Part:BBa_K4134011
BpfA (C-terminal 1000bp)
Description
BpfA stands for the biofilm-promoting protein A, a large surface protein. The 1000bp-long fragment of BpfA and the 1000bp-long fragment of AggC, the downstream genes of BpfA, are attached to our vector for homologous recombination so as to fuse the destination fragment (silver-binding protein) to the C-terminus of BpfA. This design enables the destination gene to be displayed on the surface of S. oneidensis. Considering the large size of BpfA, this fusion expression does nearly no harm to the normal physiological state of S. oneidensis and we have approved it through a biofilm growth test and bacterial density measurement.
After all the considerations above, we made the pUC57-BpfA-AtoxⅠ/AgBP2-Kana-loxP-aagC fusion vector. Its core DNA box consists of the coding sequence of a silver binding protein (either Atox1 or AgBP2) and the kanamycin resistance gene (Kana, for recombinant screening). In the following experiment, proliferated vectors were digested into two fragments before electroporation because the linearized expression vector exhibited a high site-specific recombination frequency after being inserted into the S. oneidensis genome. The digestion of the plasmid DNA (20 mg) was carried out with the restriction enzymes KpnⅠ(10 U) and EcoRⅠ(10 U) in a volume of 200 ml.
In short, we displayed a silver-binding protein AtoxⅠ onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed AtoxⅠ as a receptor to capture the silver ions in the matrix of S. oneidensis biofilms.
Usage
For the MFC design, we add a small amount of silver nitrate to the anode chamber. Silver ions are captured on the membranes and then reduced in situ by cellular metabolism-generated electrons into the transmembrane silver nanoparticles inside and traversing the cell membranes. Generally, electrons are transferred between ferric/ferrous iron redox centers through a multi-step hopping process. Since the electronic conductivity of metals is much higher than the redox center-mediated charge transfer process in cytochromes, transmembrane and outer membrane silver nanoparticles may act as metal shortcuts to bypass redox center-mediated slow electrons. During the transfer process, it is in direct contact with the external electrode to extract the charge more efficiently and improve the current density of the MFC.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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