• Created by Unknown User (jiaqixie@mit.edu), last modified by Unknown User (eersland@mit.edu) on Jun 23, 2014 16:28

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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