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1
2
3 Bacterial lawn that can be drawn on with a laser
4
5 Abstract:
6 Fast biological memory circuit
7
8 Engineering Problems
9
10 Sensitivity—1 ms is very short
11 Selectivity—Would like to use in ambient lighting
12 Noise—Do not want random lines
13 Locking Switch
14 cI434 represses
15 trpR represses ptrpL
16
17
- 17.1 Abstract
18 <img src="icons/58-bookmark.png" width="10" height="26" /> Goals
- 18.1 I. Input
- 18.2 II. Processing
- 18.3 III. Output
19 <img src="icons/58-bookmark.png" width="10" height="26" /> The SeLECT Circuit
- 19.1 SeLECT circuit
20 <img src="icons/58-bookmark.png" width="10" height="26" /> LovTAP
- 20.1 Photoswitchable proteins
- 20.2 AsLOV2
- 20.3 Structural Properties
- 20.4 Induction
- 20.5 Issues
21 <img src="icons/58-bookmark.png" width="10" height="26" /> Memory Switch
- 21.1 Genetic Switch
22 <img src="icons/58-bookmark.png" width="10" height="26" /> mRFP
- 22.1 Red Fluorescent Proteins
- 22.2 Structural Properties
- 22.3 Induction
- 22.4 Issues
23 <img src="icons/58-bookmark.png" width="10" height="26" /> Issues
- 23.1 I. Input
- 23.2 II. Processing
- 23.3 III. Output

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 </a> <a href="http://www.awkward.se" coords="200,440"> T7p activates T7 promoter </a> <a href="http://www.awkward.se" coords="600,440"> trpR represses ptrpL C </a>

<img src="icons/95-equalizer.png" width="26" height="24" /> Sensitivity:
Time it takes for (activation 1 => activation 2)

Time for enough cI434 to degrade for transcription at pRM

<img src="icons/13-target.png" width="28" height="28" /> Selectivity:
Amount of light for (deactivated => activation 1)

<img src="icons/11-clock.png" width="25" height="25" /> Speed:
Time for VioA-E synthesis & work

<img src="icons/39-spraycan.png" width="16" height="24" /> Noise:
Basal transcription at T7 <p>Close to none <p>Basal transcription at pRM <p>Minimized with low copy plasmid </div> </div> </div>

•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

</div>

Abstract

<p class="stuff">The goal is to engineer bacteria that will respond to a millisecond <a href="javascript:;" id="Link">laser</a> 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.

</td>

         </tr>

     </table>
       </blockquote>
 </tr>
 <tr>

<img src="icons/58-bookmark.png" width="10" height="26" /> 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.

 </tr>
 <tr>

<img src="icons/58-bookmark.png" width="10" height="26" /> 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.

<img src="icons/95-equalizer.png" width="26" height="24" /> Sensitivity:
How long we need to shine the laser on the bacteria to activated pRM

<img src="icons/13-target.png" width="28" height="28" /> Selectivity:
How much ambient light the bacteria can resist before activating pRM

<img src="icons/11-clock.png" width="25" height="25" /> Speed:
Once activated, how long does it take to see color

<img src="icons/39-spraycan.png" width="16" height="24" /> Noise:
How much unwanted color is generated

 </tr>
 <tr>

<img src="icons/58-bookmark.png" width="10" height="26" /> 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.

 </tr>
 <tr>

<img src="icons/58-bookmark.png" width="10" height="26" /> 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. <img src="switch_button_off.png" width="209" height="303" align="left" />

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.

 </tr>
 <tr>

<img src="icons/58-bookmark.png" width="10" height="26" /> 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.

<img src="swpics/mrfp.png" width="210" height="184" /><-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.

 </tr>
 <tr>

<img src="icons/58-bookmark.png" width="10" height="26" /> 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.

</td>

</tr>

RUiGEM

Bacterial Etch-a-Sketch

iGEM 2011

Projects

Lab Notebook

BioSafety

Acknowledgements

</td>

 </tr>

</table>

</body>

</html>

Team:Rutgers/Project

From 2011.igem.org

Contents

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

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

Abstract

<img src="icons/58-bookmark.png" width="10" height="26" /> Goals

I. Input

II. Processing

III. Output

<img src="icons/58-bookmark.png" width="10" height="26" /> The SeLECT Circuit

SeLECT circuit

<img src="icons/58-bookmark.png" width="10" height="26" /> LovTAP

Photoswitchable proteins

AsLOV2

Structural Properties

Induction

Issues

<img src="icons/58-bookmark.png" width="10" height="26" /> Memory Switch

Genetic Switch

<img src="icons/58-bookmark.png" width="10" height="26" /> mRFP

Red Fluorescent Proteins

Structural Properties

Induction

Issues

<img src="icons/58-bookmark.png" width="10" height="26" /> Issues

I. Input

II. Processing

III. Output