Part:BBa_K5109023
Deha2 surface display system
Description
This part is a modification of the expression cassette BBa K5109001 which has been improved by incorporating a sequence encoding the anchoring motif BBa K5109004, creating a display system. This expression cassette allows the display of an otherwise intracellular protein (Dehalogenase type II) on the cell surface by fusing it to a carrier.
Carrier of choice was Lpp - OmpT, with the addition of a linker, described as part: BBa K5109004. The choice of this linker compared to others was made based on previous bibliographic research to find out which pattern would offer the best surface expression. We introduced different restriction sites both upstream and downstream the carrier and the dehalogenase sequences, enabling the cassette's use in various projects. Specifically, by cutting with NdeI, the Lpp-OmpT sequence can be removed, in order to either convert the cassette to an intracellular expression one, or to allow the replacement of the carrier. In the latter case, it could be useful to compare the effectiveness of Lpp-OmpT as a carrier against other anchoring motifs. Additionally, the passenger protein can be exchanged by digesting with BsaI and BamHI.
Usage and Biology
This part is a tool for experimental bioremediation research: dehalogenases have already be indicated as putative candidate in explorative studies on PFAS degradation. Our work aims to offer a better understanding of this subject, from a new perspective: expressing the enzyme on the cell surface avoids stressing the cells with possibly toxic products that would result from the Dehalogenase work, if kept inside the cell. When expressed on the outer membrane of the cell, the dehalogenase has the possibility to work on substrates directly in the extracellular medium, releasing also the reaction products in the medium. This allows you also to more specifically monitor the degradation of compounds: when cells expressing dehalogenase on their surface are grown in a PFAS rich medium, degradation of PFAS can be monitored, as well as the formation of new compounds. Since Deha2 has been shown to be able break C-F bonds increasing the fluoride amount in the medium, as previously characterized in part BBa_K3347010 Experience, using it outside the cell would reduce the stress on the host cell by releasing fluoride outside and not inside of the microorganism.
Characterization
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 639
Illegal BamHI site found at 1372 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 667
| None |