Team:Rutgers/Etch a Sketch

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EaS writeup.docx

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 sensitive enough to respond to a short light pulse from a laser, but still “selective” to not respond to ambient lighting. We have designed a novel genetic switch to tackle these problems. If our work proves successful, it will serve as a useful model for future projects that require large 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) we decided to use mRFP1 as a color output.

LovTAP

Overview

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 Jα-helix (see figure X). In natural AsLOV2, the unfolding of the Jα-helix results 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. (Need a figure legend)

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 Y. (Need figure legend)

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 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).

Details

Thirteen successive amino terminal truncations of trpR were ligated, in frame, downstream of the region coding for the Jα-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 Jα-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.

Mechanism of Action:

Taken from Strickland et al.

Dark state-DNA dissociated: The shared helix contacts the LOV domain and trpR is in an inactive conformation.
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).
The active trpR domain of LovTAP binds the trp operator
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 initially 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 relatively small (in biological scales), the EPF-Lausanne team found that there is a significant change in transcriptional output in response to 470nm light. Using molecular dynamics simulations, the EPF-Lausanne team found two mutations that were predicted to 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 the EPF-Lausanne team.

Our Project

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

Possible issues

The UNAM team appeared to not have nearly as much success with LovTAP as the EPF-Lausanne team. 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 the conformational change of LovTAP in the presence of light is relatively unstable. Upon removal of the light source, LovTAP quickly returns to a conformation in which the Jα-helix binds to the AsLOV2 domain, inactivating the trpR DNA-binding activity. Thus, long exposure times might be necessary 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 genetic 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 had been exposed to light, so that only a short exposure to light would be necessary to draw on our bacteria (rather than keeping the light on the bacteria until color appeared). However, the switch design was not ideal because one state was not completely stable; once the switch was flipped on, it slowly decayed back to the off state. We would like our drawings to be permanent (to some degree), so we would need the activated state to be stable. We therefore redesigned the Peking bi-stable switch for this purpose.

Original Design

The Peking 2007 switch uses the pR and pRM promoters and the cI and cI434 transcriptional regulatory proteins. The cI protein activates pRM and represses pR; cI434 represses pRM; pR has high basal transcription; and pRM has low basal transcription.

Deactivated State

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.

Activated State

In the second state, cI levels are high and cI434 levels are low. This leads to high transcription from pR and low transcription from pRM, and therefore RFP is produced. Empirical evidence shows the switch will spontaneously decay back to the deactivated state. 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 ptrp, 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: ptrp and trpR. ptrp is the well-known trp promoter and operator 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.

Deactivated State

When deactivated, cI434 levels are high, trpR levels are low, and cI levels are low. This results in high transcription from ptrp, low transcription from pRM, and thus low output signal. The switch can be flipped by reducing transcription from ptrp.

Activated State

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

mRFP1

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

Engineering Problems

We have identified five major engineering problems with Etch-a-Sketch that we have called: Light, Sensitivity, Selectivity, Speed, and Noise.

Light - In order to draw on bacteria with a laser pointer, we need the bacteria to respond to light.

Sensitivity - In order to draw naturally, we need the bacteria to respond to extremely quick pulses of light; otherwise, we would have to make very slow strokes.

Selectivity - In order to use the bacteria in ambient lighting, we need to the bacteria to be able to selectively respond to just the laser pointer.

Speed - After we make our strokes with the laser pointer, we would like to see color quickly.

Noise - We do not want random spots to appear on our canvas.

We have prioritized Light and Sensitivity. This hopefully would result in something that works like an old camera; instant image capture but overnight image development.

Circuit

Overview

We have broken down the circuit into four states: Deactivated, Activation 1, Activation 2, and Activated. To recap the parts of our circuit: LovTAP represses ptrp in the light; trpR represses ptrp; cI434 repressed pRM; cI activates pRM; T7 polymerase (T7 P) transcribes at the T7 promoter; and mRFP1 generates a red color.

Deactivated

In the dark, LovTAP is inactive; transcription levels from ptrp should be high, creating a lot of cI434 which should shut down transcription from pRM.

Activation 1

After light is applied, LovTAP is activated, repressing transcription from ptrp. cI434 levels should begin to fall.

Activation 2

Once cI434 levels have dropped enough so that transcription from pRM can begin, the circuit enters an irreversible positive feedback loop. trpR levels will begin to increase, preventing the creation of cI434. cI levels will increase, further increasing transcription from pRM. Finally, T7 polymerase levels will increase, allowing the creation of mRFP1.

Activated

After entering the Activation 2 state, the light may be removed, and the cell will continue to produce mRFP1.

Mapping the circuit back to the engineering problems

Light - Our light response depends entirely on LovTAP. If it proves ineffectual, we will either need to introduce mutations that will help its function, or switch to a different system.

Sensitivity - The amount of time necessary to keep the laser pointer on the bacteria should be related to the time it takes for the locking switch to flip to the activated state and the length of time LovTAP remains in its activated state once illuminated. The former should be small by design of the switch. The latter may be improved by mutations or changing to a different light response system, if necessary.

Selectivity - The circuit should respond to only to wavelengths and intensities of light that can flip LovTAP from the dark state to the light state. Since LovTAP responds primarily to blue light, we may need to work in red ambient lighting.

Speed - The delay from the moment the locking switch is activated to the appearance of color will be related to the time it takes for the bacteria to synthesize mRFP1. This will hopefully be minimized as transcription from T7 promoters is very high when T7 polymerase is present.

Noise - Unwanted splotches will be created if there is unwanted transcription from the T7 promoter or if the locking switch is accidentally turned on by unwanted transcription from the pRM promoter. The former should be minimal as the T7 promoter is very specific to T7 polymerase. The latter should be reduced by placing the locking switch on a low-copy plasmid.

Home	Team	Official Team Profile	Etch-a-Sketch	Full Adder	MYS!S	Parts Submitted to the Registry	Modeling	Notebook	Safety	Acknowledgements

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+<html><head><title>EaS writeup.docx</title><style type="text/css">ol{margin:0;padding:0}p{margin:0}.c2{text-indent:36pt;direction:ltr;padding-bottom:15pt}.c4{text-indent:36pt;direction:ltr;padding-bottom:5pt}.c9{width:648pt;background-color:#ffffff;padding:72pt 72pt 72pt 72pt}.c15{list-style-type:upper-latin;margin:0;padding:0}.c11{height:11pt;padding-bottom:5pt}.c7{padding-left:0pt;margin-left:36pt}.c0{font-size:12pt;font-family:Times New Roman}.c13{padding-bottom:5pt}.c14{padding-bottom:15pt}.c1{font-size:12pt}.c10{text-decoration:underline}.c3{font-weight:bold}.c6{text-indent:36pt}.c12{height:11pt}.c5{direction:ltr}.c8{font-style:italic}body{color:#000000;font-size:11pt;font-family:Times New Roman}h1{padding-top:12pt;line-height:1.0;text-align:left;color:#000000;font-size:16pt;font-family:Arial;font-weight:bold;padding-bottom:3pt}h2{padding-top:12pt;line-height:1.0;text-align:left;color:#000000;font-style:italic;font-size:14pt;font-family:Arial;font-weight:bold;padding-bottom:3pt}h3{padding-top:12pt;line-height:1.0;text-align:left;color:#000000;font-size:13pt;font-family:Arial;font-weight:bold;padding-bottom:3pt}h4{padding-top:12pt;line-height:1.0;text-align:left;color:#000000;font-size:14pt;font-family:Times New Roman;font-weight:bold;padding-bottom:3pt}h5{padding-top:12pt;line-height:1.0;text-align:left;color:#000000;font-style:italic;font-size:13pt;font-family:Times New Roman;font-weight:bold;padding-bottom:3pt}h6{padding-top:12pt;line-height:1.0;text-align:left;color:#000000;font-size:11pt;font-family:Times New Roman;font-weight:bold;padding-bottom:3pt}</style></head><body class="c9"><a href="#" name="id.bda7460600d8"></a><p class="c5"><span class="c1 c10 c3">Abstract</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 sensitive </span><span class="c1">enough </span><span class="c1">to respond to a short light pulse from a laser, but still &ldquo;selective&rdquo; to </span><span class="c1">not respond to</span><span class="c1">&nbsp;ambient lighting. We have designed a novel genetic switch </span><span class="c1">to</span><span class="c1">&nbsp;tackle these problems. If our work proves successful, it will serve as a useful model for future projects that require </span><span class="c1">large</span><span class="c1">&nbsp;signal amplification. For example, researchers creating biosensors may find our work very helpful.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c3 c10">General Overview</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Our project can be broken down into three parts: light input, signal amplification, and color output. We used </span><span class="c1">LovTAP</span><span class="c1">&nbsp;to receive the light input; we designed a switch based on Peking 2007&rsquo;s Bi-stable switch to amplify the signal; and, finally, (after dabbling with some of Cambridge 2009&rsquo;s E. Chromi colors) we decided to use mRFP1 as a color output. </span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c10 c3">LovTAP</span></p><p class="c13 c5"><span class="c1 c3">Overview</span></p><p class="c4"><span class="c1">In order to draw on a bacterial</span><span class="c1">&nbsp;lawn</span><span class="c1">&nbsp;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; </span><span class="c1">it is a brilliant</span><span class="c1">&nbsp;example of genetic engineering</span><span class="c1">; and, finally, it does not require any </span><span class="c1">exotic</span><span class="c1">&nbsp;supplements or specific strains of bacteria to function. </span></p><p class="c11 c5"><span class="c1"></span></p><p class="c5 c13"><span class="c1 c3">LovTAP Parts</span></p><p class="c4"><span class="c1">As mentioned previously, LovTAP is a fusion protein. It consists of a light-response domain and a DNA binding domain</span><span class="c1">, each of which are parts of other natural proteins.</span></p><p class="c5 c11"><span class="c1"></span></p><p class="c13 c5"><span class="c1 c8">Light response</span></p><p class="c13 c5"><span class="c1 c8">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c1">The light-response domain is LOV2, the photoactive domain (i.e. the light responsive part) of AsLOV2 (</span><span class="c1 c8">Avena sativa</span><span class="c1">&nbsp;phototropin 1). AsLOV2 is a protein which allows </span><span class="c1 c8">Avena sativa </span><span class="c1">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 J&alpha;-helix (see figure X). In natural AsLOV2, the unfolding of the J&alpha;-helix results in further </span><span class="c1">downstream signalling.</span><span class="c1">&nbsp;However, we will be most interested in the fact that the J&alpha;-helix detaches when LOV2 is hit by blue light.</span></p><p class="c13 c5"><img height="160" src="images/image09.png" width="456"></p><p class="c13 c5"><span class="c1">Figure X. (Need a figure legen</span><span class="c1">d)</span></p><p class="c13 c5"><span class="c1 c8">DNA Binding</span></p><p class="c4"><span class="c1">The DNA binding domain of LovTAP is the well-known bacterial transcription factor </span><span class="c1">trpR</span><span class="c1">. In the presence of tryptophan, the trpR protein will repress transcription </span><span class="c1">of</span><span class="c1">&nbsp;t</span><span class="c1">he </span><span class="c1 c8">E. coli trp</span><span class="c1">&nbsp;operon </span><span class="c1">by binding the operator region in the </span><span class="c1 c8">trp</span><span class="c1">&nbsp;p</span><span class="c1">romoter </span><span class="c1">and, thus, blocking RNA polymerase.</span></p><p class="c13 c5"><img height="188" src="images/image10.jpg" width="408"></p><p class="c13 c5"><span class="c1">Figure Y. </span><span class="c1">(Need figure legend)</span></p><p class="c14 c12 c5"><span class="c1"></span></p><p class="c14 c5"><span class="c1 c3">LovTAP</span></p><p class="c14 c5"><span class="c1 c8">Overview</span></p><p class="c2"><span class="c1">The two seemingly unrelated parts described above share one crucial feature</span><span class="c1">:</span><span class="c1">&nbsp;an alpha helix. LOV2 binds and unbinds an alpha-helix in response to light, and one d</span><span class="c1">omain in the</span><span class="c1">&nbsp;functional trpR structure </span><span class="c1">is</span><span class="c1">&nbsp;&ldquo;bound&rdquo; to an alpha-helix. Strickland et al.&rsquo;s idea was to force them to &ldquo;fight over&rdquo;</span><span class="c1">&nbsp;</span><span class="c1">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, </span><span class="c1">when exposed to</span><span class="c1">&nbsp;light, LOV2 releases the alpha helix, allowing trpR to bind it and, thus, result in trpR activity (which of course is repression of the </span><span class="c1 c8">trp</span><span class="c1">&nbsp;promoter).</span></p><p class="c2"><img height="198" src="images/image03.gif" width="499"></p><p class="c14 c5"><span class="c1 c8">Details</span></p><p class="c4"><span class="c1">Thirteen successive </span><span class="c1">amino terminal truncations of trpR </span><span class="c1">were l</span><span class="c1">ig</span><span class="c1">ated, in frame, downstream of the region coding for the J&alpha;-helix in AsLOV2</span><span class="c1">. One construct, referred to as LovTAP (</span><span class="c1">the </span><span class="c1 c3">LOV</span><span class="c1">- and </span><span class="c1 c3">t</span><span class="c1">ryptophan-</span><span class="c1 c3">a</span><span class="c1">ctivated </span><span class="c1 c3">p</span><span class="c1">rotein</span><span class="c1">), showed increased bindin</span><span class="c1">g affinity to</span><span class="c1">&nbsp;</span><span class="c1 c8">trp</span><span class="c1">&nbsp;operator DNA</span><span class="c1">&nbsp;when illuminated</span><span class="c1">. </span><span class="c1">Further tests </span><span class="c1">showed that, upon light exposure, LovTAP binds DNA in a manner that is characteristic of the </span><span class="c1">t</span><span class="c1">rpR domain. </span><span class="c1">Additionally, mutation of the co</span><span class="c1">nserved </span><span class="c1">cysteine of the LOV2 domain, which sh</span><span class="c1">ould</span><span class="c1">&nbsp;prevent</span><span class="c1">&nbsp;the conformational change that releases the </span><span class="c1">J&alpha;-helix, was shown to</span><span class="c1">&nbsp;</span><span class="c1">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.</span></p><p class="c4 c12"><span class="c1"></span></p><p class="c4 c12"><span class="c1"></span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1">Mechanism of Action: </span></p><p class="c14 c5"><img height="404" src="images/image06.gif" width="544"></p><p class="c5 c14"><span class="c1">Taken from Strickland et al. </span></p><ol class="c15" start="1"><li class="c5 c7"><span class="c0">Dark state-DNA dissociated: The shared helix contacts the LOV domain and trpR is in an inactive conformation. </span></li><li class="c7 c5"><span class="c0">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).</span></li><li class="c7 c5"><span class="c0">The active trpR domain of LovTAP binds the trp operator</span></li><li class="c7 c5"><span class="c0">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. </span></li></ol><p class="c12 c5"><span class="c0"></span></p><p class="c5"><span class="c0 c3">iGEM &amp; LovTAP </span></p><p class="c5"><span class="c0 c8">EPFL</span></p><p class="c6 c5"><span class="c0">LovTAP was initially cloned into a </span><span class="c1">B</span><span class="c0">io</span><span class="c1">B</span><span class="c0">rick and characterized by the EPF-Lausanne 2009 team. Strickland et al. measured only a 5-fold change in DNA</span><span class="c1">-</span><span class="c0">binding affinity from dark state to light state in LovTAP. Though this change is </span><span class="c1">relatively small </span><span class="c0">(in biological scales), the EPF-Lausanne team found that there is a significant change in transcriptional output in response to 470nm light. Using molecular dynamics simulations, the EPF-Lausanne team found two mutations </span><span class="c1">that were predicted to </span><span class="c0">improve LovTAP&rsquo;s function by increasing the stability of the light state and decreasing the time it takes to flip from dark state to light state.</span></p><p class="c6 c12 c5"><span class="c0"></span></p><p class="c5"><span class="c0 c8">UNAM</span></p><p class="c5"><span class="c0">A new LovTAP part was synthesized by the UNAM 2010 team via the alteration of EPF-Lausanne&rsquo;s LovTAP. Primary changes include the removal of two 2&nbsp;PstI restriction sites from the coding region of LovTAP and the addition of one of the point mutations that was proposed by the EPF-Lausanne team. </span></p><p class="c14 c12 c5"><span class="c0"></span></p><p class="c14 c5"><span class="c1 c3">Our Project</span></p><p class="c6 c5"><span class="c1">For our circuit we will be using UNAM&rsquo;s modified LovTAP, K360127. For the </span><span class="c1 c8">trp </span><span class="c1">promoter and operator, we will be using our own ptrp, based on ptrpL K360023. </span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c8">Possible issues</span></p><p class="c5"><span class="c1 c8">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c1">The</span><span class="c1 c8">&nbsp;</span><span class="c1">UNAM team appeared to not have nearly as much success with LovTAP as the EPF-Lausanne team. Whether this was due to the mutation that was introduced or some other factor is unclear. We hoped to study UNAM&rsquo;s LovTAP and possibly introduce other mutations to improve LovTAP&rsquo;s function. </span><span class="c1">For example, Strickland et al. identified mutations that increased LovTAP&rsquo;s dynamic range (i.e. the difference in repression in the dark state vs. the light state) from 5 to 70.</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Another issue is that the conf</span><span class="c1">ormational change of </span><span class="c1">LovTAP</span><span class="c1">&nbsp;in the presence of</span><span class="c1">&nbsp;light is relatively unstable. Upon removal of the light source, LovTAP quickly returns to a con</span><span class="c1">formation in which the J&alpha;-helix binds to the AsLOV2 domain, inactivating the trpR DNA-binding activity. &nbsp;</span><span class="c1">Thus, long exposure times might be necessary for the circuit to react to the light, which would be undesirable for drawing with a laser pointer.</span><span class="c1">&nbsp;We may have to search for mutations that stabilize the light state, and thus allow </span><span class="c1">LovTAP to remain in the light state for a longer time after being activated</span><span class="c1">.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c10 c3">Peking Bi-stable Switch </span></p><p class="c5"><span class="c1 c3">Overview</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The Peking 2007 iGEM team designed a genet</span><span class="c1">ic</span><span class="c1">&nbsp;switch that can be flipped between two stable states (on and off), a so</span><span class="c1">-</span><span class="c1">called bi-stable switch. Such a switch would be desirable in the Etch-a-Sketch circuit because it would allow the bacteria to &ldquo;remember&rdquo; if it </span><span class="c1">had been</span><span class="c1">&nbsp;exposed to light, </span><span class="c1">so that</span><span class="c1">&nbsp;only a short exposure to light would be necessary to draw on our bacteria (rather than keeping the light on the bacteria until color appeared). However, </span><span class="c1">the switch </span><span class="c1">design was not ideal </span><span class="c1">because</span><span class="c1">&nbsp;one state was not completely stable; once the switch </span><span class="c1">wa</span><span class="c1">s flipped on, it slowly decayed back to the off state. We would like our drawings to be permanent (to some degree), so we would need the </span><span class="c1">activated</span><span class="c1">&nbsp;state to be stable. We therefore redesigned the Peking bi-stable switch for this purpose.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c3">Original Design</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The Peking 2007 switch uses the pR</span><span class="c1">&nbsp;and</span><span class="c1">&nbsp;pRM promoters</span><span class="c1">&nbsp;and the</span><span class="c1">&nbsp;cI and cI434 </span><span class="c1">transcriptional regulatory</span><span class="c1">&nbsp;proteins. </span><span class="c1">The</span><span class="c1">&nbsp;cI protein activates pRM and represses pR; cI434 represses pRM; pR has high basal transcription; and pRM has low basal transcription. </span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c8">Deactivated State</span></p><p class="c5"><img height="146" src="images/image05.png" width="792"></p><p class="c5"><span class="c1">In</span><span class="c1">&nbsp;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 </span><span class="c1">levels.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c8">Activated State</span></p><p class="c5"><img height="149" src="images/image07.png" width="792"></p><p class="c5"><span class="c1">In the second state, cI levels are high and cI434 levels are low. This leads to high transcription from pR</span><span class="c1">&nbsp;and</span><span class="c1">&nbsp;</span><span class="c1">low </span><span class="c1">transcription from pRM, </span><span class="c1">and therefore</span><span class="c1">&nbsp;RFP i</span><span class="c1">s </span><span class="c1">produc</span><span class="c1">ed</span><span class="c1">. Empirical evidence shows the switch will spontaneously decay back to </span><span class="c1">the deactivated state</span><span class="c1">. This is probably due to the high basal transcription levels of pR.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c3">Our </span><span class="c1 c3">D</span><span class="c1 c3">esign: Locking Switch</span></p><p class="c5"><span class="c1">For our switch, we desired a simple circuit with a stable on state. The parts we used are ptrp, 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: ptrp and trpR. ptrp is the well-known </span><span class="c1 c8">trp</span><span class="c1">&nbsp;</span><span class="c1">promoter and operator</span><span class="c1">&nbsp;sequence. trpR is the corresponding</span><span class="c1">&nbsp;</span><span class="c1">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. </span></p><p class="c5 c12"><span class="c1"></span></p><p class="c5"><span class="c1 c8">Deactivated State</span></p><p class="c5"><img height="154" src="images/image02.png" width="775"></p><p class="c5"><span class="c1">When deactivated</span><span class="c1">, cI434 levels are high, trpR levels are low, and cI levels are low. This results in high transcription from ptrp, low transcription from pRM, and thus low output signal. The switch can be flipped by reducing transcription from </span><span class="c1">ptrp.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c8">Activated State</span></p><p class="c5"><img height="154" src="images/image11.png" width="775"><span class="c1"><br>In the </span><span class="c1">active </span><span class="c1">state, cI434 levels are low, trpR levels are high, and cI levels are high. This results in low transcription from ptrp, high transcription from pRM, and thus high output signal. Since trpR is a strong repressor for ptrp, we expect state 2 to be stable.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c10 c3">mRFP1</span></p><p class="c6 c5"><span class="c1">For our output signal, we decided to use mRFP1. </span><span class="c1">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. </span><span class="c1">It has many attractive qualities: the color is visible in normal lighting; no special supplements are necessary for the color to develop; the coding sequence for the protein is short (as compared to </span><span class="c1">other pigment generating proteins</span><span class="c1">); finally, the protein itself generates the color, which hopefully will result in faster image development than a protein which catalyzes a reaction that generates color.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c10 c3">Engineering Problems</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We have identified five major engineering problems with Etch-a-Sketch that we have called: Light, Sensitivity, Selectivity, Speed, and Noise.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c8">Light - </span><span class="c1">In order to draw on bacteria with a laser pointer, we need the bacteria to respond to light. </span></p><p class="c5"><span class="c1 c8">Sensitivity - </span><span class="c1">In order to draw naturally, we need the bacteria to respond to extremely quick pulses of light; otherwise, we would have to make very slow strokes.</span></p><p class="c5"><span class="c1 c8">Selectivity</span><span class="c1">&nbsp;- In order to use the bacteria in ambient lighting, we need to the bacteria to be able to selectively respond to just the laser pointer.</span></p><p class="c5"><span class="c1 c8">Speed</span><span class="c1">&nbsp;- After we make our strokes with the laser pointer, we would like to see color quickly.</span></p><p class="c5"><span class="c1 c8">Noise - </span><span class="c1">We do not want random spots to appear on our canvas.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We have prioritized Light and Sensitivity. This hopefully would result in something that works like an old camera; instant image capture but overnight image development.</span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c10 c3">Circuit</span></p><p class="c5"><span class="c1 c3">Overview</span></p><p class="c5 c6"><span class="c1">We have broken down the circuit into four states: Deactivated, Activation 1, Activation 2, and Activated. To recap the parts of our circuit: LovTAP represses ptrp in the light; trpR represses ptrp; cI434 repressed pRM; cI activates pRM; T7 polymerase (T7 P) transcribes at the T7 promoter; and mRFP1 generates a red color.</span></p><p class="c5"><img height="498" src="images/image12.png" width="800"></p><p class="c5"><span class="c1 c3">Deactivated</span></p><p class="c6 c5"><span class="c1">In the dark, LovTAP is inactive; transcription levels from ptrp should be high, creating a lot of cI434 which should shut down transcription from pRM.</span></p><p class="c5"><img height="502" src="images/image00.png" width="800"></p><p class="c12 c5"><span class="c1"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c5"><span class="c1 c3">Activation 1</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After light is applied, LovTAP is activated, repressing transcription from ptrp. cI434 levels should begin to fall.</span></p><p class="c5"><img height="502" src="images/image08.png" width="800"></p><p class="c12 c5"><span class="c1"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c5"><span class="c1 c3">Activation 2</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Once cI434 levels have dropped enough so that transcription from pRM can begin, the circuit enters an irreversible positive feedback loop. trpR levels will begin to increase, preventing the creation of cI434. cI levels will increase, further increasing transcription from pRM. Finally, T7 polymerase levels will increase, allowing the creation of mRFP1.</span></p><p class="c5"><img height="502" src="images/image01.png" width="800"></p><p class="c12 c5"><span class="c1"></span></p><p class="c12 c5"><span class="c1"></span></p><p class="c5"><span class="c1 c3">Activated</span></p><p class="c5"><span class="c1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After entering the Activation 2 state, the light may be removed, and the cell will continue to produce mRFP1.</span></p><p class="c5"><img height="502" src="images/image04.png" width="800"></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c12 c5"><span class="c1 c3"></span></p><p class="c5"><span class="c1 c3">Mapping the circuit back to the engineering problems</span></p><p class="c5"><span class="c1 c8">Light - </span><span class="c1">Our light response depends entirely on LovTAP. If it proves ineffectual, we will either need to introduce mutations that will help its function, or switch to a different system.</span></p><p class="c5"><span class="c1 c8">Sensitivity - </span><span class="c1">The amount of time necessary to keep the laser pointer on the bacteria should be related to the time it takes for the locking switch to flip to the activated state and the length of time LovTAP remains in its activated state once illuminated. The former should be small by design of the switch. The latter may be improved by mutations or changing to a different light response system, if necessary.</span></p><p class="c5"><span class="c1 c8">Selectivity - </span><span class="c1">The circuit should respond to only to wavelengths and intensities of light that can flip LovTAP from the dark state to the light state. Since LovTAP responds primarily to blue light, we may need to work in red ambient lighting.</span></p><p class="c5"><span class="c1 c8">Speed - </span><span class="c1">The delay from the moment the locking switch is activated to the appearance of color will be related to the time it takes for the bacteria to synthesize mRFP1. This will hopefully be minimized as transcription from T7 promoters is very high when T7 polymerase is present.</span></p><p class="c5"><span class="c1 c8">Noise - </span><span class="c1">Unwanted splotches will be created if there is unwanted transcription from the T7 promoter or if the locking switch is accidentally turned on by unwanted transcription from the pRM promoter. The former should be minimal as the T7 promoter is very specific to T7 polymerase. The latter should be reduced by placing the locking switch on a low-copy plasmid.</span></p></body></html>
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-<p class=MsoNormal><b><u><span style='font-size:12.0pt'>Abstract</span></u></b></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><span style='mso-tab-count:
-'>            </span>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.<o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><b><u><span style='font-size:12.0pt'>General Overview<o:p></o:p></span></u></b></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><span style='mso-tab-count:
-'>            </span>Our project can be broken down into three parts: light
-input, signal amplification, and color output. We used <span class=SpellE>LovTAP</span>
-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. <span class=SpellE>Chromi</span> colors) decided to use
-mRFP1 as a color output. <o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span class=SpellE><b><u><span style='font-size:12.0pt'>LovTAP</span></u></b></span><b><u><span
-style='font-size:12.0pt'><o:p></o:p></span></u></b></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><b><span style='font-size:12.0pt'>Overview<o:p></o:p></span></b></p>
-<p class=MsoNormal style='margin-bottom:5.0pt;text-indent:.5in'><span
-style='font-size:12.0pt'>In order to draw on bacteria with a laser pointer, we
-need bacteria to be able to respond to light. Previous <span class=SpellE>iGEM</span>
-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 <span
-class=SpellE>LovTAP</span>. 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.
-<o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><span class=SpellE><b><span
-style='font-size:12.0pt'>LovTAP</span></b></span><b><span style='font-size:
-.0pt'> Parts<o:p></o:p></span></b></p>
-<p class=MsoNormal style='margin-bottom:5.0pt;text-indent:.5in'><span
-style='font-size:12.0pt'>As mentioned previously, <span class=SpellE>LovTAP</span>
-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.<o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><i><span style='font-size:12.0pt'>Light
-response<o:p></o:p></span></i></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><i><span style='font-size:12.0pt'><span
-style='mso-tab-count:1'>            </span></span></i><span style='font-size:
-.0pt'>The light-response domain is LOV2, the photoactive domain (i.e. the
-light responsive part) of AsLOV2 (<span class=SpellE>Avena</span> sativa <span
-class=SpellE>phototropin</span> 1). AsLOV2 is a protein which allows <span
-class=SpellE>Avena</span> sativa to respond to 470 nm light. It does this by
-undergoing a major conformational change upon being struck by a photon with a <span
-class=SpellE>wavelenght</span> near 470 nm. In detail, the absorption of the
-photon leads to the formation of a covalent bond between a <span class=SpellE>flavin</span>
-mononucleotide (FMN) cofactor and a conserved cysteine residue. This new bond
-distorts the conformation of the protein, causing the detachment and unfolding
-of the J&#945;-helix (see figure X). Detachment and unfolding of the
-J&#945;-helix can result in further downstream <span class=SpellE>signalling</span>.
-However, we will be most interested in the fact that the J&#945;-helix detaches
-when LOV2 is hit by blue light.<i><o:p></o:p></i></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shapetype
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-style='font-size:12.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><span style='font-size:12.0pt'>Figure
-X. <o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><i><span style='font-size:12.0pt'>DNA
-Binding<o:p></o:p></span></i></p>
-<p class=MsoNormal style='margin-bottom:5.0pt;text-indent:.5in'><span
-style='font-size:12.0pt'>The DNA binding domain of <span class=SpellE>LovTAP</span>
-is the well-known bacterial transcription factor <span class=SpellE>trpR</span>.
-As part of E. <i>coli</i>’s natural <span class=SpellE>trp</span> operon, in
-the presence of tryptophan, <span class=SpellE>trpR</span> will repress
-transcription at the <span class=SpellE>trp</span> promoter by binding the <span
-class=SpellE>trp</span> operator region and, thus, blocking RNA polymerase.<o:p></o:p></span></p>
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-style='font-size:12.0pt'><o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:5.0pt'><span style='font-size:12.0pt'>Figure
-Y.<o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:15.0pt'><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal style='margin-bottom:15.0pt'><span class=SpellE><b><span
-style='font-size:12.0pt'>LovTAP</span></b></span><b><span style='font-size:
-.0pt'><o:p></o:p></span></b></p>
-<p class=MsoNormal style='margin-bottom:15.0pt'><i><span style='font-size:12.0pt'>Overview<o:p></o:p></span></i></p>
-<p class=MsoNormal style='margin-bottom:15.0pt;text-indent:.5in'><span
-style='font-size:12.0pt'>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, <span class=SpellE>trpR’s</span>
-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 <span
-class=SpellE>trpR</span>, there is no <span class=SpellE>trpR</span> activity
-in the dark. However, in the light, LOV2 releases the alpha helix, allowing <span
-class=SpellE>trpR</span> to bind it and, thus, result in <span class=SpellE>trpR</span>
-activity (which of course is repression of the <span class=SpellE>trp</span>
-promoter).<o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:15.0pt;text-indent:.5in'><span
-style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal style='margin-bottom:15.0pt'><i><span style='font-size:12.0pt'>Details<o:p></o:p></span></i></p>
-<p class=MsoNormal style='margin-bottom:5.0pt;text-indent:.5in'><span
-style='font-size:12.0pt'>AsLOV2 was ligated via its J&#945;-helix to a
-succession of 13-amino terminal truncations of <span class=SpellE>trpR</span>
-(11-108 residues). One construct, referred to as <span class=SpellE>LovTAP</span>
-(the <b>LOV</b>- and <b>t</b>ryptophan-<b>a</b>ctivated <b>p</b>rotein), preferentially
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-illuminated, suggesting that <span class=SpellE>LovTAP</span> can bind the <span
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-the J&#945;-helix of the LOV domain to the middle of the amino-terminal helix
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-light exposure <span class=SpellE>LovTAP</span> binds DNA in a manner that is
-characteristic of the <span class=SpellE>TrpR</span> 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).<o:p></o:p></span></p>
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-     <p class=MsoNormal>Operator DNA<span style='mso-spacerun:yes'>  </span></p>
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-<p class=MsoNormal style='margin-bottom:15.0pt'><span class=GramE><span
-style='font-size:12.0pt'>Taken from Strickland et al.</span></span><span
-style='font-size:12.0pt'> <o:p></o:p></span></p>
-<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo2;
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-style='mso-list:Ignore'>A.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-</span></span></span><![endif]><span style='font-size:12.0pt'>Dark state-DNA
-dissociated: The shared helix contacts the LOV domain and <span class=SpellE>trpR</span>
-is in an inactive conformation. <o:p></o:p></span></p>
-<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo2;
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-</span></span></span><![endif]><span class=SpellE><span style='font-size:12.0pt'>Photoexcitation</span></span><span
-style='font-size:12.0pt'> occurs at 470nm (represented by the FMN <span
-class=SpellE>chromophore</span> going from yellow to white) causing the contact
-between the LOV domain and the shared helix to be disrupted. <span
-class=SpellE><span class=GramE>trpR</span></span> now binds the helix and
-assumes its active conformation (represented by the helix going from blue to
-red).<o:p></o:p></span></p>
-<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo2;
-tab-stops:list 0in .5in'><![if !supportLists]><span style='font-size:10.0pt'><span
-style='mso-list:Ignore'>C.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-</span></span></span><![endif]><span style='font-size:12.0pt'>The active <span
-class=SpellE>trpR</span> domain of <span class=SpellE>LovTAP</span> binds the <span
-class=SpellE>trp</span> operator<o:p></o:p></span></p>
-<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo2;
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-</span></span></span><![endif]><span style='font-size:12.0pt'>Cessation of
-light causes the LOV domain to return to its dark state, dissociating <span
-class=SpellE>LovTAP</span> from the DNA and reinstating contact between the LOV
-domain and shared helix. <o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><span class=SpellE><span class=GramE><b><span
-style='font-size:12.0pt'>iGEM</span></b></span></span><b><span
-style='font-size:12.0pt'> &amp; <span class=SpellE>LovTAP</span> <o:p></o:p></span></b></p>
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-<p class=MsoNormal style='text-indent:.5in'><span class=SpellE><span
-style='font-size:12.0pt'>LovTAP</span></span><span style='font-size:12.0pt'> was
-cloned into a <span class=SpellE>biobrick</span> 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 <span class=SpellE>LovTAP</span>.
-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 <span class=SpellE>LovTAP’s</span> function by increasing
-the stability of the light state and decreasing the time it takes to flip from
-dark state to light state.<o:p></o:p></span></p>
-<p class=MsoNormal style='text-indent:.5in'><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'>UNAM<o:p></o:p></span></i></p>
-<p class=MsoNormal><span style='font-size:12.0pt'>A new <span class=SpellE>LovTAP</span>
-part was synthesized by the UNAM 2010 team via the alteration of EPF-Lausanne’s
-<span class=SpellE>LovTAP</span>. Primary changes include the removal of two
-&nbsp;<span class=SpellE>PstI</span> restriction sites from the coding region
-of <span class=SpellE>LovTAP</span> and the addition of one of the point
-mutations that was proposed by EPF-Lausanne. <o:p></o:p></span></p>
-<p class=MsoNormal style='margin-bottom:15.0pt'><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal style='margin-bottom:15.0pt'><b><span style='font-size:12.0pt'>Our
-Project<o:p></o:p></span></b></p>
-<p class=MsoNormal style='text-indent:.5in'><span style='font-size:12.0pt'>For
-our circuit we will be using UNAM’s modified <span class=SpellE>LovTAP</span>,
-K360127. For the <span class=SpellE>trp</span> promoter and operator, we will be
-using <span class=SpellE>ptrpL</span>, K360023. <o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'>Possible issues<o:p></o:p></span></i></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'><span style='mso-tab-count:
-'>            </span></span></i><span style='font-size:12.0pt'>UNAM appeared
-to not have nearly as much success with <span class=SpellE>LovTAP</span> 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 <span class=SpellE>LovTAP</span>
-and possibly introduce other mutations to improve <span class=SpellE>LovTAP’s</span>
-function. For example, Strickland et al. identified mutations that increased <span
-class=SpellE>LovTAP’s</span> dynamic range (i.e. the difference in repression
-in the dark state vs. the light state) from 5 to 70.<i><o:p></o:p></i></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><span style='mso-tab-count:
-'>            </span>Another issue is that <span class=SpellE>LovTAP’s</span>
-light state is unstable. Upon removal of the light source, <span class=SpellE>LovTAP</span>
-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 <span class=SpellE>LovTAP</span>
-to remain in the light state for a longer time after being activated.<o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><b><u><span style='font-size:12.0pt'>Peking Bi-stable Switch
-<o:p></o:p></span></u></b></p>
-<p class=MsoNormal><b><span style='font-size:12.0pt'>Overview<o:p></o:p></span></b></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><span style='mso-tab-count:
-'>            </span>The Peking 2007 <span class=SpellE>iGEM</span> 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.<o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><b><span style='font-size:12.0pt'>Original Design<o:p></o:p></span></b></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><span style='mso-tab-count:
-'>            </span>The Peking 2007 switch uses <span class=SpellE>pR</span>,
-<span class=SpellE>pRM</span>, <span class=SpellE>cI</span>, and cI434.
-Avoiding unnecessary detail: <span class=SpellE>cI</span> activates <span
-class=SpellE>pRM</span> and represses <span class=SpellE>pR</span>; cI434
-represses <span class=SpellE>pRM</span>; <span class=SpellE>pR</span> has high
-basal transcription; and <span class=SpellE>pRM</span> has low basal
-transcription. <o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'>State 1<o:p></o:p></span></i></p>
-<p class=MsoNormal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
- id="Picture_x0020_4" o:spid="_x0000_i1026" type="#_x0000_t75" style='width:468pt;
- height:156pt;visibility:visible;mso-wrap-style:square'>
- <v:imagedata src="EaSwriteup_files/image009.png" o:title=""/>
-</v:shape><![endif]--><![if !vml]><img width=624 height=208
-src="EaSwriteup_files/image010.gif" v:shapes="Picture_x0020_4"><![endif]></span><i><span
-style='font-size:12.0pt'><o:p></o:p></span></i></p>
-<p class=MsoNormal><span style='font-size:12.0pt'>In the first state, cI434
-levels are high and <span class=SpellE>cI</span> levels are low. This leads to
-high transcription from <span class=SpellE>pR</span> and low transcription from
-<span class=SpellE>pRM</span>, which, in this particular circuit, results in
-GFP production. The switch can be flipped by increasing <span class=SpellE>cI</span>
-levels.<o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'>State 2<o:p></o:p></span></i></p>
-<p class=MsoNormal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape
- id="Picture_x0020_5" o:spid="_x0000_i1025" type="#_x0000_t75" style='width:468pt;
- height:156pt;visibility:visible;mso-wrap-style:square'>
- <v:imagedata src="EaSwriteup_files/image011.png" o:title=""/>
-</v:shape><![endif]--><![if !vml]><img width=624 height=208
-src="EaSwriteup_files/image012.gif" v:shapes="Picture_x0020_5"><![endif]></span><i><span
-style='font-size:12.0pt'><o:p></o:p></span></i></p>
-<p class=MsoNormal><span style='font-size:12.0pt'>In the second state, <span
-class=SpellE>cI</span> levels are high and cI434 levels are low. This leads to
-high transcription from <span class=SpellE>pR</span>, low transcription from <span
-class=SpellE>pRM</span>, 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 <span class=SpellE>pR.</span><o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><b><span style='font-size:12.0pt'>Our design: Locking Switch<o:p></o:p></span></b></p>
-<p class=MsoNormal><span style='font-size:12.0pt'>For our switch, we desired a
-simple circuit with a stable on state. The parts we used are <span
-class=SpellE>ptrpL</span>, cI434, <span class=SpellE>pRM</span>, <span
-class=SpellE>trpR</span>, and <span class=SpellE>cI</span>. As in the Peking
-switch, <span class=SpellE>cI</span> activates <span class=SpellE>pRM</span>
-and cI434 represses <span class=SpellE>pRM</span>. We cloned two additional
-parts for our switch: <span class=SpellE>ptrpL</span> and <span class=SpellE>trpR</span>.
-<span class=SpellE><span class=GramE>ptrpL</span></span> is the well-known <span
-class=SpellE>trp</span> operon/promoter/leader sequence. <span class=SpellE><span
-class=GramE>trpR</span></span> is the corresponding repressor which is induced
-by tryptophan. In our system, we will assume there are constantly high levels
-of tryptophan so that <span class=SpellE>trpR</span> will always act as a
-repressor. <o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'>State 1<o:p></o:p></span></i></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></i></p>
-<p class=MsoNormal><span style='font-size:12.0pt'>In the first state, cI434
-levels are high, <span class=SpellE>trpR</span> levels are low, and <span
-class=SpellE>cI</span> levels are low. This results in high transcription from <span
-class=SpellE>ptrpL</span>, low transcription from <span class=SpellE>pRM</span>,
-and thus low output signal. The switch can be flipped by reducing transcription
-from <span class=SpellE>ptrpL</span>.<o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><i><span style='font-size:12.0pt'>State 2<o:p></o:p></span></i></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><br>
-In the second state, cI434 levels are low, <span class=SpellE>trpR</span>
-levels are high, and <span class=SpellE>cI</span> levels are high. This results
-in low transcription from <span class=SpellE>ptrpL</span>, high transcription from
-<span class=SpellE>pRM</span>, and thus high output signal. Since <span
-class=SpellE>trpR</span> is a strong repressor for <span class=SpellE>ptrpL</span>,
-we expect state 2 to be stable.<o:p></o:p></span></p>
-<p class=MsoNormal><span style='font-size:12.0pt'><o:p>&nbsp;</o:p></span></p>
-<p class=MsoNormal><b><u><span style='font-size:12.0pt'>mRFP1<o:p></o:p></span></u></b></p>
-<p class=MsoNormal style='text-indent:.5in'><span style='font-size:12.0pt'>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.<o:p></o:p></span></p>
-<p class=MsoNormal style='text-indent:.5in'><span class=GramE><span
-style='font-size:12.0pt'>mRFP1</span></span><span style='font-size:12.0pt'> is
-a red fluorescent protein based on <span class=SpellE>DsRed</span>. Natural <span
-class=SpellE>DsRed</span> requires <span class=SpellE>tetramerization</span> in
-order to fluoresce. <span class=GramE>mRFP1</span> contains 33 mutations that,
-one, allow it to function as a monomer and, two, allow it to fold more quickly.
-<o:p></o:p></span></p>
-</div>
-</body>
-</html>
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