Introduction
In order to draw on a bacterial lawn with a laser pointer, we need bacteria to be able to respond to light. Previous iGEM teams have worked on many different ways to do this each with different advantages and disadvantages. We were struck by the simplicity and beauty of LovTAP. In particular, it functions by itself as a single protein; it is a brilliant example of genetic engineering; and, finally, it does not require any exotic supplements or specific strains of bacteria to function.
LovTAP Parts
As mentioned previously, LovTAP is a fusion protein. It consists of a light-response domain and a DNA binding domain, each of which are parts of other natural proteins.
Light Response
The light-response domain is LOV2, the photoactive domain (i.e. the light responsive part) of AsLOV2 (Avena sativa phototropin 1). AsLOV2 is a protein which allows Avena sativa to respond to 470 nm light. It does this by undergoing a major conformational change upon being struck by a photon with a wavelength near 470 nm. The absorption of the photon leads to the formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine residue.
This new bond distorts the conformation of the protein, causing the detachment and unfolding of the Ja-helix (see figure 1). In natural AsLOV2, the unfolding of the Ja-helix results in further downstream signalling. However, we will be most interested in the fact that the Ja-helix detaches when LOV2 is hit by blue light.
Figure 1.
DNA Binding
The DNA binding domain of LovTAP is the well-known bacterial transcription factor trpR. In the presence of tryptophan, the trpR protein will repress transcription of the E. coli trp operon by binding the operator region in the trp promoter and, thus, blocking RNA polymerase.
Figure 2
Overview
The two seemingly unrelated parts described above share one crucial feature: an alpha helix. LOV2 binds and unbinds an alpha-helix in response to light, and one domain in the functional trpR structure is “bound” to an alpha-helix. Strickland et al.’s idea was to force them to “fight over” a single alpha helix. Thus, since LOV2 has a higher affinity for the helix in the dark than trpR, there is no trpR activity in the dark. However, when exposed to light, LOV2 releases the alpha helix, allowing trpR to bind it and, thus, result in trpR activity (which of course is repression of the trp promoter).
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Figure 3 |
Details
Thirteen successive amino terminal truncations of trpR were ligated, in frame, downstream of the region coding for the Ja-helix in AsLOV2. One construct, referred to as LovTAP (the LOV- and tryptophan-activated protein), showed increased binding affinity to trp operator DNA when illuminated. Further tests showed that, upon light exposure, LovTAP binds DNA in a manner that is characteristic of the trpR domain. Additionally, mutation of the conserved cysteine of the LOV2 domain, which should prevent the conformational change that releases the Ja-helix, was shown to abolish binding to DNA in the presence of light. As this cysteine is crucial in the function of LOV2, this result suggests that the observed light sensitivity of the DNA-binding activity is due to LOV2.
Taken from Strickland et al.
A. Dark state-DNA dissociated: The shared helix contacts the LOV domain and trpR is in an inactive conformation.
B.
Photoexcitation occurs at 470nm (represented by the FMN chromophore going from yellow to white) causing the contact between the LOV domain and the shared helix to be disrupted. trpR now binds the helix and assumes its active conformation (represented by the helix going from blue to red).
C.
The active trpR domain of LovTAP binds the trp operator
D.
Cessation of light causes the LOV domain to return to its dark state, dissociating LovTAP from the DNA and reinstating contact between the LOV domain and shared helix.
Figure 4.
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