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 humanBacterial and plantViral 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 endsWhen 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 sequenceOpen DNA Recombine with DNA cut with the

same restriction enzymeUse ligase to seal the cuts and

rejoin the fragments

Restriction enzymes

Experimental Design

Recombinant DNA

Examples of Products of Genetic Engineering using microbes

Factor VIIIErythropoetinInsulinInterferonEpidermal 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 cropsInsecticide resistant cropsHerbicide 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

pGlo

pGlo

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

Crop damage due to frost

P syringae on surface of leaves

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

Agrobacterium tumefaciens and nature’s genetic engineering

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

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

Genetic Engineering and Ti