The Bacterial Etch-a-Sketch

Abstract The Etch-a-Sketch project aims to create a lawn of bacteria that can be drawn on with a laser pointer. This seemingly inconsequential task actually presents many interesting engineering challenges. In particular, the bacteria need to be 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 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.

Mechanism of Action
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. 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.

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.   The MYS!S walkthrough is located here! https://2011.igem.org/Team:Rutgers/MYS!S_WT

Features
I. Protocols In the protocol tab is a BioCoder compatible C++ file that contains the lab procedures for mutating the original Lovtap DNA into a standard safe form. The C++ file can be compiled with the BioCoder software available here. http://research.microsoft.com/en-us/um/india/projects/biocoder/   II. Rna Structure Analysis In the RNA structure tab there are images of the unmodified and modified RNA structure. We hope that this will help the user decide whether the changes are structurally advantageous. Hopefully in the future more advanced RNA structure modeling algorithms can be implemented to help the user make an informed decision.

Future of MYS!S
beta Unfortunately, we’re talking about the capabilities of the future MYS!S v.10, for now all we have is the beta edition. So for the time being, these are the imminent improvements we would like to make for the second version of MYS!S. Better algorithms for modifying DNA Currently, when determining how to modify DNA MYS!S does not take into account the eventual RNA structure and whether the changes will inhibit protein production. We would like to incorporate algorithms that make changes to DNA in a way that will increase the amount of protein formed by translation. On the same note, it might also be helpful for a synthetic biologist not just to increase protein production but maybe to limit it.   Not just support site directed mutagenesis Right now MYS!S for a codon optimization creates a large number of primers for a sequence of say 700bp. We’re talking about upwards of 50 primers making site directed mutagenesis realistically impossible. We would like MYS!S to support other methods of manipulating physical DNA.   Better visualization methods for RNA structure We want the user to be able to visually check whether the RNA structure is acceptable. If it is not acceptable the user should be able to manually modify the DNA sequence to improve the RNA structure. Preference for lab protocols : Not all labs do things the same, MYS!S should be able to customize lab protocols to how the user’s lab gets things done.

Where can I get MYS!S?

Github MYS!S is currently available as an Eclipse download. It will very soon be available as a java application. The downloads can be obtained from our GitHub site. https://github.com/RutgersGEARS/iGEM-Rutgers-Software MYS!S is still a work in progress. Please feel free to report any bugs or crashes that occur as issues on our github page. https://github.com/RutgersGEARS/iGEM-Rutgers-Software/issues