Gene expression optimization

The performance of a biotreatment system ultimately depends on optimization of the activity of microbes and the ability to control the process parameters of the treatment system [157]. In this respect, the ability to monitor gene copy numbers and gene expression is highly useful for real time optimization of the efficiency of a biotreatment system. Advanced molecular techniques as well as low cost methods (e.g., antibody detection of enzymes based on color reaction strips fluorescence i.e., GFP marked organisms with UV light detection) can also be applied to monitor the microbial community structure, persistence of the added bacteria, and their interactions with indigenous populations. 

We optimized the cultivation protocol with respect to aqueous medium composition, organic phase, and phase ratio (the ratio of the volume of the organic phase to the total liquid volume). The best system consists of a defined mineral medium, with glucose as the carbon source and diethylhexylphthalate as the organic phase at a phase ratio of 0.5. The organic phase contained 2 vol % styrene and 1 vol % of octane, which we added as an inducer for gene expression. 

The EST has continued to be a popular test subject to further optimization, especially through the addition of alternative pathways of differentiation and the addition of molecular markers of effects. Besides cardiac muscle differentiation, other cell types such as neurons (29-31), osteoblasts (32), adipocytes (33), and hepato-cytes (34) have been generated in dedicated differentiation protocols, providing additional opportunities for testing the interference of differentiation pathways with chemical exposures. The addition of transcriptomics approaches to measure differential gene expression, both as influenced by the differentiation process as well as due to chemical exposure, has proven informative and may enhance the predictability of EST assays (35). The extension of this concept to human embryonic stem cell lines is still in its infancy, but has opened a new realm of options that bypasses the need for interspecies extrapolation (36). 

Optimization of gene expression may be applied at every step of the process, from initial cloning and characterization to initiation of clinical trials. Often several rounds of optimization will be required to select the expression system that produces the highest yield with the lowest cost fermentation and purification schemes. Significant resources are allocated for optimization because the best protein expression system can lead to several hundred- to a thousandfold increased efficiency in protein produced. Under these conditions, as much as 10% of total proteins produced by the expression system are target proteins. 

---