Part:BBa_K2450501

SpyCatcher sfGFP quencher

SpyCatcher, sfGFP, TEV cleavable linker, reach quencher

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
  

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, BBa_K1319002 , from team Aachen 2014, 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:

Figure 1: Graph to show efficacy of quencher. Cells were grown overnight in minimal media (M9) in a plate reader and GFP was measured. Fluorescence was corrected against a blank of M9 and then the starting fluorescence was subtracted from end fluorescence to give absolute response.

The data in figure 1 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 8-10-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 (figure 2) and time-course (figure 3) 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.

Figure 2: End-point analysis of induction and cleavage. Cells were grown overnight in M9 media and subcultured down before being induced with arabinose at 0.1% w/v and IPTG. They were then allowed to run in a BMG CLARIOSTAR platereader for 10 hours. Three biological and technical repeats were averaged and the SEM used to make the error bars.

Figure 3: Graph showing the time-course of induction and cleavage. Conditions were the same as for Figure 4, above. The second graph shows error bars in dotted lines above and below each line, in the same colour.

As expected, induction of the protease relieved quenching of sfGFP. The time-course graph shows that the different levels of induction of our plasmid lead to different rates of increase of fluorescence, a story which is not clear from the end-point graph. The level of TEV-protease produced may reach a different steady-state in each strain, and leakiness of the lac promoter could lead to the increase in fluorescence even in the uninduced strain. The 5uM strain takes longer than the 500uM strain to reach the same level of fluorescence, even if the end result shows there is not a significant difference.

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