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Recombinant DNA, Biotechnology, and Microbes. Microbiology 221. Overview – Putting microbes to Work – Molecular Cloning.
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Recombinant DNA, Biotechnology, and Microbes Microbiology 221
Overview – Putting microbes to Work – Molecular Cloning • Recombinant DNA technology utilizes the power of microbiological selection and screening procedures to allow investigators to isolate a gene that represents as little as 1 part in a million of the genetic material in an organism. • The DNA from the organism of interest is divided into small pieces that are then placed into individual cells (usually bacterial). • These can then be separated as individual colonies on plates, and they can be screened through rapidly to find the gene of interest.
Recombinant DNA( natural and manipulative) • Combination of DNA from organisms from two different sources • Bacterial and human • Bacterial and plant • Viral and human
Basics of Restriction enzymes • Isolated from various bacteria, restriction enzymes recognize short DNA sequences and cut the DNA molecules at those specific sites. • (A natural biological function of these enzymes is to protect bacteria by attacking viral and other foreign DNA.)
Process • Restriction endonucleases cut at defined sequences of (usually) 4 or 6 bp. They cut on both strands of DNA • This allows the DNA of interest to be cut at specific locations. The physiological function of restriction endonucleases is to serve as part of system to protect bacteria from invasion by viruses or other organisms • Cuts yield either "staggered" or "sticky" ends (see figure) or "blunt" ends. • Two pieces of DNA cut with the same enzyme, can be pasted together using another enzyme called "DNA ligase".
Sticky ends Sticky ends • When the ends of the restriction fragments are complementary, EcoRI – recognition sequence 5'‑‑‑G ‘AATTC‑‑‑3' • 3'‑‑‑CTTAA ‘G‑‑‑5'
Blunt ends • (1) The restriction endonuclease cleaves in the center of the pseudopalindromic recognition site to generate blunt (or flush) ends. • HaeIII GG'CC • HincII GTY'RAC
Restriction enzymes generate fragments that facilitate recombination
Process • Cut ends in recognition sequence • Open DNA • Recombine with DNA cut with the same restriction enzyme • Use ligase to seal the cuts and rejoin the fragments
Examples of Products of Genetic Engineering using microbes • Factor VIII • Erythropoetin • Insulin • Interferon • Epidermal growth factor
Industrial applications • Oil “ eating” microbes – Prince William Sound – Alaska • Degradation of mercury in the environment – Clean up of contaminated sites
Agricultural applications • Frost resistant crops • Insecticide resistant crops • Herbicide resistant crops
Transformation with pGlo • PRE-INCUBATIONThe recipent E. coli cells will be exposed to positively charged calcium chloride (CaCl2) ions. This treatment is meant to stress the bacterium in order to render its cell membrane and cell wall permeable to the donar plasmid. This process will make the recipient E. coli "competent" to uptake the plasmid.* INCUBATIONThe plasmid (with amp+ gene) is added to a recipient E. coli suspension, which will now be called E. coli + because it is the one which is being transformed. Another E. coli suspension will act as a control, called E. coli - because it will not be exposed to the plasmid; therefore, it will NOT inherit the gene.* HEAT SHOCKThe recipient cells plus plasmids and the control cells not exposed to the plasmids are briefly exposed to 42 degrees C. This step will maximize the uptake of the plasmid through the wall and membrane of the cells.
Plasmid Vectors • Ori( origin of replication) • Polylinker cloning sites • Regulatory region • ( lac operon) • Antibiotic resistance gene(s) • Reporter gene for protein – color or fluorescent molecule
pGlo • Ori • Polylinker cloning region • Amp ( beta lactamase for resistance) • araC( arabinose operon) • pBad • Green fluorescent protein - reporter
Gene fusion • Transposition of genes from one location to another on a chromosome • Also can result in the deletion of a section of a chromosome • Gene fusion has been used with Pseudomonas syringae, a bacterium that grows on plants
Pseudomonas syringae • Produce a protein that forms a nucleus for ice crystals • Ice crystals damage leaves and stems • Removing the gene, prevents damage to crops when the crops are sprayed with the resistant forms
Protoplast fusion • Removal of the cell wall of organisms of two strains can result in the recombination of their genetic material. • Can select for desirable features of both strains • Effectively used in yeast, molds, and plants
Protoplast fusion • Nocardia lactamdurans – produces the antibiotic cephalomycin • New strains increase the yield of this important antibiotic
Gene amplification • Bacteriophages or plasmids are introduced into cells to repliicate or reproduce at rapid rates • This is used particularly in strains of bacteria that produce antibiotics • Amplification can also be used to increase the yield of amino acids, vitamins, and enzymes
Nature of the Microbe • A. tumefaciens is a Gram-negative, non-sporing, motile, rod-shaped bacterium, • Closely related to Rhizobium which forms nitrogen-fixing nodules on clover and other leguminous plants. • Possesses a large, natural plasmid called Ti
Agrobacterium tumefaciens • Attracted to wounds or openings in the plant cell wall • Uses acetosyringone to inject into the plant cells • Ti plasmid enters the plant cell and integrates randomly into the host • Plasmid codes or opines and nopalines two distinctive gene products that lead to tumor production in infected plants
Ti plasmid and genes • ori--replication controlled sites • tra region--responsible for mobility from bacteria to plant cell • vir--induce uncontrolled cell division in the host plant • t region (tDNA)--group of genes that control the transfer of the tDNA to the host chromosome
