Selection guided evolution

Figure 26. Dimerization of butadiene in the crystalline phase. Lower panel Logarithmic plots of the room-temperature evolution of the integrated absorption of characteristic vinylcyclohexene absorption bands at different pressures. The linear evolution unambiguously demonstrates the first-order kinetics of the reaction. Upper panel Evolution of the natural logarithm of the dimerization rate constant as a function of pressure (full squares, left axis the dotted line is intended as a guide for the eye) and evolution of the intensity ratio between selected polymer and dimer (vinylcyclohexene) bands (empty dots, right axis).

Many workers have in fact used density matrix methods for the calculation of line shapes and intensities in multiple resonance experiments, and two excellent reviews of the background theory are available. (49, 50) In addition there is also a simple guide (51) to the actual use of the method which is capable of predicting the results of quite elaborate experiments. Major applications have included the calculation of the complete double resonance spectrum from an AX spin system which gives 12 transitions in all (52) an extremely detailed study of the relaxation behaviour of the AX2 systems provided by 1,1,2-trichloroethane and 2,2-dichloroethanol (53) the effects of gating and of selective and non-selective pulses on AB and AX spin systems and the importance of the time evolution of the off-diagonal elements of the density matrix in repetitively pulsed FT NMR and spin-echo work (54) the use of double resonance to sort out relaxation mechanisms and transient responses (55) the calculation of general multiple resonance spectra (56) and triple resonance studies of relaxation in AB and AX spin systems. (57)... 

The selection of improved variants from a library of genotypes is crucial because it guides the development of a protein toward a certain functionality. In other words, the precise definition and implementation of the selection criteria comprise the most important steps in any directed evolution project When, for example, improper library generation techniques are chosen, a directed evolution project will run slower and less efficiently. In the extreme, there will be no progress at all. However, if improper selection processes are chosen, the directed evolution project will run in the wrong direction generating useless protein phenotypes. Careful consideration and evaluation of available selection processes is therefore key to successful directed evolution projects. 

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