Integration of native and engineered photosynthetic microorganisms into artificial assemblies for the development of promising biosensors targeted to environmental monitoring
Author(s)
Rodio, Giuseppe
Date Issued
March 16, 2012
Type
Doctoral Thesis
Abstract
The aim of this PhD project is the development and optimization of photosynthetic biosensors for
monitoring environmental contaminants (e.g. herbicides).
Were simultaneously followed two research lines: the aim of the first one was the production of an
electrochemical biosensor based on whole algal cells activity of the genus Chlamydomonas, the
second line to develop a chimeric biomedietor consisting union of protein sequences derived from
purple bacterium photosynthetic Reaction Centers (RCs), Rhodobacter sphaeroides and
Chlamydomonas algae for the development of an electrochemical biosensor. The second research
line aspired to create sensing element with the sensitivity characteristics of a typical eukaryotic
reaction center, and the bacterial reaction center stability.
About the electrochemical biosensor based on photosynthetic activity of C. reinhardtii whole cells
immobilized on Screen Printed Electrode (SPEs) after two alternative immobilization procedures
have been tested on the selected biomediator: a first one based on coating with a polymer called
Nafion® was deeply explored, while the second one consisting in the biomediator entrapment
within a calcium-alginate gel. Particular attention has been dedicated to the above mentioned
alginate immobilization procedure, revealed to be much more promising than the Nafion® one.
Dose-response curves were carried out for two different herbicide, a triazine type, the other type of
urea and determined some important parameters of the biosensor. Very sensitive analyte Limit of
Detection (LOD) were obtained, reaching a LOD equal to 6x10-9 M for the herbicide Linuron and
9x10-8M for the herbicide simazine. The data analysis obtained from the dose-response curves and
the recovery experiments, suggested the existence of a second binding site for plastochinone in
Chlamydomonas, in agreement with recent literature data that indicate the presence in some species
of plants and cyanobacteria.
The electrode with immobilized algae can be stored for a month at room temperature without losing
functionality.
About the second research line a feasibility bioinformatic study was carried out to check what was
the optimal molecular biology approach to improve the sensitivity of Rhodobacter sphaeroides
toward herbicides, and we decided to try to create a Chimera to melt the sensitivity characteristic
toward herbicides of algae Photosystem II (PSII), with the stability characteristic of bacterial RC.
Regarding the chimera, construction, we proceeded with the nucleotide sequence selection and
identification of the encoding region of Chlamydomonas interest protein by bioinformatics analysis,
and its subsequent synthesis by PCR and cloning into plasmid vector pCR®2.1. Once cloned and
amplified, the interest sequence was excised and then cloned into two different Rhodobacter
expression vectors: a gene coding sequences containing only the photosynthetic reaction center (LM, H), which also contains other gene coding sequences of the LH1 antenna. The recombinant
strains were then grown and used for the extraction of photosynthetic membranes. The visible
absorption spectrum showed that the chimeric reaction center is present at low concentrations,
although in different quantities in both transformants. The low concentrations may be due to
assembly problems and/or stability problems that will require further analysis to determine the
functionality, stability and the ability to bind herbicides for its possible future use as biomediator.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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