Part:BBa_K5109023

Deha2 surface display system

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

Enzymatic tests on BBa_K5109023

After successfully cloning Dehalogenase II inside pJUMP29-1A ∆NdeI (BBa_K5109060), before testing our part on PFAS molecules, we performed a control enzymatic test. Dehalogenase II is known to successfully degrade fluoroacetic and chloroacetic acid, therefore we decided to set some tests that would use chloroacetic acid in order to test the correct functioning of the protein: the chloroacetate substrate can be converted into glycolate after the removal of the chlorine done by the dehalogenase. We tested both wild type TOP10 F' as a control and TOP10 F' cells containing our expression vector. A part of the cells containing the vector were grown in addiction with IPTG 5 uM to induce protein expression, meanwhile another part was grown without IPTG as a control. All the cells were then incubated with LB medium + chloroacetic acid 2.5mM, and monitored for 48h. After the 48h we obtained the following results:

- F’ are wild type cells

- DeHa2 are induced cells

- DeHa2 NI are not induced

After 48h, the concentration of chloroacetic acid the medium decreased for 98% in cells that were induced with IPTG, while chloroacetic acid was degraded of 90% in cells without IPTG. As expected, wild type cells did not show any particular change in chloroacetate concentration over time Colonies expressing DeHa2 (#9 refers to colony number 9 we choose during colony picking) showed degradation both under IPTG induction and without IPTG: this may suggest a possible leaky activity of IPTG promoter.

The results lead us to consider part K5109023 a possible display system for the expression of an outer membrane protein. Further investigation is needed in order to confirm the correct functioning of the protein, and to subsequently test it on PFAS molecules.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 639
  Illegal BamHI site found at 1372
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 667