Difference between revisions of "Part:BBa K1319001"

Revision as of 22:40, 16 October 2014

RFC[25]-compatible dark quencher based on K1319000 (E0030)

This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP)..

Two mutations were introduced that eliminated fluorescence:

• L90I
• Y145W

References

• Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]

Usage and Biology

This protein is designed to be a dark quencher for GFP (E0040) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick K1319013 this is realised and the proteins are fused with the linker K1319016 which includes a specific TEV protease (available as K1319004) cleavage site. The fusion of the proteins bring GFP and REACh 1 in proximity to each other which allows GFP and REACh 1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh 1 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows for a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh 1 cancelling the FRET interaction and providing a GFP fluorescence response.

Characterization

In order to characterize K1319001 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319013, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319001 and not based on a faulty expression K1319008 was also introduced into the cells on a pSB1C3 backbone to allow for a double plasmid system. Both constructs were put into E. coli BL21 (DE3) compared to I20260 and B0015. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319013 and having the same promoter, RBS, Terminator and plasmid backbone.

The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.

Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319013 + K1319008 After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 9-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.

The strong fluorescence response (9-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319001. The cutting results in a separation of GFP and REACh 1 resulting in a collapse of the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is longer absorbed by REACh 1 and emitted as heat but rather as fluorescence with a wavelength of 511 nm.

The very low fluorescence in the non induced double plasmid system of K1319013 and K1319008 shows the functionality of K1319001. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319001. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 12,5. This also includes the slight leakiness of the TEV protease.

Sequence and Features