The REACh Construct

On this page, we present our biosensor on the molecular level. Explore the different parts of our genetic device:

A Fluorescence Answer Faster Than Expression

Biosensors often work with a system that is comprised of a reported gene under the control of a promoter directly induced by the chemical that the sensor is supposed to detect. In the case of our 2D biosensor for Pseudomonas aeruginosa, the expression of our reporter gene, GFP, would be directly induced by the activity of the bacterium's quorum sensing molecules. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we wanted to use a system that gives a fluorescent answer fast than just expressing the fluorescent protein.

Approach of a traditional biosensor In this model, the expression of GFP is directly controlled by a promoter whose activator binds to a molecule secreted by the pathogen.

Instead of the traditional approach, we constitutively express our reporter gene in a quenched form. As GFP-REACh fusion protein, fluorescence is suppressed. Our biosensor gives a response when homoserine lactones of Pseudomonas aeruginosa are taken up by our sensor cells where the autoinducer activates the expression of the TEV protease by binding to the LasI promoter in front of the protease gene.

Our novel biosensor approach Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elicit a fluorescence response.

This approach has two advantages:

• When Pseudomonas aeruginosa is detected by our cells, the reporter protein has already been expressed and only waits to be cleaved off the REACh quencher. The cleavage reaction catalyzed by the TEV protease is a faster process than expression and correct folding of GFP. This way we hope for an earlier response by our sensor cells.

• While a certain concentration of homoserine lactone will produce the same number of gene read-outs, one TEV protease can cleave many GFP-REACh constructs. Through the cleavage step, we introduce an amplification step into our system. With the TEV protease, we will be able to produce a much stronger signal in a short time interval.

Model of the molecular approach and the output over time The molecular setup of the novel biosensor (left) yields results indicating a strong and fast fluorescence output after induction (right). A directly inducable system was modeled and added to the plot for comparison. Tinkercell was used for modeling.

The novel biosensor approach was modeled as shown above. The plotted results also include a model of a direct expression of GFP as it appears in traditional biosensors. The strength of the promotor used for the traditional approach is twice as high as the strength of the promotor upstream of the TEV coding sequence in our novel approach. Despite the stronger promotor, a higher GFP concentration is generated in the model of the novel biosensor, proving the stronger and faster output of our construct in theory.

The FRET (Förster Resonance Energy Transfer) System

Förster resonance energy transfer (FRET), sometimes also called fluorescence resonance energy transfer, is a physical process of energy transfer. In FRET, the energy of a donor chromophore, whose electrons are in an excited state, is passed to a second chromophore, the acceptor. The energy is transferred without radiation and is therefore not exchanged via emission and absorption of photons. The acceptor then releases the energy received from the donor, for example, as light of a longer wavelength.

In biochemistry and cell biology, fluorescent dyes, which interact via FRET, are applied as "optical nano metering rules", because the intensity of the transfer is dependend on the spacing between donor and acceptor, and can be observed up to distances of 10 nm. This way, protein-protein interactions and conformational changes of a variety of tagged biomolecules can be observed. For efficient FRET to occur, there must be a substantial overlap between the donor fluorescence emission spectrum and the acceptor fluorescence excitation (or absorption) spectrum ([http://www.nature.com/nprot/journal/v8/n2/full/nprot.2012.147.html Broussard et al., 2013]), as described in the figure below.

FRET betweeen donor and acceptor On the left: Jablonski diagram showing the transfer of energy between donor and acceptor; on the right: For successful FRET, the emission spectrum of of the donor has to overlap with the absorption spectrum of the acceptor.

However, fluorescence is not an essential requirement for FRET. This type of energy transfer can also be observed between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence, and fit acceptors. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching instead. Thus, this kind of acceptors are also referred to as dark quenchers. In our project, we use a FRET system with a dark quencher, namely our REACh construct.

REACh Proteins - Dark Quenchers of GFP

In 2006, [http://www.pnas.org/content/103/11/4089.full Ganesan et al.] were the first to present a previously undescribed FRET acceptor, a non-fluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). YFP can be used as a FRET acceptor in combination with GFP as the donor in FRET microscopy and miscellaneous assays in molecular biology. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption - as illustrated in previous section - but have separated emission spectra to allow their selective imaging.

To optimize the spectral overlap of this FRET pair, the group obtained a genetically modified YFP acceptor. Mutations of amino acid residues that stabilize the excited state of the chromophore in enhanced YFP (EYFP) resulted in a non-fluorescent chromoprotein. Two mutations, H148V and Y145W, reduced the fluorescence emission by 82 and 98 %, respectively. Ganesan et al. chose the Y145W mutant and the Y145W/H148V double mutant as FRET acceptors, and named them REACh1 and REACh2, respectively. Both REACh1 and REACh2 act as dark quenchers of GFP.

Producing a GFP-REACh Fusion Protein

In our project, we reproduced the REACh1 and REACh2 proteins by subjecting an RFC-25 compatible version of the BioBrick [http://parts.igem.org/Part:BBa_E0030 E0030] (EYFP) to a QuikChange mutation, creating the BioBricks [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319002 K1319002]. Subsequently, we fused each REACh protein with GFP (mut3b) which is available as BioBrick [http://parts.igem.org/Part:BBa_E0040 E0040]. The protein complex was linked via a protease cleavage site, [http://parts.igem.org/Part:BBa_K1319016 K1319016]. As constitutive promoter we use [http://parts.igem.org/Part:BBa_J23101 J232101]. When GFP is connected to either REACh quencher, GFP will absorb light but the energy will be transferred to REACh via FRET and then emitted in the form of heat; the fluorescence is quenched. Our cells also constitutively express the [http://parts.igem.org/Part:BBa_C0179 LasR] activator. Together with the HSL molecules from P. aeruginosa, LasR binds to the [http://parts.igem.org/Part:BBa_J64010 LasI] promoter that controls the expression of the TEV protease, which we make available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]. When the fusion protein is cleaved by the TEV protease, REACh will be separated from GFP. The latter will then be able to absorb and emit light as usual.

Composition of our biosensor For our biosensor, we use a mix of already available and self-constructed BioBricks.

The resulting fusion proteins were labelled as [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP fused with REACh1) and [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP fused with REACh 2) and the linker between the proteins is labelled as [http://parts.igem.org/Part:BBa_K1319013 K1319016].

Cutting the Fusion Protein with the TEV Protease

To cut the GFP_REACh fusion protein off, we chose Tobacco Etch Virus (TEV) protease, a highly sequence-specific cysteine protease, that is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo. The native protease also contains an internal self-cleavage site. This site is slowly cleaved to inactivate the enzyme. The physiological reason for the self-cleavage is unknown, however, undesired for our use. Therefore, our team uses a variant of the native TEV protease that contains the mutation S219V which results in an alteration of the cleavage site so that self-inactivation is diminished.

TEV protease with a bound peptide This picture shows the TEV protease with a peptide chain bound in the binding pocket ready to be cleaved. The bound peptide chain has the recognition sequence inside the binding pocket. It is randered with POV-Ray.

Though quite popular in molecular biology, the TEV protease is not avaiable as a BioBrick yet. Hence, the Aachen team introduces a protease with anti-self cleavage mutation S219V and codon optimized for E. coli [http://parts.igem.org/Part:BBa_K1319004 to the Parts Registry this year.]

Achievements

Characterization of GFP-REACh1 and GFP-REACh 2 together with an IPTG inducible TEV protease

The characterization of the TEV protease and the REACh 1 and REACh 2 dark quencher proteins was performed by introducing both simultaneously into E. coli. The resulting double plasmid cells therefore contained [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP-REACh 1 fusion protein) and [http://parts.igem.org/Part:BBa_K1319008 K1319008] (IPTG inducible TEV protease) or [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP-REACh 2 fusion protein) and K1319008 respectively. K1319013 and K1319014 were situated on the pSB3K3 plasmid backbone and K1319008 on the pSB1C3 backbone, two standard plasmids with different oris allowing their simultaneous use in one cell.

As a positive control [http://parts.igem.org/Part:BBa_I20260 I20260] was used. I20260 contains the same promoter ([http://parts.igem.org/Part:BBa_J23101 J23101]] and RBS ([http://parts.igem.org/Part:BBa_B0032 B0032]) as well as the same fluorescence protein (GFP, [http://parts.igem.org/Part:BBa_E0040 E0040]) and is located on the same plasmid backbone (pSB3K3). Therefore it is expected that when all fusion proteins are successfully cut by the TEV protease, the fluorescence level of the double plasmid constructs reaches the same level as the positive control of I20260. As a negative control we used [http://parts.igem.org/Part:BBa_B0015 B0015], a coding sequence of a Terminator which shouldn't show any sign of fluorescence.

To better evaluate the fluorescence we adjusted the fluorescence reading with the observed OD to achieve a fluorecence reading independent of the amount of cells present but rather representative of the amount of fluorescence per cell. With this the fluorescence of a constitutive expression, no expression and both of the REACh + TEV constructs induced and not induced are observed. All measurements were done in a biological triplicate.

Comparison of K1319013 + K1319008, K1319014 + K1319008, I20260 (positive control) and B0015 (negative control) Fluorescence was normalized by dividing by the optical density.

The negative control B0015 does not exhibit any significant fluorescence as expected. The positive control I20260 shows a steady increase in fluorescence for the first X hours. After that the fluorescence stays constant due to the end of the exponential growth phase and the cells becoming stationary and not producing any more GFP. The production is also indpendent od the induction with IPTG as it is expected.

Both double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 don't exhibit a strong fluorescence before induction with IPTG. In the non induced constructs the fluorescence stays low and only increases slightly over time. It is severely weaker than the fluorescence reached by the induced constructs or the positive control but also higher then the negative control. This shows that the promoter system used is not completely shut down without induction but significantly weaker compared with the induced constructs. It is also a partly due to an imperfect dark quenching of GFP by the REACh 1 and REACh 2 proteins.

The induced double plasmid constructs exhibit a fast rise in fluorescence after induction up to an increase of over 10 fold compared to the non induced constructs. K1319013 + K1319008 reaches the the same level of fluorescence as I20260 indicating a complete cutting of the fusion proteins by the TEV protease. K1319014 + K1319008 doesn't reach the same level of fluorescence but the nearly 10 fold increase in fluorescence is a clear indicator for the TEV protease cutting the fusionprotein prodiced by K1319014. the same level of fluorescence as a the positive control is not achieved probably due to generally lower expression level of K1319014 in the cells.

Summary

The double plasmid systems of K1319013 + K1319008 as well as K1319014 + K1319008 clearly demonstrate the Quenching ability of the REACh 1 and REACh 2 proteins as well as the funcionality of the TEV protease. Both REACH 1 and REACH 2 show a significant quenching ability of GFP shown in the difference of fluorescence between the positive control I20260 and the non induced double plasmid systems. This is also comfirmed by the resulting fluorescence after induction showing that the TEV protease is successfully able to cut the fusion proteins as well as the proper expression of both fusion proteins. Combined this characterization shows a validation of the functionality of the REACh 1 protein ([http://parts.igem.org/Part:BBa_K1319001 K1319001]), the REACh 2 protein ([http://parts.igem.org/Part:BBa_K1319002 K1319002]) and the TEV protease ([http://parts.igem.org/Part:BBa_K1319004 K1319004]).

Comparing the kinetic of the GFP-REACh fusion proteins with a standard lacI inducible GFP epression

To assess the kinetic of the fusion proteins K1319013 (GFP-REACh 1) and K1319014 (GFP-REACh 2) the double plasmid systems of K1319013 + K1319008 and K1319014 + K1319008 were compared to a standard expression of GFP under the control of a lacI promoter in [http://parts.igem.org/Part:BBa_K731520 K731520] made by the iGEM Team TRENTO in 2012 to evaluate the kinetic prediction of an a faster fluorescence response with our construct compared to a normal expression.

Therefore K731520 and the same double plasmid construct that was described earlier (K1319013 + K1319008 and K1319014 + K1319008) were cultivated in E. coli BL21(DE3) and fluorescence and OD were measured. Once again the fluorescence was adjusted for the OD to show a relative fluorescence on a cell per cell basis. Also it was especially looked at the difference between the induced and not induced state. This difference (fluorescence quotient) gives a better indicator for a system which is used as a sensor because the difference between an on and off state is more important for a clear and unmistakable signal compared to the overall fluorescence. Hence the OD adjusted fluorescence quotient for both double plasmid constructs (K1319013 + K1319008 and K1319014 + K1319008) and K731520 was obtained and plotted in the following graphic.

Fluorescence was normalized by dividing by the optical density. The fluorescence of induced cells was additionally divided by the fluorescence of not induced cells.

The graphic clearly shows the faster kinetic of the cut GFP-REACh fusion protein compared to a standard GFP expression. Both fluorescence signals of the double plasmid constructs achieve a higher difference in fluorescence signal netween induced and non induced state as well as at a faster rate. This proves the earlier made hypothesis of the kinetic of the GFP-REACh fusion protein combined with a TEV protease.

summary

The kinectic of the fusion protein combined with the TEV protease exhibits the exact characteristics as predicted earlier. The response is clearly faster than normal expression by accumulating a reservoir of fusion proteins who are not fluorescing due to the dark quencher attaches to it. This reservoir is then activated by the induction of the TEV protease which results in the cutting of the fusion protein, releasing GFP from the dark quencher and disturbing the FRET mechanims between it and GFP. This results in the observed faster fluorescence reaction due to multiplicating effect by the TEV protease in which every one TEV protease can account for many fluorescence proteins being activated.

Characterizing the GFP-REACh constructs in sensor chips

K1319013 + K1319008 and K1319014 + K1319008 uninduced (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 and these then induced with 2 µl IPTG of 100 mM concentration. Then roughly every 10 minutes we measured fluorescence (GFP, excitation 496 ± 9 nm, emission 516 ± 9 nm) in the Platereader. The results were mapped in a heatmap as seen 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 uninduced chips. This again shows the constructs working as intended with the TEV protease cutting the linker so that the fusion protein gets separated into GFP and a dark quencher which are no longer in range of each other for a FRET style quenching. Therefore GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease.

Outlook

The system of the GFP-REACh fusion proteins with an inducible TEV protease has been establsihed and shows clearly the desired results of being faster than normal expression. The next step will be to engineer the TEV protease to be inducible by the HSL instead of IPTG and then to incorporate both the inducible TEV protease and the fusion protein on one plasmid backbone. This would also allow us to choose a high copy plasmid for both inserts, instead of a high copy plasmid for the TEV protease and a low to mid copy plasmid for the fusion protein which should yield an overall higher fluorescence readout.

Afterwards we would then like to characterize this construct the same way we have done with the double plasmid system and explore option for other fluorescence proteins than GFP to incorporate into our system with different quenchers to be able to have multiple fluorescence responses readable at the same time while being fast than the normal expression.

Also finding and testing different promoters to induce the TEV protease is planned to be able to detect not only Pseudomonas aeruginosa but also other pathogens or other relevant molecules in general so that we can establish a concept for faster recognition for a variety of uses.

References

• Broussard, Joshua A, Benjamin Rappaz, Donna J Webb, and Claire M Brown. "Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt." Nature protocols 8.2 (2013): 265-281. doi:10.1038/nprot.2012.147.

• Ganesan, Sundar, Simon M. Ameer-beg, Tony T. C. Ng, Borivoj Vojnovic, and Fred S. Wouters. " A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP." Proceedings of the National Academy of Sciences of the United States of America 103.11 (2006): 4089–4094. doi: 10.1073/pnas.0509922103

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