Part:BBa_K4134066:Design
BpfA-Atox1-Linker-KanR-loxP-AggC
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The pUC57-BpfA-AtoxⅠ/AgBP2-Kana-loxP-aggC fusion vector contains a core DNA box consisting of the coding sequence of a silver binding protein (either Atox1 and AgBP2, hereinafter referred to as AgBP) and the kanamycin resistance gene (Kana). The core DNA box enables BpfA to bind silver ions and helps us screen the recombinants. The upstream and downstream of our core DNA box are two sequence fragments of the MR-1 homologous gene BpfA (3' end) and aggC (5' end), both of which are 1000 bp in length, used to realize the recombination transformation of MR-1 and the localization of the silver-binding protein. The entire design is based on the pUC57 (mini) vector, which carries the ampicillin resistance gene (AmpR). S. onedensis has no apparent codon usage bias, so genetic fusion of AgBP and BpfA can be performed without much codon optimization. To create the AgBP knock-in construct, using the wild-type S.onedensis MR-1 genome as models, the homology arm sequences were amplified using BpfA-F/R and AggC-F/R as primers, each 1000 bp in size. The AgBP sequence was synthesized by Qingke Biotechnology, and the Kana resistance gene fragment was amplified from the prokaryotic expression vector pET28a by the primer Kana-F/R. After PCR amplification to obtain BpfA, AggC, Atxo1, and KanR sequences, the sequence size was verified through agarose gel electrophoresis, and the fragments were recovered and inserted into the pUC57mini vector to construct pUC57mini-Atox1-Kana-loxP-AggC. To fuse Atox1 in-frame with the C-terminus of BPFA, the vector pUC57mini-Atox1-Kana-loxP-AggC was double digested with EcoRI and KpnI, and the vector-free linear fragment was purified for subsequent electroporation into MR- 1.
Source
Reference: Sivakumar K, Mukherjee M, Cheng H I, et al. Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms[J]. Biotechnology and Bioengineering, 2015, 112(3): 512-520.