Difference between revisions of "Part:BBa K2450501"

Revision as of 19:58, 21 October 2017

SpyCatcher sfGFP quencher

SpyCatcher, sfGFP, TEV cleavable linker, reach quencher

Usage and Biology

This part was designed to be targeted to the outer membrane. It has a TorA leader sequence which targets it to the periplasm through the Tat translocase system, which translocates folded proteins. The spycatcher domain forms an isopeptide bond with a spytag domain on another protein. This part is intended to bond to an outermembrane protein with a spytag domain such as [BBa_K2450401] in order to target this part to the outer membrane and subsequently outer membrane vesicles (OMVs).

The sfGFP fluorescence is quenched by the dark quencher which converts the energy that would of been emitted as light and converts it into vibrational (heat) energy. This quenching is relieved by cleavage of the TEV cleavage site by the TEV protease which cleaves the dark quencher from sfGFP. When not in close proximity to the sfGFP the dark quencher can no longer quench fluorescence, resuulting in a measurable increase in fluorescence.

The aim of this part is to be able to detect lysis by the TEV protease in loacalised areas, specifically OMVs. The TorA leader sequence ensures the protein is targeted to the intermembrane space. The binding of this protein through the spycatcher/spytag system localises the protein to a particular protein/ region of the intermembrane space, such as the spytag-OmpA part [BBa_K2450451]. When this protein is taken up into the OMV and these are extracted from the cell, the part is protected from cleavage from the TEV protease, by the OMV membrane. On lysis of the OMV, the part can be cleaved and fluorescence observed, which can be used to measure the fraction of OMVs lysed by a particular technique. It can also be used to measure rates of cleavage at the outer membrane or in lysed OMVs, which can be used as a measure of the rates of macromolecular interaction at these membranes, which may be different to the rates of interaction in the cytoplasm.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 4
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 397
Illegal SapI.rc site found at 1291

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-This part was designed to be targeted to the outer membrane. It has a TorA leader sequence which targets it to the periplasm through the Tat translocase system. This translocates folded proteins. The spycatcher domain forms an isopeptide bond with a spytag on  another domain. This has been designed to bond to an outermembrane protein with a spytag domain such as [BBa_K2450401] in order to target this part to the outer membrane and subsequently outer membrane vesicles (OMVs).
+This part was designed to be targeted to the outer membrane. It has a TorA leader sequence which targets it to the periplasm through the Tat translocase system, which translocates folded proteins. The spycatcher domain forms an isopeptide bond with a spytag domain on another protein. This part is intended to bond to an outermembrane protein with a spytag domain such as [BBa_K2450401] in order to target this part to the outer membrane and subsequently outer membrane vesicles (OMVs).
-The sfGFP fluorescence is quenched by the dark quencher which converts the energy that would of been emitted as light and converts it into vibrational (heat) energy. This quenching is relieved by cleavage by the TEV protease which cleaves the dark quencher from sfGFP.
+The sfGFP fluorescence is quenched by the dark quencher which converts the energy that would of been emitted as light and converts it into vibrational (heat) energy. This quenching is relieved by cleavage of the TEV cleavage site by the TEV protease which cleaves the dark quencher from sfGFP. When not in close proximity to the sfGFP the dark quencher can no longer quench fluorescence, resuulting in a measurable increase in fluorescence.
-The aim of this part is to be able to detect lysis by the TEV protease in loacalised areas, specifically OMVs. The TorA leader sequence ensures the protein is targeted to the intermembrane space. The binding of this protein through the spycatcher/spytag system localises the protein to a particular protein/ region of the intermembrane space, such as the spytag-OmpA part [BBa_K2450451]. When this protein is taken up into the OMV and these are extracted from the cell, the part is protected from cleavage from the TEV protease, by the OMV membrane. On lysis of the OMV, the part can be cleaved and fluorescence observed, which can be used to measure the fraction of OMVs lysed by a particular technique.
+The aim of this part is to be able to detect lysis by the TEV protease in loacalised areas, specifically OMVs. The TorA leader sequence ensures the protein is targeted to the intermembrane space. The binding of this protein through the spycatcher/spytag system localises the protein to a particular protein/ region of the intermembrane space, such as the spytag-OmpA part [BBa_K2450451]. When this protein is taken up into the OMV and these are extracted from the cell, the part is protected from cleavage from the TEV protease, by the OMV membrane. On lysis of the OMV, the part can be cleaved and fluorescence observed, which can be used to measure the fraction of OMVs lysed by a particular technique. It can also be used to measure rates of cleavage at the outer membrane or in lysed OMVs, which can be used as a measure of the rates of macromolecular interaction at these membranes, which may be different to the rates of interaction in the cytoplasm.
-It can also be used to measure rates of cleavage at the outer membrane or in lysed OMVs, which can be used as a measure of the rates of macromolecular interaction at these membranes, which may be different to the rates of interaction in the cytoplasm.
 <span class='h3bb'>Sequence and Features</span>