THESIS
2014
xiv, 107 pages : illustrations ; 30 cm
Abstract
MtrCAB (metal reducing protein complex of MtrC, MtrA and MtrB), a key electron transfer component in Shewanella oneidensis MR-1 for extracellular metal reduction, is able to pump electrons directly from periplasm to external environment. In an ongoing project of our research group, MtrCAB was heterologously expressed in a photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1, for the purpose of constructing a photosynthetic microbial fuel cell through direct electron transfer from the bacterium to the outside electrode. However, the secretory expression level of MtrCAB is low, which could potentially limit the electron transfer efficiency of engineered R. sphaeroides. One of the possible reasons lies in the inadequate recognition of signal peptides that directs MtrCAB across the cytop...[
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MtrCAB (metal reducing protein complex of MtrC, MtrA and MtrB), a key electron transfer component in Shewanella oneidensis MR-1 for extracellular metal reduction, is able to pump electrons directly from periplasm to external environment. In an ongoing project of our research group, MtrCAB was heterologously expressed in a photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1, for the purpose of constructing a photosynthetic microbial fuel cell through direct electron transfer from the bacterium to the outside electrode. However, the secretory expression level of MtrCAB is low, which could potentially limit the electron transfer efficiency of engineered R. sphaeroides. One of the possible reasons lies in the inadequate recognition of signal peptides that directs MtrCAB across the cytoplasmic membrane for subsequent localization.
A number of studies have revealed the decisive role of signal peptide in translocation efficiency of non-cytoplasmic proteins. Heterologous expression of non-cytoplasmic proteins often results in low yield at the right location, probably due to inefficient recognition of non-native signal peptides by recognition systems in the host. Previous studies show that modification of heterologous proteins with host homologous signal peptides offers a promising solution to this problem. Therefore, this project aims to improve the expression level of MtrCAB at their specific locations and enhance the electron conducting capacity of the engineered R. sphaeroides, by replacing MtrCAB signal peptides with native ones from R. sphaeroides.
In this thesis, each gene of mtrC, mtrA and mtrB was modified with DNA sequence of a native signal peptide from R. sphaeroides by gene cloning. The recombinant gene was integrated into an inducible-expression plasmid and expressed in R. sphaeroides. Quantitative western blot results indicate that the secretion amount of modified MtrC doubles in anaerobic condition and quintuples in aerobic condition, compared with that of unmodified MtrC. Also the modified MtrA shows an approximate one fold improvement in localization efficiency under both culture conditions. Yet no improvement is detected in the case of MtrB. Overall, the electron transfer efficiency of engineered R. sphaeroides with modified MtrCAB is one fold higher than that with the unmodified MtrCAB.
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