THESIS
2013
xiv, 81 pages : illustrations (some color) ; 30 cm
Abstract
The photosynthetic microorganisms, Rhodobacter species, have attracted increasing interests
as expression host in bioproduction. Their unique metabolism has made these bacteria suitable
for producing membrane proteins and proteins modified with metal cofactors. Meanwhile,
their ability to utilize solar energy offers attractive benefits which cannot be enjoyed by
traditional hosts, e.g. E. coli. In recent years, structural biologists have been using
Rhodobacter sphaeroides for the production of membrane proteins.
In this thesis research, we aim to provide a detailed characterization study on Rhodobacter
sphaeroides 2.4.1 as an expression host for heterologous proteins and to explore its usage in
the expression of a cytochrome component protein (i.e., MtrA). The thesis work starte...[
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The photosynthetic microorganisms, Rhodobacter species, have attracted increasing interests
as expression host in bioproduction. Their unique metabolism has made these bacteria suitable
for producing membrane proteins and proteins modified with metal cofactors. Meanwhile,
their ability to utilize solar energy offers attractive benefits which cannot be enjoyed by
traditional hosts, e.g. E. coli. In recent years, structural biologists have been using
Rhodobacter sphaeroides for the production of membrane proteins.
In this thesis research, we aim to provide a detailed characterization study on Rhodobacter
sphaeroides 2.4.1 as an expression host for heterologous proteins and to explore its usage in
the expression of a cytochrome component protein (i.e., MtrA). The thesis work started with
the expression of MtrA protein, which involves in an extracellular electron transfer pathway
in Shewanella oneidensis, in Rhodobacter sphaeroides 2.4.1. Using the expression vector,
pIND4, MtrA protein was successfully expressed in Rhodobacter sphaeroides 2.4.1 and
confirmed by Western blotting. The intracellular location of MtrA was verified by periplasmic
extraction in the preparation of Western blotting samples. The function of MtrA was also
verified through ferrozine assay by quantifying the ability to reduce ferric ions in Rhodobacter sphaeroides 2.4.1 expressing MtrA.
To facilitate the function of MtrA in a complete electron transfer pathway, other components
in an MtrCAB pathway need to be expressed. Thus a multi-protein expression vector is
needed. In order to construct a bicistronic expression vector for the Rhodobacter system, the
vector pRK442(H) was chosen as a vector backbone. The expression system constructed on
the basis of this backbone suffers from the phototoxicity of tetracycline due to its usage for
maintaining the plasmid under phototrophic condition. To overcome this difficulty, pHW1
was constructed using the protein expression cassette on an inducible plasmid, pIND4, and
ampicillin resistant gene was used to replace the native tetracycline resistant gene carried by
pRK442(H).
After the construction of pHW1, features about this plasmid were characterized including the
segregational plasmid stability, protein expression level and so on. The data showed that
anaerobic culture, dark condition and relatively high inducer concentration enhanced the
plasmid stability. The effect of different stages at which IPTG induction was performed on
protein expression level was also explored.
This part is a component of a project to construct a novel photosynthetic micro fuel cell. Now
the focus is mainly on construct electron pathway. The first component has been expressed
and new plasmid pRKBic without phototoxicity problem will be used in the expressing of
multi-cytochromes from Shewanella oneidensis in Rhodobacter sphaeroides 2.4.1.
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