Team:EPF-Lausanne/LOVTAP

Brief overview

For organisms to thrive in a changing light environment, they sense and respond to light: the sensory information allows them to move in response to that stimulus. These responses are mediated by phototropins, which are photosensory proteins consisting of a serine-threonine kinase domain and a pair of nonidentical Light, Oxygen, or Voltage (LOV) sensitive domains which contain the noncovalently bound chromophore flavin mononucleotide (FMN).

In the article of Strickland et al., an allosteric switch was created by joining two domains, as expained below. The resulting protein has a domain-domain overlap with a shared helix, this shared helix acting as a rigid lever arm. The disruption of the helical contacts causes a shift in the conformation. Thus, photoexcitation would change the conformation of the protein, and that will change the stability of the helix-domain contacts. This will change the affinity of the shared helix for the two domains, and a signal could be then propagated.

The system is called LovTAP system. The light-sensitive input module is the LOV2 domain of Avena sativa phototropin 1 (AsLOV2). Absorption of a photon by AsLOV2 triggers the formation of a covalent adduct between FMN (the flavin mononucleotide) cofactor and a conserved cysteine residue. This formation leads to the displacement and unfolding of an helix in the LOV domain, which is likely to mediate a signal propagation. LOV domains absorb light through a flavin cofactor, photochemically form a covalent bond between the chromophore and a cysteine residue in the protein, and proceed to mediate activation of an attached kinase domain.

The output module was the bacterial transcription factor trp repressor (TrpR). TrpR can bind its operator DNA as a homodimer.

By ligating AsLOV2 to TrpR, they were able to construct an allosteric switch called LovTAP : LOV- and tryptophan-activated protein. This protein protects DNA from digestion when illuminated.

Phototropin switching mechanism is regulated at the stage of LOV2-Jalpha interactions, which are mostly intact in the dark. Illumination leads to the formation of a protein-flavin adduct which distorts the LOV2 structure sufficiently to substantially weaken the LOV2-Jalpha interactions, thus freeing the Jalpha-helix and allowing it to unfold. Taken from the article: Estimation of the available free energy in a LOV2-Jalpha photoswitch Xiaolan Yao, Michael K Rosen & Kevin H Gardner, Nature Chemical Biology 4, 491 - 497 (2008)

LovTAP can thus be found in two states : dark-state and light-state. The ground state is referred to as the ‘‘dark’’ state, and its photoactivated state as the ‘‘light’’ state. The TrpR domain associates with the shared helix (shared from AsLOV2 and TrpR).

In the dark state (A in the image below), a few residues of the shared helix presumably dissociate from the TrpR domain and dock against the LOV domain. The steric overlap is then relieved, which decreases the TrpR domain's DNA-binding affinity (when the shared helix contacts the LOV domain, the TrpR domain is in an inactive conformation).

In the light state, the residues reassociate with the TrpR domains (because the photoexcitation disrupts contacts between the shared helix and the LOV domain), which restores DNA-binding affinity and the system is in the active form (B). LovTAP can then bind DNA. But this system is not stable, and the LOV domains returns to the dark state (C), which triggers the dissociation of LovTAP from the DNA (D).

In our project, the idea is to implement such a system in E. coli, using synthetic biology and then caracterise it in vivo (in the article they have only done the caracterisation in vitro). First of all, we want to clone the LovTAP gene into an IPTG inductible BioBrick using a LacI promoter from the registry as well as a RBS and a terminator. Following this, we will clone a readout that would express RFP upon ligation of the LovTAP (after illumination) to its promoter sequence (the trp promoter). As we want to have RFP expression upon illumination (and thus binding of the LovTAP), we will need to create an inverter.

Cloning strategy

Our aim was to create a biobrick containing LovTAP under the influcence of an inducible promoter. This part needed to contain (in order) the inducible promoter (LacI), RBS (ribosome binging site), the LovTAP gene (that we recovered from Dr. ?? Sosnick’s lab) and finally a terminator (Term). This entire part was of course to be flanked by the standard prefix (E,X) and suffix (S,P). In order to synthesize this part, we proceeded in three steps :

1. We ligated LovTAP with the terminator (we used a double terminator ?).

2. We also ligated LacI with RBS.

3. Finally we ligated these two parts together to obtain our biobrick.

So finally we have :

With the biobrick, we have an inducible system : when we add IPTG, the promoter activates the expression of the LovTAP gene.

Read out system

Once we have our protein LovTAP produced, we needed a read out system to assess whether the protein was functional. We designed two different read out systems :

1. The read out 1 (RO1) contains the tryptophan operon followed by RBS, RFP and Term. In normal conditions, RFP should be expressed (so we should see some red fluorescence). This is because TRP repressor (TrpR) is expressed only if there is tryptophane available in the medium and then it inhibits TRP operon. The idea is that when LovTAP is produced (and in light state, that is when we add blue light), it will bind to the TRP operon and repress the RFP gene. We should therefore observe a decrease in red fluorescence. In order to characterize the read out, we tested it in different conditions : with or without TRP, with different amount of TRP, with or without light. If add TRP, we expect that the fluorescence will decrease.

2. The read out 2 (RO2) is composed of TRP op, RBS, TetR, Term and then TetRP, RBS, RFP and Term. It is a double repressor system : TrpR/LovTAP binds to the TRP operon, so repressing the expression of TetR. On the other hand, TetR (if expressed) inhibits the prodcution of RFP by acting on the TetR promoter. The final result is that if LovTAP is active, red fluorescence should increase as RFP is expressed. TRP has the same effect as LovTAP but we also used ATC (… ??) : ATC has the same effect as TetR, it binds to ???? qq un qui a compris comment marche le ATC complète svp ! When we add TRP and ATC, we should therefore have the same effect as with an active LovTAP : production of red fluorescence.

So all this function like this :

Results

The biobrick parts (inducible LovTAP, RO1 and RO2) have been produced. We are currently sequencing them to confirm that there are no errors in the insert.

We characterized RO1 and partly RO2 by culturing cells containing one of the read out systems under different conditions :

- With TRP

- With ATC

- With both

- Without anything

And at different concentrations.

We had some trouble with the media, as we usually cultured the cells in LB, but it contains some TRP so it pertubated the results. Therefore we had to find a medium in which there was no TRP but on which cells could grow. We did a lot of different tests and we finally settled on a medium containing M9/minimal, amino acids and thiamine. At the beginning we also compared the results between growth on LB or M9 to see the difference.

In the following graphics, we can see that RO1 effectively shows a decrease in fluorescence after of TRP :

This is a graphic of RO1#1 without any added TRP :

And here we add some TRP (one dose, see note book for the details of the experiment) :

So it is functional !

For RO2, it wasn’t as clear : if we add some ATC it seems to increase a little bit RFP fluorescence, if we add ATC + TRP also but if we add only TRP we don’t see any significant change. Here we only added a dose and a half of ATC :

Here we added TRP and ATC (one dose each) :

And there we added only TRP (1.5 dose):

Media we used

An interesting course on TrpR and Trp operon: [http://www2.hawaii.edu/~scallaha/SMCsite/475%20Lectures/475Lecture34.pdf TrpR]

Biobricks

Characterizes E.Coli Trp repressor, and the Trp operon sequence :
Summary of what characterizes E.Coli Trp repressor : The tryptophan biosynthetic pathway
One good article : RNA-based regulation of genes of tryptophan synthesis and degradation
Protein sequence from NCBI here‎‎
Design a biobrick that coexpresses LOVTAP and RFP (after Trp operon) when LOVTAP binds the Trp operon. Design a switch on/off read out.

Wet lab objectives

in vitro

- Redo the experiment they did in the LOVTAP article (2)

Major problem, the conformational change of LOVTAP is weak and the protection assay results show a small difference of LOVTAP binding on DNA between dark state and light state !!! ----> try to improve this