An alternative approach to the use of antibiotic selection markers for maintenance of recombinant plasmid vectors in Escherichia coli based on an aminoacid auxotrophy complementation has been developed. An E. coli M15-derivated glycine-auxotrophic strain of has been constructed by means of a PCR-based approach. This mutant strain contains a deletion in the glyA gene, which encodes for serine hydroxymethyl transferase, an enzyme involved in the main glycine biosynthesis pathway in E. coli. Also, we have constructed the complementation plasmid pQEalphabetarham derived from the commercially available expression vector pQE40 (QIAGEN) containing the glyA homologous gene under the control of the constitutive weak promoter P3. By using the E. coli M15DeltaglyA strain combined with the pQEalphabetarham plasmid, a successful complementation system was achieved, allowing transformants to grow on minimal media without glycine supplementation. The capability of the new system E. coli M15DeltaglyA/pQEalphabetarham for recombinant overproduction of rhamnulose 1-phosphate aldolase was evaluated in antibiotic free fed-batch cultures at controlled specific growth rate, obtaining high cell density cultures and high RhuA production and productivity levels comparable to those obtained with the conventional system. The new selection marker based on glycine-auxotrophy is a promising genetic tool, not only for recombinant protein production, but also for plasmid DNA production processes, where antibiotics can not be present in the medium formulation.

No abstract

No abstract

No abstract

Water saving techniques such as drip irrigation are important for rice (Oriza sativa L.) production in some areas. Subsurface drip irrigation (SDI) is a promising alternative for intensive cropping since surface drip irrigation (DI) requires a higher degree of labor to allow the use of machinery. However, the semi-aquatic nature of rice plants and their shallow root system could pose some limitations. A major design issue when using SDI is to select the dripline depth to create appropriate root wetting patterns as well as to reduce water losses by deep drainage and evaporation. Soil texture can greatly affect soil water dynamics and, consequently, optimal dripline depth and irrigation frequency needs. Since water balance components as deep percolation are difficult to estimate under field conditions, soil water models as HYDRUS-2D can be used for this purpose. In the present study, we performed a field experiment using SDI for rice production with Onice variety. Simulations using HYDRUS-2D software successfully validated soil water distribution and, therefore, were used to predict soil water contents, deep drainage, and plant water extraction for two different dripline depths, three soil textures, and three irrigation frequencies. Results of the simulations show that dripline depth of 0.15 m combined with one or two daily irrigation events maximized water extraction and reduced percolation. Moreover, simulations with HYDRUS-2D could be useful to determine the most appropriate location of soil water probes to efficiently manage the SDI in rice.