is crucial

  • genes of these enzyme have been cloned into E. Coli for commercial use

PCR applications + RT-PCR

• valuable for cloning or sequencing, comparative + phylogenetic studies

• reverse transcription PCR (RT-PCR)

  • makes DNA from mRNA template

  • detect gene expression or to prod. intron-free eukaryotic gene for bacterial expression

  • use enzyme RT to convert RNA to complementary DNA (cDNA)

Manipulating DNA → Gel Electrophoresis + Nucleic Acid Hybridization

Gel Electrophoresis → employs an agarose gel to separate nucleic acid by size + charge

• nucleic acid move towards + electrode bc of negative charged phosphate grps

• small molecule moves faster than large

• gel sustained via dyes like ethidium bromide (causes nucleic acid to fluoresce)

• DNA fragment can be purified + used for other purposes

Nucleic acid hybridization → complementary base pairing of SS of DNA or RNA from 2 different sources to give a hybird double helix

• widely used in detecting, characterizing, identifying DNA + RNA segments

• segments of known ssDNA used in hybridization are nucleic acid probes

• detection via radioactivity or colored/fluorescent products

Southern Blot → hybridization procedure where DNA is THE TARGET and probe is RNA / DNA

Northern Blot → hybridization procedure where RNA is THE TARGET and probe is RNA / DNA

FISH (Fluorescent In Situ Hybridization) → uses fluorescent probes to target specific nucleic acid sequences in cells

Molecular Cloning

is the movement of a gene from original source to small + manipulable genetic element (vectors)

• results in recombinant DNA (molecule containing DNA from different sources)

• gene can be manipulated

• cloned DNA replicated in appropriate host

• foundation for much of genetic engineering

Overview of Gene Cloning + Restriction Enzymes

• Isolation + fragmentation of source DNA (from polymerase chain rxn)

• Insertion of DNA into cloning vector

  • small, independently replicating genetic element that can carry + replicate cloned DNA

  • designed to allow insertion of foreign DNA

• inserting vector into host

Restriction enzymes w/ different sequence specificities widespread among BACTERIA

• PROTECTS against viral DNA attack

DNA ligase joins / anneals DNA

• if source + vector have compementary sitcky ends from being cut by the same restriction enzyme, DNA ligase can anneal them

• if source DNA is PCR generated, DNA ligase can join amplified DNA to specialized vectors

Recombinant DNA introduced into suitable host for replication

• often yields mixture of recombinant constructs in which ONLY some cells contain the desired gene

Recombineering

this allows for foreign DNA to be inserted into vectors / chromosomes

• specialized strains of E. Coli have been designed for cloning using homologous recombination

• design insert DNA to possess short homologous seq. with target DNA

• activate recombinase enzyme that flips DNA insert into vector

• Originally developed in S. Cerevisiae ; naturally low efficiency in E. Coli

• revolutionized cloning bc compatible restriction enzyme site not needed

Cloning Vectors

are several types of viruses, cosmids (plasmids w. lambda phage cos site), artificial chromosomes

• use dependent on size of fragment to be cloned + host

• contains MULTIPLE cloning sites (MCS) w. lacZ gene encoding lactose-degrading beta- galactosidase

  • DOESNT inactivate lacZ

Use of pUC19

• restriction enzyme w cut site within MCS chosen

• vectors + foreign DNA cut w. the enzyme

• DNA inserted into cute site + ligated with DNA ligase

• X-gal to detect Beta- galactosidase activity

• insertional inactivation of gene within lacZ used to detect cloned DNA

Yeast Artificial chromosomes (YACs)

• linear vectors that replicate like normal chromosomes but have sites for insertion of large DNA fragments

most useful are NON-pathogenic, easy to grow + transform w. engineered DNA

E. Coli, Bacillus Subtilis, + S. Cerevisiae used as hosts

Molecular Methods for Mutagenesis

Conventional mutagens produce mutations at random

• site-directed mutagenesis (in vitro mutagenesis) uses synthetic DNA and cloning techniques to introduce mutations at precise cites

  • Can chAndy 1 or few bases

  • Can insert large DNA segments

Site-Directed Mutagenesis

• Easy to obtain 12-40 base DNA oligonucleotides through chemical synthesis

• PCR can used to obtained gene with specific mutation depending on position of desired change

• ALLOWS ANY BASE PAIR IN A GENE TO BE CHANGED

• Used to change a specific amino acid in active site and compare modified and wild-type enzyme

• Link catalysis, resistance, susceptibility to chemical and physical agents, protein interactions w. Specific amino acids

• Improve properties with genetic engineering

Cassette Mutagenesis and Gene Disruption

• DNA cassettes/cartridges → synthetic fragments that can make more than a few base pair change or replace sections of a gene via cassette mutagenesis

  • Can be synthesized by PCR or direct synthesis

  • Replace sections using restriction sites or recombineering

• Gene disruption inserts cassettes in middle of a gene, disrupting coding sequence

  • Loss of function of gene in which cassettes is inserted is a knockout mutation

  • Cassettes encoding antibiotic resistance are common

Somatotropin and Other Mammalian Proteins

Biotech → use of living organisms for production of valuable products

Somatotropin

• insulin was 1st human protein made commercially via genetic engineering

• Human somatotropin (growth hormone) is a single polypeptide encoded by a single gene

  • Deficiency of this → dwarfism

  • Treat stunt growth via recombinant human somatotropin

  • Recombinant bovine somatotropin (rBST) commonly used in dairy industry ; stimulates milk production in cows

Somatotropin + Other Mammalian Proteins

• Tissue Plasminogen activator (TPA) dissolves blood clots

• Blood clotting factors (Hemophiliacs)

• Human DNAse to treat mucus buildup in cystic fibrosis patients

Transgenic Organisms in Agriculture + Aquaculture

Transgenic organisms (GMOs) : genetically engineered organism that contains a gene (transgene) from another organism

• “Genetically modified” → engineered whether or not they contain foreign DNA

Agrobacterium tumefaciens (plant pathogen)

• contains Ti plasmid that can be used to transfer DNA directly into certain plants

• Responsible for virulence ; encodes genes that mobilize DNA for transfer to plant

• T-DNA → Ti plasmid segments transferred to plant; sequences at ends essential for transfer, foreign DNA for transfer must resides between these ends

The Ti Plasmid and Transgenic Plants

• binary vector → common Ti-vector system for gene transfer to plants + consisting of cloning vectors plus helper plasmid

• vector transformed into E. Coli and conjugated into A. tumefaciens

• helper plasmid (D-Ti) allows for transfer to plant

Herbicide- and Insect-Resistant Plants

• main genetically mod crops are soybeans, corn, cotton, canola

• herbicide rez engineered to protect crop plants (e.g. soybeans) from herbicides that kill weeds

  • Ex: glyphosate → inhibit aromatic amino acid biosyn.

• helps w. rez to dmg by some insects

  • EX: Bt toxin from bacillus thuringiensis → toxic to beetles + fly larvae + mosquitoes

  • insect rez to Bt toxin emerging → rootworms

• Pseudomonas chloraphis produces small insecticidal peptides

Transgenic Fish

• microinjection for delivery + recombination of foreign DNA into fertilized egg genome

  • EX: AquAdvantage salmon

    • reaches market size in 18 months compared to 3 yrs

    • can be grown + harvested quicker than non-GMO salmon

Engineered Vaccines + Therapeutics

Recombinant Vaccines, Vaccinia Virus, + Subunit Vaccines

• vaccines elicit immunity to disease when injected

• can mod a pathogen w. genetic engineering to delete virulence factor + retain those that elicit immune responses → making a less virulent vaccine

• can add genes from pathogenic virus to genome of a harmless carrier virus → making vector vaccine

• Polyvalent vaccine → single vaccine that immunizes against 2 different diseases

Vaccinia virus widely used to prep recombinant vaccines

• cloning req selective marker → thymidine kinase

• genes 1st inserted into E. Coli plasmid containing thymidine kinase (tdk) gene inactivating tdk

• select for viruses whose tdk gene contains insert

• can be engineered to form polyvalent vaccines

• Subunit vaccines → contains only specific protein or proteins from a pathogenic organism

  • EX: coat protein of a virus

  • is popular bc they produce a lot of immune-triggering proteins and can be given in high doses, offering a safer option than weak or dead viruses that might still cause infection

• if glycosylation req. , subunit vaccine is prod in eukaryotic host (yeast)

  • EX: Hep B subunit vaccine

Commensal Bacteria and Therapeutic Delivery

• delivery to target tissues/cells = problematic bc degrade bloodstream or stomach acid

• normal gut flora have been used to prod + release drugs in a host

  • this doesn’t elicit immune response + well tolerated

• anaerobic microbes of interest for anticancer therapeutic delivery + to release tumor-specific to immune cells to facilitate tumor targeting

• target delivery of beneficial drug

  • engineered commensal bacteria to convert intestinal cells into glucose-responsive insulin-secreting cells

  • Lactobacillus engineered to deliver glucagon-like peptide 1 (GLP-1) to intestinal epithelial cells con.

• engineered probiotics used to sense + kill biofilm-forming strains of P. Aeruginosa

Pathogens as Engineered Anticancer Therapies

Listeria monocytogenes

• intracellular pathogenic bacterium that causes listeriosis (foodborne illness)

• engineered recombinant strain of this bacterium that express antigens on tumor cells

• triggers immune system to prod tumor-specific antibodies, destroys bacterium + tumor

• turned into anticancer vehicles to deliver drugs or radioisotopes to tumor cells to kill pancreatic tumor cells

Specific Delivery of Antibodies as an Anticancer Therapy

• use antibodies (immune proteins that atk foreign substances)

  • binding of antibodies to intracellular target in cancer cells can trigger immune system to kill those cells

• Bacillus anthracis → protective antigen has been engineered to carry synthetic anticancer antibody

• Protective antigen → antibody complex binds to receptor + is taken up by cancer cell

  • immune system triggered, kills cell

Environmental Gene Mining

Metagenome → collective genome of an environment

Gene mining → process of identifying + isolating potentially useful genes from environment w/o culturing the organism that contains them

• DNA (or RNA then cDNA) directly isolated from environment then cloned into ap