Ajuste de datos con modelo de caja negra

Several problems hamper the discovery of novel genes and gene products from metagenomic clone libraries. These include the choice of heterologous host, detection of activities and appropriate screens for the detection of activities, which can all prove to be problematic.

E.coli is the heterologous host of choice in most cases (Ekkers at al., 2012), with Uchiyama and colleagues having reported that ~40% of foreign genes are expressed in E. coli. However the expression of foreign genes can be impeded by host codon usage preferences, problems with gene promoter recognition, transcription initiation factors, improper protein folding and the inability to export gene products from the host cell (Ekkers et al., 2012). In addition expression of foreign gene products can sometimes be toxic to the heterologous host (Uchiyama & Miyazaki, 2009). The abundance of genes of interest in the source environment and the cloned insert size and library size also has an effect on the probability of cloning particular genes (Uchiyama & Miyazaki, 2009). Ekkers and colleagues have described the ‘great screen anomaly’, where gene and product discovery from clone libraries is disappointingly low compared to what might be expected (Ekkers et al., 2012).

Efforts to increase the rate of gene and product discovery can possibly be improved by the use of multiple heterologous host expression systems. Shuttle vectors that can be transformed from E. coli to hosts such as Streptomyces or

46

Pseudomonas may increase the chances of heterologous expression (Ekkers et

al., 2012). Enhanced detection methods such as the inclusion of reporter genes

(e.g. green fluorescent protein (GFP), β-lactamase or tetracycline resistance on vectors may allow for detection of activities which is below detection thresholds from phenotypic assays alone (Uchiyama & Miyazaki, 2009). Uchiyama and colleagues also suggest that improvements in synthetic biology can lead to the design and synthesis of novel genes based on gene sequences in curated databases which may then be cloned into expression systems. Finally, the design of novel functional screens to detect activities of interest will be required if functional based metagenomic approaches are to lead to an increased discovery of genes and gene products of industrial or pharmaceutical interest (Steele et al., 2009).

1.7 Summary

Marine sponges host a remarkable diversity of symbiotic microorganisms. These symbionts appear to play vital physiological roles in the host, including cycling of vital nutrients – carbon, nitrogen and sulfur, and may also play an important role in host defence through the production of bioactive secondary metabolites of varied chemical classes, which in themselves may display wide ranging activities of biotechnological interest. The vast genetic diversity associated with individual sponges can be exploited through culture dependent and culture independent techniques. Exploitation of sponge associated microbial genes has led to the development of commercially available pharmaceutical products while others are in clinical trials. Increased efforts to sample, characterize, analyse and screen sponge derived microbial products offers hope for the development of many more such products for the marketplace.

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