BACTERIOPHAGES AND THEIR DIVISION CELL CYCLES

STRUCTURE OF T-4 BACTERIOPHAGE

LIFE CYCLE OF BACTERIOPHAGE

APTAMER

Aptamers are oligonucleic acid or peptide molecules selected from a large random sequence pool to bind to specific target molecule. They can be used for both basic research and clinical purposes as macromolecular drugs.
More specifically, aptamers can be classified as:
DNA or RNA aptamers. They consist of (usually short) strands of oligonucleotides.
Peptide aptamers. They consist of a short variable peptide domain, attached at both ends to a protein scaffold.
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RNA and DNA aptamers
Aptamers are nucleic acid binding species that have been evolutionary engineered through in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even organisms. Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
In 1990, two labs independently developed the technique of selection with the Gold lab utilizing the term SELEX for their process of selecting RNA ligands against T4 DNA polymerase and the Szostak lab, coining the term in vitro selection, selecting RNA ligands against various organic dyes. The Szostak lab also coined the term aptamer (from the Latin, aptus, meaning ‘to fit’) for these nucleic acid-based ligands. Two years later, the Szostak lab and Gilead Sciences, independent of one another, utilized in vitro selection schemes to evolve single stranded DNA ligands for organic dyes and human coagulant, thrombin, respectively.
Interestingly enough, the notion of selection in vitro was actually preceded twenty-plus years prior when the infamous Sol Spiegelman utilized a Qbeta replication system as a means for the evolution of a self-replicating molecule. In addition, a year before the publishing of in vitro selection and SELEX, Gerald Joyce utilized a system that he termed ‘directed evolution’ to evolve a nucleic acid enzyme, a ribozyme.
Over the course of the next six years, many researchers have been utilizing aptamer selection as a means for application and discovery. Recently, in 2001, the process of in vitro selection has been automated by the Ellington lab at The University of Texas at Austin, reducing the duration of an in vitro selection experiment from six months to three days.
While the process of artificial engineering of nucleic acid ligands is highly interesting to biology and biotechnology, the notion of aptamers in the natural world had yet to be uncovered until 2002 when Ronald Breaker and his coworkers discovered a nucleic acid-based genetic regulatory element called a riboswitch that possesses similar molecular recognition properties to the artificially made aptamers. In addition to the discovery of a new mode of genetic regulation, this adds further credence to the notion of an ‘RNA World,’ a postulated stage in time in the origins of life on Earth.
While the development of aptamer evolution is mildly amusing, its application is truly monumental. Recent developments in aptamer-based therapeutics have been rewarded in the form of the first FDA approved aptamer-based drug in treatment for age-related macular degeneration (AMD), called Macugen offered by Eyetech Pharmaceuticals and Pfizer. In addition, companies such as Cambridge, MA-based Archemix have been examining the pharmacological properties of aptamers against targets such as the coagulant thrombin for use in postoperative treatments, complement c5 implicated in inflammatory-related tissue injuries, platelet-derived growth factor that is involved in the diabetic retinopathy disease state, and a few others.
In addition to the development of aptamer-based therapeutics, many researchers such as the Ellington lab and the Boulder, CO-based SomaLogic have been developing diagnostic techniques for whole cell protein profiling called proteomics, and medical diagnostics for the distinction of disease versus healthy states.
As a resource for all in vitro selection and SELEX experiments, the Ellington lab has developed the Aptamer Database cataloging all published experiments. This is found at http://aptamer.icmb.utexas.edu/.
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Peptide aptamers
Peptide aptamers are proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range).
The variable loop length is typically comprised of 10 to 20 amino acids, and the scaffold may be any protein which have good solubility and compacity properties. Currently, the bacterial protein Thioredoxin-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two Cysteins lateral chains being able to form a disulfide bridge.
Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system.
Selection of Ligand Regulated Peptide Aptamers (LiRPAs) has been demonstrated. By displaying 7 amino acid peptides from a novel scaffold protein based on the trimeric FKBP-rapamycin-FRB structure, interaction between the randomized peptide and target molecule can be controlled by the small molecule Rapamycin or non-immunosuppressive analogs.

http://en.wikipedia.org/wiki/Aptamer

LAL TEST

http://www.horseshoecrab.org/med/med.html = best link=

http://www.cambrex.com/content/bioscience/article.class.126.id.1317
To obtain the lysate required for the LAL test, horseshoe crabs are taken from the ocean floor, and a small amount of their blood is drawn. The animals are then returned unharmed to the sea. Next, the crabs’ blood cells (amebocytes) are separated and lysed to obtain the cellular proteins.

http://www.ocean.udel.edu/horseshoecrab/Research/lal.html

The Limulus amebocyte lysate (LAL) test is in vitro assay for detection and quantitation of bacterial endotoxin. Validated test methods performed at Nelson Laboratories includes the gel clot technique and the kinetic turbidimetric and chromogenic (colorimetric) assays.

http://en.wikipedia.org/wiki/Limulus_Amebocyte_Lysate
Limulus Amebocyte Lysate (LAL) is an aqueous extract of blood cells (amebocytes) from the horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantitation of bacterial endotoxins.

There are three basic LAL test methodologies: gel-clot, turbidimetric, and chromogenic. The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebral spinal fluid

GROWTH OF MICROBES

http://books.google.co.in/books?vid
=ISBN0521439809&id=DrHQtIbiunkC&pg=PA193&lpg=PA193&dq=
microbiology+growth+of+microorganisms&sig=XsOOWNIAeOGvgxui5kXwq-qK7l0

General MicrobiologyBy Hans G. Schlegel