the MYSIS Project

View As One PageView As Slideshow     The MYS!S Project: BioBrick Optimizer Abstract MYS!S is a stand alone software tool designed to assist an “iGEMer” when designing a new BioBrick part or modifying an existing one. MYS!S handles several problems that a synthetic biologist might encounter in the lab that can be solved with site directed mutagenesis. The utilities that MYS!S supports to modify DNA sequences on paper, can be “actualized” in the lab using site directed mutagenesis to change physical DNA “stored” in a backbone. MYS!S will determine the most efficient modified sequence by optimizing for the least number of rounds of site directed mutagenesis. Once the changes are finalized primers are designed. In addition to primer design, MYS!S provides lab protocols for both site directed mutagenesis and transformation into competent cells. Currently the lab protocols are supplied as a BioCoder compliant C++ file, that can then be compiled with the BioCoder library.

Operations
I. DNA Optimization First, it performs DNA codon optimization based on the target organism by removing rare tRNAs and replacing them with more prevalent tRNAs. (We noticed that many teams decided to utilize sequences from a variety of organisms such as firefly luciferase to ultimately be transformed into an experimentally “stable” organism.)   II. Processing Secondly, the program checks the coding sequence for any restriction sites that are not allowed by the format specified by the user. After checking the sequence, MYS!S removes the restriction sites while ensuring that the modified sequence is still codon optimized. In addition, it was pointed out that frequently a synthetic biologist would like to change specific amino acids in the DNA coding sequence. MYS!S decides what difference in nucleotide sequence are required to best make the amino acid change.   III. Restriction site analysis Alternatively, an iGEMer might also want to add restriction sites to a DNA sequence in order to remove a particularly useful sequence. The program can handle this by either allowing the user to specify where the restriction site should be or by choosing what restriction site is desired and how sites need to be added to the sequence.

How Mys!S Does it
The MYS!S Environment One of the main ideas behind MYS!S was to make synthetic biology more accessible to a wider “spectrum” of people. We want the program to provide an environment that enables the user to understand and explore the changes being made to the DNA in a more intuitive manner. Custimization In addition, MYS!S is designed to be highly customizable by providing the user with the ability to add and modify components such as an organism’s codon usage table and new assembly standards. The ultimate goal is to create a program that can be customized by the user for their specific work and the lab protocols they are comfortable with.

MYSIS: An Introduction

Walk through One of the main ideas behind MYS!S was to make synthetic biology more accessible to a wider “spectrum” of people. We want the program to provide an environment that enables the user to understand and explore the changes being made to the DNA in a more intuitive manner. In addition, MYS!S is designed to be highly customizable by providing the user with the ability to add and modify components such as an organism’s codon usage table and new assembly standards. The ultimate goal is to create a program that can be customized by the user for their specific work and the lab protocols they are comfortable with. To showcase the capabilities of MYS!S we would like to walk you through an analysis of a current BioBrick in the registry. For this example we are going to use part BBa_K191006 which is the protein coding sequence for LovTAP. LovTAP was used by both of our laboratory projects Etch-a-Sketch and Full Adder. One of the issues with LovTAP is that it contains restriction sites not allowed by some BioBrick assembly standards. Lets say we want to transform the LovTAP coding region into e-coli After opening MYS!S, navigate to the screen to manage components. To do this go to the MYS!S menu and click “Manage Components”. It will open up a screen that will allow the user to manage which organisms and assembly standards the program handles.

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.