This project is now in cold storage. As of 2014-06-23 Erik Ersland has sent all needed info to the Harvard team (that traitor) since they are doing something that they want to change colors.
If anyone wants to send someone this project or is trying to figure out what the heck this is, use(read) an email like this and attach "shift register design, view this first.pptx" and "Genetic shift register function.pptx" from the attachments to this page. The power points should contain all the information you need.
This circuit has n distinct states it can be in, these states are in a list (additionally, there is an order, and each one has a unique "reference"), the circuit takes some molecule in as an advance signal that causes it to change from one state to the next (at the end, it may either stay in the final state or loop back to the first). This allows for some pretty neat stuff (I have a list of brainstormed applications if you're interested)
This circuit is really complicated though, like its own iGEM project. The smallest circuit has 3 or 4 states. Each state has 5 promoter-protein pairs(with a DNA reference that can be bound by a protein that binds to a specific DNA region, like CAS9 or a TAL protein). This means that the smallest functional circuit has 15 promoter-protein-reference pairs, which makes it quite a task to implement (especially since most of these proteins are repressing other expression units, so tuning is complicated). We never got into specifics, but the design works logically if you have two advance signals (if you only use 1 signal, than it could advance and then advance again depending on timing, but if you have every even number state use advance A and every odd number state use advance B, then it works because the signal to go from 1->2 is not the same as the signal to got from 2-> 3. You can get a separate circuit that will take an input C and on a transition from C low to C High, output A or B in a way that alternates A then B then A... so that C is the only input and its much more stable like this. We figured we'd use TAL repressors, you need 2+2n for n states (2 advance signals and 2 for each state). With repressors, the advance molecule is inverted, so you normally have it and you remove it to advance, not normally not have it and add it to advance. You could also use inducible promoters, so adding the inducer makes it advance.
The picture should be bellow. Each color is a state, the states are red, orange, green, blue. Each arrow is a constitutive promoter (it should be one promoter per gene, but that gets more confusing when drawn), and each x is an end of transcription. The colored notch beneath the arrow is the unique reference sequence that a TAL R binds to. The triangles are the TAL R's that bind to the same color notch. The circles are reporters, in your case, it would be the color displaying protein. The rectangle with a square cut out next to the colored notch on the far right piece is where the advance signal enters, the repressor for this must be absent and the advance signal must be present for the protein on this to be produced. Each row is a state, the pieces are, from left to right: Represses next state unless the next state is active (so all regions are off by default, and only 1 should be on at a time) | Active beheavior, the pattern of genes expressed when this state is active, it produces the promoter, represses the previous state's left most part (so by repressing the thing that represses it, it keeps itself turned on), and represses the middle part (active behavior) of the previous state, so if 2 states right next to each other are active, it will automatically proceed to only the last one being active (this is important for state advances) | State advance, if the state is active and it receives the advance signal, then it turns on the next state. Since this makes 2 states active, the system automatically proceeds to only the second being active.
I've attached power points that should explain this more clearly. If you use this, please contact the MIT iGEM team and cite Erik Ersland for the idea. <Circuit diagram here> |
The old log of meeting notes follows, as transcribed by Jiaqi. All of the old documents are attached to this page
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Members: (Add Your Name if interest post cutoff) Erik Ersland Shinjini Saha Jiaqi Xie Alex Leffell ====================================================================================================== Just setting a page down to see what you guys have and to share resources. Linked list circuit if you add a timer to produce the advance signal, it will be a clock This uses only cas9 repressors and a small molecule signal to handle states. Key: each line is a node circuit of nodes (there isn't supposed to be a cross out, I don't know why its there) ->0'(1),0 payload,>0(3'),>0(3),->A0(0') ->1'(2),1 payload,>1(0'),>1(0),->A1(1') ->2'(3),2 payload,>2(1'),>2(1),->A2(2') ->3'(0),3 payload,>3(2'),>3(2),->A3(3') explanation of components (same comma convention): initial state: [(0),(1),(2),(3)] present (blocking active behaviour) state change: (0') introduced state change: (A) introduced ,now [(0),(2),(3),(0'),(A)] present future state changes: each addition of (A) will move state forward one slot ====================================================================================================== 14 Feb 9 Applications: Time progression, biological clock. Any multi-step chemical/biological process moderated by a single organism.Beer brewing. Ethanol/Biofuel production. Drugs/Enzyme production. Chemical processes with steps that would normally interfere with one another moderated locally. Safety precaution, self-termination. Biological CS circuits. Large AND and OR Gates Add back command to create Turing Machine. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Implementation: Cas9, Guide RNA for repression. mRNA sensing repression. ====================================================================================================== 14 Feb 12 Updated Powerpoint with improved sprites and arrow pushing. V2 ====================================================================================================== 14 Feb 13 Brainstorming session. Forgot some repression arrows. Message was clear enough such that no one cared. Will fix if not lazy. -JX Cell that allows for post processing.Yeast that has the capability to creating a variety of beer. ====================================================================================================== 14 Feb 20 Victory. Our brave soldiers have triumphed over the forces of ??? in the battle of the first cutoff. Todo: Make a mailing list and post on wiki and igem-2014. Coordinate meeting times for preliminary experiments and circuit design. Human(e) practices? Monsanto? Control? Termination? Transfer? - JX ====================================================================================================== 14 Feb 27 Pre-meeting Pow Wow: Anschluss failed. Middle segment can be one piece or separate, need testing. Activating small molecule: rtTA (Tet-ON) system: http://www.tetsystems.com/science-technology/scientific-figures/#c22 (first diagram to the right.) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Testing: Primary Chassis Considered: Yeast Middle segment can be one piece or separate, optimization? Precise mechanism for the AND of the last part needs testing. (NOT A) && B Gate Degradation rate of Repressors, measured and controlled. State change time sensitive. Look up circuitry. 6.004 ocw materials. Propagation times and state thresholds (determined by cooperativity and binding kinetics, and etc. ). ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Motivation: Technological Advance. It's big. Large system testing. (Lot's of TAL_Rs) State based combinational system. Memory System. Beer. Drugs. Vat Production. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ From Ron: ====================================================================================================== 14 Mar 6 Annexation! Alzheimer has annexed XNA! The international community will not stand for this! Alzheimer has annexed Turing Machines! The international community will not stand for this! ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Human Practices Pre-Brainstorming: #Safety Mechanism# Any safety mechanism/self termination system will be heavily selected against. Production of toxins that kill neighboring cells. Cells that fail to undergo suicide will be eliminated by their more competent neighbors. Transfer crucial genes into shift register. The deactivation of the register and it's associated gene will lead to cell death. Prevent logic failures by making every state after a critical threshold program for death. Make the forward signal a signal from cell division, advancing the state with every generation of cell division. #Vaccine# Immunizations can have their effect maximized by 1: having a larger imune response (duration and quantity of antgen), 2: have multiple antigens for a given disease, and 3: have only one antigen at a time (apparently if you have too many antigen, the immune system only deals with a few). Our project is perfect for this. I read somewhere about implanting a colony of microbes in a physical barrier that prevented cells from getting through but not molecules. We could have a colony of yeast (or whatever is the easiest to kill should it escape) that produces a large set of antigens over a long time Counting circuit. One injection/implantation. Over time, the implanted colony cycles through the antibodies, increasing effectiveness of immunization by exposing the body to only one antigen at a time. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Brainstorming: Logic Gates/better visuals needed. Get someone on it in the near future. Need alternating advance molecules, or a shift register to alternate so that machine doesn't advance too many states. Need to look into biological shift registers, and their ability to resist changing states multiple times in the presence of the signal. iGEM meet: Ron's ideas: Pulse generator switching on S_even and S_odd alternately. Need to look into how this could be achieved, possibly in a simpler way than the toggle switch.
14 Mar 13 New Member! 14 Mar 20 Where is in the world is Carm... Modeling talk I'm not quite sure of. PROGRESS! Debugging Galore! ====================================================================================================== 14 Apr 10 You wish your circuit diagrams look this good.... Who am I kidding. The top part represents a toggle switch that filters the step signal to prevent an oscillation. The four repeating units represent the primary design of the current project. Bright green represent active paths and dark green represent inactive paths. ======================================================================================================
2014-Apr-22 Allocation of responsibilities. How does current project fit in with Alzheimer's project? Implementation? Human practices? Transition to dosage control? Titration project? Combine with Alzheimer.
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