Team:Rutgers/Etch a Sketch

Abstract

            The Etch-a-Sketch project aims to create a lawn of bacteria that can be drawn on with a laser pointer. This seemingly inconsequential task actually presents many interesting engineering challenges. In particular, the bacteria need to be extremely sensitive in order to respond to a short light pulse from a laser, but they still must be “selective” enough to use in ambient lighting. We have designed a novel genetic switch that we hope will tackle these problems. If our work proves successful, it will serve as a useful model for future projects that require massive signal amplification. For example, researchers creating biosensors may find our work very helpful.

General Overview

            Our project can be broken down into three parts: light input, signal amplification, and color output. We used LovTAP to receive the light input; we designed a switch based on Peking 2007’s Bi-stable switch to amplify the signal; and finally (after dabbling with some of Cambridge 2009’s E. Chromi colors) decided to use mRFP1 as a color output.

LovTAP

Overview

In order to draw on bacteria 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 brilliantly engineered fusion protein; and, finally, it does not require any exotic supplements or strains of bacteria in order 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 wavelenght near 470 nm. In detail, 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 Jα-helix (see figure X). Detachment and unfolding of the Jα-helix can result in further downstream signalling. However, we will be most interested in the fact that the Jα-helix detaches when LOV2 is hit by blue light.

Figure X.

DNA Binding

The DNA binding domain of LovTAP is the well-known bacterial transcription factor trpR. As part of E. coli’s natural trp operon, in the presence of tryptophan, trpR will repress transcription at the trp promoter by binding the trp operator region and, thus, blocking RNA polymerase.

Figure Y.

LovTAP

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, trpR’s functional structure has one domain “bound” to an alpha-helix. Strickland et al.’s brilliant idea was to force them to “fight over” (since share implies that both can use the alpha helix simultaneously) 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, in the 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).

Details

AsLOV2 was ligated via its Jα-helix to a succession of 13-amino terminal truncations of trpR (11-108 residues). One construct, referred to as LovTAP (the LOV- and tryptophan-activated protein), preferentially protected trp operator DNA from digestion when illuminated, suggesting that LovTAP can bind the trp operator in the light. (The particular construct joins the Jα-helix of the LOV domain to the middle of the amino-terminal helix of trpR at Phe22). Further tests showed that upon light exposure LovTAP binds DNA in a manner that is characteristic of the TrpR domain. Additionally, mutation of the previously mentioned conserved cysteine of the LOV2 domain abolished the light sensitivity of DNA protection. As this cysteine has been shown to be crucial in the function of LOV2, this suggests that the observed light sensitivity is due to LOV2 (rather than some black magic).

Mechanism of Action:

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.

iGEM & LovTAP

EPFL

LovTAP was cloned into a biobrick and characterized by the EPF-Lausanne 2009 team. Strickland et al. measured only a 5-fold change in DNA binding affinity from dark state to light state in LovTAP. Though this change is miniscule (in biological scales), EPF-Lausanne found that there is a significant change in transcriptional output in response to 470nm light. Using molecular dynamics simulations, EPF-Lausanne found two mutations that may help improve LovTAP’s function by increasing the stability of the light state and decreasing the time it takes to flip from dark state to light state.

UNAM

A new LovTAP part was synthesized by the UNAM 2010 team via the alteration of EPF-Lausanne’s LovTAP. Primary changes include the removal of two 2 PstI restriction sites from the coding region of LovTAP and the addition of one of the point mutations that was proposed by EPF-Lausanne.

Our Project

For our circuit we will be using UNAM’s modified LovTAP, K360127. For the trp promoter and operator, we will be using ptrpL, K360023.

Possible issues

            UNAM appeared to not have nearly as much success with LovTAP as EPF-Lausanne. Whether this was due to the mutation that was introduced or some other factor is unclear. We hoped to study UNAM’s LovTAP and possibly introduce other mutations to improve LovTAP’s function. For example, Strickland et al. identified mutations that increased LovTAP’s dynamic range (i.e. the difference in repression in the dark state vs. the light state) from 5 to 70.

            Another issue is that LovTAP’s light state is unstable. Upon removal of the light source, LovTAP quickly returns to its inactive dark state. Thus, long exposure times might be necessary in order for the circuit to react to the light, which would be undesirable for drawing with a laser pointer. We may have to search for mutations that stabilize the light state, and thus allow LovTAP to remain in the light state for a longer time after being activated.

Peking Bi-stable Switch

Overview

            The Peking 2007 iGEM team designed a switch that can be flipped between two stable states (on and off), a so called bi-stable switch. Such a switch would be desirable in the Etch-a-Sketch circuit because it would allow the bacteria to “remember” if it was exposed to light, and, thus, only a short exposure to light would be necessary in order to draw on our bacteria (rather than keeping the light on the bacteria until color appeared). However, Peking’s original design was not ideal because one state was not completely stable; once the switch is flipped on, it will slowly decay back to the off state. We would like our drawings to be permanent (to some degree), so we would need the on state to be stable. We redesigned the Peking bi-stable switch for this purpose.

Original Design

            The Peking 2007 switch uses pR, pRM, cI, and cI434. Avoiding unnecessary detail: cI activates pRM and represses pR; cI434 represses pRM; pR has high basal transcription; and pRM has low basal transcription.

State 1

In the first state, cI434 levels are high and cI levels are low. This leads to high transcription from pR and low transcription from pRM, which, in this particular circuit, results in GFP production. The switch can be flipped by increasing cI levels.

State 2

In the second state, cI levels are high and cI434 levels are low. This leads to high transcription from pR, low transcription from pRM, and thus RFP production. Empirical evidence shows the switch will spontaneously decay back to state 1. This is probably due to the high basal transcription levels of pR.

Our design: Locking Switch

For our switch, we desired a simple circuit with a stable on state. The parts we used are ptrpL, cI434, pRM, trpR, and cI. As in the Peking switch, cI activates pRM and cI434 represses pRM. We cloned two additional parts for our switch: ptrpL and trpR. ptrpL is the well-known trp operon/promoter/leader sequence. trpR is the corresponding repressor which is induced by tryptophan. In our system, we will assume there are constantly high levels of tryptophan so that trpR will always act as a repressor.

State 1

In the first state, cI434 levels are high, trpR levels are low, and cI levels are low. This results in high transcription from ptrpL, low transcription from pRM, and thus low output signal. The switch can be flipped by reducing transcription from ptrpL.

State 2

In the second state, cI434 levels are low, trpR levels are high, and cI levels are high. This results in low transcription from ptrpL, high transcription from pRM, and thus high output signal. Since trpR is a strong repressor for ptrpL, we expect state 2 to be stable.

mRFP1

For our output signal, we decided to use mRFP1. It has many attractive qualities: the color is visible in normal lighting; no special supplements are necessary in order for the color to develop; the sequence is short (as compared to other pigment generating proteins); the protein itself generates the color, which hopefully will result in faster image development than a protein which catalyzes a reaction that generates color.

mRFP1 is a red fluorescent protein based on DsRed. Natural DsRed requires tetramerization in order to fluoresce. mRFP1 contains 33 mutations that, one, allow it to function as a monomer and, two, allow it to fold more quickly.