Difference between revisions of "Part:BBa K5109334"
| Line 1: | Line 1: | ||
| + | __NOTOC__ | ||
| + | <partinfo>BBa_K5109001 short</partinfo> | ||
| + | |||
| + | The coding sequence of a truncated version of OmpT, to be used as a carrier in a C-terminal display system. OmpT, which stands for Outer Membrane Protease, is an outer membrane protease of E. coli that contributes to bacterial pathogenicity. Since we isolate only 3 transmembrane domains, its catalytic function is lost; in our project, we only exploit its structure as a membrane anchoring domain for enzymes that would otherwise be intracellular. | ||
| + | A linker was added to provide the necessary flexibility for the display system; this linker contains a thrombin cleavage site, which can be used when isolating the enzyme expressed on the surface is desired. | ||
| + | NdeI restriction sites were incorporated at both ends of the sequence to enable easy insertion into a specifically designed vector. In particular, we designed it to insert into the expression cassette BBa_K5109001, which already contains the enzyme to be expressed on the bacterial surface. | ||
| + | As OmpT is an endogenous gene of E. coli, we extracted the sequence directly from its genome by performing PCR, using primers with tails containing the NdeI and thrombin cleavage sites, incorporating them directly into the sequence. | ||
Revision as of 08:15, 28 September 2024
Dehalogenase type II intracellular expression tool
The coding sequence of a truncated version of OmpT, to be used as a carrier in a C-terminal display system. OmpT, which stands for Outer Membrane Protease, is an outer membrane protease of E. coli that contributes to bacterial pathogenicity. Since we isolate only 3 transmembrane domains, its catalytic function is lost; in our project, we only exploit its structure as a membrane anchoring domain for enzymes that would otherwise be intracellular. A linker was added to provide the necessary flexibility for the display system; this linker contains a thrombin cleavage site, which can be used when isolating the enzyme expressed on the surface is desired. NdeI restriction sites were incorporated at both ends of the sequence to enable easy insertion into a specifically designed vector. In particular, we designed it to insert into the expression cassette BBa_K5109001, which already contains the enzyme to be expressed on the bacterial surface. As OmpT is an endogenous gene of E. coli, we extracted the sequence directly from its genome by performing PCR, using primers with tails containing the NdeI and thrombin cleavage sites, incorporating them directly into the sequence.