Mutational analysis of structure - function interactions within selected sites on the Escherichia coli ribosome

Master of Science - Adult EducationMutations were sought in Escherichia coli ribosomal RNA and ribosomal proteins that confer dependence to the antibiotic streptomycin, using both newly available as well as well-established genetic systems. I found that a classical ribosomal mutant, Sm-D3, was streptomycin dependent and had an additional mutation in another ribosomal component – protein L7/L12. The double mutant had an 8-fold lower streptomycin requirement as compared to Sm-D3 with a wild-type rplL. This supported a functional involvement of L7/L12 in the decoding center of the ribosome

Studies on the functional interaction of translation initiation factor IF1 with ribosomal RNA

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L).  This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association.  Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p

Self-powered programmable microfluidic platform for POC diagnostics

We present in this paper a new concept of a self-powered infusion microfluidic pump for lab on a chip (LOC) applications, called (i)SIMPLE. By combining infusion and withdraw mode of pumping a whole new world for designing and implementing complex multi-step bio-assay protocols on a lab on a chip opens up. The pumping system is robust, easy to fabricate, inexpensive, user-friendly, and suited for mass replication technologies addressing most of the LOC requirements. Here we present the implementation of a bio-assay for the detection of creatinine in serum samples of patients with chronic kidney disease.status: publishe

Biocatalysis on the surface of Escherichia coli : melanin pigmentation of the cell exterior

Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.QC 20161031</p

Self-powered infusion microfluidic pump for ex vivo drug delivery

In this work, we present a new iSIMPLE concept (infusion Self-powered Imbibing Microfluidic Pump by Liquid Encapsulation), which requires no external power for activation nor liquid manipulation, it is easy to use while its fabrication method is extremely simple, inexpensive and suited for mass replication. The pump consists of a working liquid, which is - after finger activation - absorbed in a porous material (e.g. filter paper). The air expelled from the porous material increases the pressure in the downstream outlet channel and propels the outlet liquid (i.e. the sample) through the channel or ejects it. Here we investigated the influence of different filter papers on the iSIMPLE flow rates, achieving a wide range from 30 down to 0.07 μL/min. We also demonstrated the versatility of the iSIMPLE in terms of the liquid volume that can be manipulated (from 0.5 μL up to 150 μL) and the working pressure reaching 64 kPa, unprecedented high for a self-powered microfluidics pump. In addition, using a 34 G microneedle mounted on the iSIMPLE, we successfully injected liquids with different viscosities (from 0.93 up to 55.88 cP) both into an agarose matrix and a skin-like biological ex vivo substrate (i.e. chicken breast tissue). This work validated the compatibility of the iSIMPLE with drug delivery in a controlled way into a skin-like matrix, envisioning a whole new scenario for intradermal injections using self-contained skin patch. In addition, due to the extreme flexibility of the design and manufacturing, the iSIMPLE concept offers enormous opportunities for completely autonomous, portable and cost effective LOC devices.status: Published onlin