Coding
REACh1

Part:BBa_K1319001

Designed by: Michael Osthege, Florian Gohr   Group: iGEM14_Aachen   (2014-09-03)

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) called REACh1.

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. 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 realized and the proteins are fused together with the linker K1319016 which includes a specific TEV protease (available as K1319004) cleavage site. The fusion of the proteins bring GFP and REACh1 in proximity to each other which allows GFP and REACh1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh1 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows the release of a strong fluorescence signal, if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh1 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 create a double plasmid system. Both constructs were put into E. coli BL21 (DE3) and 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 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 REACh1 collapsing the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh1 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.

To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319001, K1319013 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).

Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319013
The expressed fusion protein K1319013 exhibits a fluorescence more than 30 fold smaller as the positive control of I20260.


This experiments shows that the fluorescence of the fusion protein GFP-REACh1 is more than 30-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of >96 %!

To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319013.

Check PCR for K1319013
The length of the PCR product matches the length of the control plasmid.


The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319013 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.

Check PCR for K1319008
The length of the PCR product matches the length of the control plasmid.


This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for K1319008 and K1319013 can be found in the parts registry.

characterization of K1319001 with the iGEM Team Aachen 2D Biosensor

K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells
The induced double plasmid systems K1319013 + K1319008 and K1319014 + K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG.


To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2 µL IPTG with a concentration of 100 mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496 ± 9 nm, emission 516 ± 9 nm) roughly every 10 min in the plate reader. The results were plotted in the heatmap shown on the left.

The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



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