the Bacterial Etch-a-Sketch

View As One PageView As Slideshow     Real: Bacterial lawn that can be drawn on with a laser   Abstract: Fast biological memory circuit   Engineering Problems   Input: ~1 ms laser pulse Sensitivity—1 ms is very short Selectivity—Would like to use in ambient lighting Output: Violacein Speed—Would like to see colors quickly Noise—Do not want random lines lovTAP. Activated by 470nm light. When active, acts as trpR which represses ptrpL. Stays active for about 1 minute. Locking Switch   Based on Peking 2009 memory circuit cI434 represses pRM cI activates pRM trpR represses ptrpL   By default, ptrpL is “on” and pRM is “off” A drop in cI434 should reverse permenantly lovTAP represses pTrpL T7p activates T7 promoter trpR represses ptrpL C Sensitivity: Time it takes for (activation 1 => activation 2) Time for enough cI434 to degrade for transcription at pRM Selectivity: Amount of light for (deactivated => activation 1) Speed: Time for VioA-E synthesis & work Noise: Basal transcription at T7 Close to none Basal transcription at pRM Minimized with low copy plasmid •Complete LovTAP Plasmid—K360127—Ordered, 887bp –pSB1C3, 2070bp, Chl •T7 Promoter—I712074—Plate 1—6N, 46bp –pSB1AK8, 3426bp, Amp, Kan •VioA-E—K274002—Plate 3—12B, 7345bp –pSB1T3, 2463bp, Tet •ptrpL—K360023—get synthesized, 49bp + prefix/suffix = 108bp –Will put into pSB1C3, 2070bp, Chl •cI434 + LVA—C0052—Plate 1—4G, 669bp –pSB1A2, 2079bp, Amp •cI + LVA—C0051—Plate 1—4E, 750bp –pSB1A2, 2079bp, Amp •Modified pRM—I12040—Plate 1—20D, 91bp –pSB2K3, 4425bp, Kan •RBS+T7 P+NLS—I712069—Plate 2—13K, 2678bp –pSB1AK3, 3189bp, Amp, Kan •(Strong) RBS—B0034—Plate 1—2M, 12bp –pSB1A2, 2079bp, Amp   Abstract The goal is to engineer bacteria that will respond to a millisecond laser pulse. This is of concern because no living thing needs to worry about a stimulus that lasts for such a short amount of time. Additionally, in order for the light to elicit a response from the bacteria, the circuit must amplify the tiny light signal and translate it to a molecule that can regulate transcription. The light itself is not able to activate or repress a promoter. It is important that the bacteria only respond to the laser pulse and not all light sources. The bacteria must either have a threshold level for the amount of light needed to induce color or respond to only certain wavelengths of light. <Contents>

Goals
I. Input The goal is to engineer bacteria that will respond to a millisecond laser pulse. This is of concern because no living thing needs to worry about a stimulus that lasts for such a short amount of time. Additionally, in order for the light to elicit a response from the bacteria, the circuit must amplify the tiny light signal and translate it to a molecule that can regulate transcription. The light itself is not able to activate or repress a promoter. It is important that the bacteria only respond to the laser pulse and not all light sources. The bacteria must either have a threshold level for the amount of light needed to induce color or respond to only certain wavelengths of light.   II. Processing The circuit must be able to hold memory, so that it does not return to its original state once the light signal is removed.   III. Output The goal is to make the bacteria produce color in response to the light signal. The bacteria would be grown out into a lawn and then drawn on with the laser pointer and this drawing would appear in color, similar to drawing on a sketch pad. We are literally etching a sketch on the bacteria. A concern is how long the bacteria will take to respond to the light and produce color. Moreover the bacteria should be sensitive enough so that only the bacteria that are exposed to the light should respond. Essentially there should be as little noise as possible in the coloring. A successful engineered Bacterial Etch-a-Sketch would allow a lawn of bacteria to be used as a sketch pad where we can draw on with a household laser pointer. Depending on the application, this system has the ability to produce intracellular pigment when exposed to 477nm (blue) light. In the future, our bacteria could help solve problems that require a rapid response to light with a quick? visual output.

The SeLECT Circuit
SeLECT circuit SeLECT: Sensitive, Light-Effected Circuit with Threshold (SeLECT) The SeLECT circuit uses LovTAP that was developed by many teams and labs around the world, a memory switch based on work done by Peking University, and color generators developed by Cambridge. Sensitivity: How long we need to shine the laser on the bacteria to activated pRM Selectivity: How much ambient light the bacteria can resist before activating pRM Speed: Once activated, how long does it take to see color Noise: How much unwanted color is generated

LovTAP
Photoswitchable proteins Photoswitchable proteins offer the unique ability to perturb living cells, tissues and intact organisms with high spatial and temporal precision1. In particular, genetically encoded photoswitches such as LOV (light, oxygen, voltage) and phytochrome domains can be conveniently used in many different experi­mental contexts2–7. The LOV2 domain of Avena sativa phototropin 1 (AsLOV2) has proven especially useful for controlling functionally diverse effectors including DNA-binding proteins, enzymes and small GTPases2,3,5.   AsLOV2 The current design uses LovTAP to sense light. LovTAP is a fusion protein of AsLOV2 and trp repressor at a common alpha helix. AsLOV2 stands for Avena sativa phototropin 1. It is a LOV (Light, oxygen, voltage) domain that was discovered in phototropins, which are light-activated serine-threonine kinases that facilitate blue light responses in plants and algae. LOV domains carry a flavin chromophore (either FMN or FAD) that broadly absorbs light at 447nm (cite).   Structural Properties In the functional conformation of the trp repressor, the protein is “loosely” bound to the alpha helix (of what?). If LovTAP cannot bind the alpha helix, then the repressor will not function. AsLOV2 on the other hand, “tightly” binds a similar alpha helix in the dark. However, when exposed to 477 nm (blue) light, AsLOV2 undergoes a conformational change and cannot bind the alpha helix. Thus, LovTap is a trp repressor in the light and is not active in the dark.   Induction We will need to shine the blue light on the bacteria to initiate LovTap detaching from the Trp Repressor. However, the length of the exposure time before it takes for the bacteria to effectively undergo the reaction is unknown. We plan on testing the LovTap protein with the color genes in a single plasmid first to determine the speed of this reaction. From there, we will decide if LovTap is or is not the best light-activated system for our design and may seek a new one ?????????   Issues Another issue to avoid is UV light’s effect on LovTap. We will need to test to make sure that LovTap does not detach from TrpR by just regular sunlight so that activation of our pathway occurs only when we want it to. Otherwise, we may see random color spots.

Memory Switch
Genetic Switch The memory switch, designed Pecking University 2007, will be used in order to allow the bacteria to remember if it has been exposed to laser light or not. This should allow the input signal to be amplified in some sense. The memory uses a modified pRM promoter from lambda phage. When the bacterium is not yet exposed to light, we repress the pRM promoter with cI434. CI434 is located on a transcript with a ptrpL promoter. In the Select circuit, LovTap will repress the ptrpL promoter upon light exposure. This will stop repression of the pRM promoter, allowing transcription. Included in the transcript is cI, which is an activator of the pRM promoter, and trpR to repress ptrpL and thus the production of cI434. Thus even though upon discontinuing light exposure, repression of pRM via cI4343 should occur, the switch represses cI434 and allows for the pRM promoter to continue being transcribed, in conjunction with the pRM activator cI. pRM transcription should stay on via this positive feedback loop.

mRFP
Red Fluorescent Proteins mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. he latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. <-filler Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies. http://www.mendeley.com/research/improved-monomeric-red-orange-yellow-fluorescent-proteins-derived-discosoma-sp-red-fluorescent-protein/   Structural Properties In the functional conformation of the trp repressor, the protein is “loosely” bound to the alpha helix (of what?). If LovTAP cannot bind the alpha helix, then the repressor will not function. AsLOV2 on the other hand, “tightly” binds a similar alpha helix in the dark. However, when exposed to 477 nm (blue) light, AsLOV2 undergoes a conformational change and cannot bind the alpha helix. Thus, LovTap is a trp repressor in the light and is not active in the dark.   Induction In order to produce color a signal needs to be attached to pRM. This signal will be a T7 polymerase, which will activate a strong T7 promoter. Included on the transcript, along with the T7 promoter will be modified RFP (mRFP). Once the bacteria are exposed to light and the Select circuit is activated, the exposed bacteria should produce modified red fluorescent protein, which can be seen via the unaided eye. mRFP1 was derived from the Discosoma sp. fluorescent protein "DsRed"by direction evolution.   Issues Basal transcription can also be a problem at the T7 promoter. T7 is a very strong promoter, so if basal transcription occurs we predict we will get intense color randomly. We can test other, weaker promoters in T7’s place to see if we still get significant color intensity. Putting a weaker promoter should not significantly affect the color intensity because we have placed this promoter on a high copy plasmid to amplify as much as possible.

Issues
I. Input Writing speed is probably the most important concern we will have in this project. We will need to test how long it takes for the bacteria to transcribe the genes and create the color. Seeing as how bacteria replicated very quickly (within half an hour), we predict that transcription occurs even faster since it is a process necessary for bacteria to replicate. Our guess is that this will not be too significant of a problem, envisioning the color to appear within a few minutes.   II. Processing ?   III. Output The goal is to make the bacteria produce color in response to the light signal. The bacteria would be grown out into a lawn and then drawn on with the laser pointer and this drawing would appear in color, similar to drawing on a sketch pad. We are literally etching a sketch on the bacteria. A concern is how long the bacteria will take to respond to the light and produce color. Moreover the bacteria should be sensitive enough so that only the bacteria that are exposed to the light should respond. Essentially there should be as little noise as possible in the coloring. A successful engineered Bacterial Etch-a-Sketch would allow a lawn of bacteria to be used as a sketch pad where we can draw on with a household laser pointer. Depending on the application, this system has the ability to produce intracellular pigment when exposed to 477nm (blue) light. In the future, our bacteria could help solve problems that require a rapid response to light with a quick? visual output.