NCBI BLAST enzyme nucleotide homologies.

Enzymes can catalyze reactions under very mild conditions (like a neutral aqueous solution at room temperature and pressure), yet provide exceptional specificity. Being proteins folded into complex three-dimensional shapes, enzymes create an active site on which the substrates can react, lowering the activation energy by binding to transition states of the given reaction. As the total output of free energy remains constant, the thermodynamics of the reaction are unchanged, but the reaction is generally accelerated. Substrates are attracted together with electrostatic forces and furthermore the enzymes themselves are not consumed in the reaction. Enzyme kinetics is a dynamical analysis of the chemistry of enzymes, working with reaction rates, equilibrium conditions and energy profiles. The Michaelis-Menten Equation is particularly important, describing the relationship between rate and substrate concentration for the reaction:

Temperature and acidity (optimal pH) are principal controls. The speed of an enzyme steadily increases with the temperature up to the point of thermal denaturation. At this point the enzyme unravels. Without the active site the enzyme ceases to function. In conditions of substrate saturation, Vmax is indicative of reaction rates and can be assumed as the specificity constant, a means of comparing enzymatic catalysis of substrate.

The P. putida bacterium, the ‘workhorse’ of microbiology labs, is certified for manipulations with a fully elaborated genome. Selectively transferring to (therefore transforming) a target cell with the genome sequences of another is a starting point for commencing the procedural testing of mutations in a genome to obtain a desired effect - in this project, production of an effective enzyme.

To understand the extent to which sequences of deoxyribonucleic acid (DNA) may correspond and interact, nucleotide data from the National Center for Biotechnology Information (NCBI) is available on the Internet. BLAST is an NCBI tool to find matching strings in genome databanks. VectorNTI is a software package for analyzing operon organization and designing primers from NCBI output. Long genome sequences are sometimes classified into percentages of GC pairs present (DNA is based on pairs of adenine, cytosine, guanine, or thymine nucleotides), an approximate signal of gene expression in a fragment visualized with staining. Indicative for the presence of a gene, high GC content DNA is more difficult to denature due to an extra hydrogen bond.

Gel Electrophoresis separates fragments to analyze amounts of DNA product captured in a dense gel, and can be used to study the effect of restriction digestion (“cutting”) and ligations (“joining”). A platebased assay pertains to measurement of the rate at which bacteria break down a thin layer of substrate on the surface of agar plates, pointing to particular colonies that were able to produce enzymes more or less effective at breaking down the substrate.

Primers are complementary to a specific part of bacterial DNA. When they are bound, the cell’s enzyme Taq polymerase can copy this part of the genome. The Polymerase Chain Reaction (PCR) for obtaining workable quantities of a sequence in the lab, also known as amplifying, is a practical three-step method to duplicate the length of the strand from two oligonucleotide primers bound to opposing bases on a source template fragment of DNA or RNA denatured (‘unwound’) by meticulous heating, using polymerase to match and assemble a copy with corresponding nucleotides as it travels along the length of the strand. This is repeated, now denaturing the new copies. A DNA shuffling technique (digestion – reassembly - amplification) can be used to reduce random mutation errors from PCR.