Difference between revisions of "Part:BBa K2450501"

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===Characterisation===
 
===Characterisation===
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<b>Summary</b>: As this part was based on a previous part,we improved its characterisation by demonstrating that its functions of quenching GFP fluorescence and relieving this quenching upon addition of TEV protease were not affected by: <html></br></html>
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<b>Summary</b>: As this part was based on a previous part, we improved its characterisation by demonstrating that the functions of a) quenching GFP fluorescence and b) relieving this quenching upon addition of TEV protease were not affected by: <html></br></html>
 
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<html> <ol>
 
<li>Adding upstream leader sequences, the TorA and Spy-Tag </li>
 
<li>Adding upstream leader sequences, the TorA and Spy-Tag </li>

Revision as of 18:26, 31 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.

Characterisation

Summary: As this part was based on a previous part, we improved its characterisation by demonstrating that the functions of a) quenching GFP fluorescence and b) relieving this quenching upon addition of TEV protease were not affected by:

  1. Adding upstream leader sequences, the TorA and Spy-Tag
  2. Adding a poly-his tag on the c-terminus of the quencher
  3. Replacing GFP with sfGFP, allowing it to be used in the periplasm

For more details on why this was important for our project, please visit the results page of our wiki.
Results: We used PCR to create a part that did not contain the quencher, but still contained the upstream sequences of our part, and compared the fluorescence of this with the quenched sfGFP part: (placeholder graph) The above data demonstrate the efficiency of the quencher to quench sfGFP, and to quench when there is a poly his-tag and upstream leader sequences added, something which was previously unknown. There is a 2-3-fold increase in fluorescence when the quencher is removed, and the quenched sfGFP shows no higher fluorescence than a non-fluorescent control.


We also ran both end-point analysis and time-course to see the effectiveness of relief and the dynamics of the reaction. Here we co-transformed our part with a plasmid containing IPTG-inducible TEV protease.

As can be seen from the above graphs, when the plasmid containing our part (BBa_K2450501) (sfGFP with the Reach 2 quencher) was co-transformed with a plasmid containing an IPTG-inducible TEV protease, induction of the protease relieved quenching.



Please visit our wiki page to learn more about the specific experiments and the context of the part.

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