This work deals with the characterization of a phosphorothioate anchoring strategy for aptamer molecules linked to gold, in the context of electrochemical sensors, using adenosine aptamer as model system. Surface density of immobilized phosphorothioate oligonucleotide sequences has been explored for a range of oligonucleotide concentrations (0.055 -55 mM), finding a consequent variation of molecular surface density (3.5 Â 10 11 -2.8 Â 10 13 molecules/cm 2 ). Most suitable aptamer concentration for adenosine recognition was also explored and found to be around 5.5 mM. As proof of concept of phosphorothioate strategy, electrochemical response to adenosine concentration was measured using a ferrocene-labeled oligonucleotide sequence, and phosphorothioate anchoring thermal stability was compared to thiol immobilization.

No abstract

Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO2 through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted.

Alginates are polymers used in the food and pharmaceutical industries as stabilizing, thickening, gel or film-forming agents. Food coatings formed from alginate have shown to have excellent properties such as flexibility and resistance to tearing. These polysaccharides are extracted from natural brown algae, but they can also be produced by a bio-process. One strategy to produce alginates is through the manipulation of the culture conditions during fermentation using Azotobacter vinelandii. This work proposes the development of a PI control strategy to improve the production of alginate from Azotobacter vinelandii in a laboratory bio-reactor scale.

No abstract