Berk2006-LogicGatesTeam

From OpenWetWare
Revision as of 18:53, 29 July 2006 by Bosworth (talk | contribs)
Jump to navigationJump to search

Return to main page

wrb 07/29/06

  • miniprepped overnights. i put too much bufferP1 into part P0412. then I pippetted the extra out (and took some cells with me). woops. the next steps looked normal though....
  • was going to digest Spe, Xba, AlwN1...but we're out of alwn1. going to digest with Bgl I instead... F2620 & R0040 digest Spe & Bgl, P0412 & F1610 digest Xba + Bgl. f2620+p0412 and r0040 + f1610 ligated.
  • gel came out clean. r0040 piece were 1140bp to 99bp so resolution was not great, but i think i pulled the right band out.

wrb 07/28/06

  • spun down overnights and put in freezer. went whitewater rafting.

wrb 07/27/06

  • some overinghts did not grow. did them again.

wrb 07/26/06

  • ran test digest + gel on constructs (and backbones)
  • the gel was pretty murky (note: protocol on test digest says run digest for 30 minutes. i think that is bs. run a test digest for 2 hours) but the stuff we could see indicated that we got parent vector, not contruction vector. bummer
  • started overnights of construction pieces: P0412, F2620, F1610, R0040

dlk 07/25/06

  • made mini preps of the overnight cultures pSB1a2-F2620+P0412 and pSB1A2-R0040+F1610
  • mini preps are sitting in a blue holder above the lgoic drawer awaiting some good ol' testin' digestin'
  • Made -80 stocks of pSB1a2-F2620+P0412 and pSB1A2-R0040+F1610 and put into box2

dlk 07/24/06

  • looked at the cell culture plates from sunday, a handful of colonies on each plate, good sign
  • used ape to plan out a test digest
  • we can digest both R0040+F1610 and F2620+P0412 with enzyme BG11 (Or Bgi1, can't tell I's from 1's)
  • made overnight stocks of R0040+F1610 and F2620+P0412 using LB Carb, put into spinner incubator
  • appropriated a drawer to store all our nifty digrams in
  • quest for simulation initated, more thoughts to come later, two inital posablities, doing a cell level simulation (simulate conjugation) and doing a simulation one level higher with cell types (simulate info flow)

wrb 07/23/06

  • ligated constructs. regular procedure.
  • transformed into TG1. Included saving for an hour in LBmedia.
  • plated both constructs onto seperate LBcarb plates. plates went in at 2.30pm
  • started overnights of T9002 and I13601, so that NAND and AND gates can be made as early as tomorrow (if our transforms work). overnights went in at 2pm.
  • modified elevator pitch on front page to include logic gates.
  • Daniel said the magic words: computer simulation. We will now quest to write simulations of our logic gate systems.

wrb 07/22/06

  • digested R0040 (spe, awln1), F1610 (xba, awln1), F2620 (spe, awln1), and P0412 (xba, awln1). digest ran for 2 hours.
  • gel and gel-cut was a little bothersome. <attach picture>. many bands exhibited only single cuts. ...we'll see how it goes. Ran the gel with small wells. the 35uL of digest + ladder was loaded into 4 (10uL) wells for each digest type.
  • gel purified. Some of the gels were so big that we went into 2 tubes. Otherwise, protocol was straightforward. eluted into 10uL bufferEB, not 2mM Tris.
  • Daniel worked some more on presenting the logic gate topic to a.) our team and b.) everyone else. That work is posted: Logic&Info processing intro

wrb 07/21/06

  • miniprepped the overnights
  • made -80 stocks of overnights
  • the results of the T9002 test were nonconclusive. The plate was not expressing gfp, but the control did not grow at all. The cells that should have made Lux to trigger T9002 did not grow.

need to:

  • set up digests for parts that need cutting and pasting to make new parts.
  • possibly, transform new parts
  • test parts without conjugation; insert parts into plasmids that do conjugation and test that.

wrb 07/20/06

  • started overnights for AND and NAND gates controlled via small molecules (Lux system): T9002, R0040, F1610, F2620, P0412, I13601, I13603. Grown in LBcarb
  • grew T9002 with LuxI producing cell srain (given from Chris) with the intent of showing that T9002 expresses GFP in the presence of Lux.

wrb 07/19/06

  • Spec'd parts for AND and NAND gates, designing for the scenario that we have Lock&Keys and for the scenario that we don't. More on this laiter, but the locks and keys make the gates more scalable into a complex system.

a NAND logic gate. In the system suggested here cell1 and cell2 provide input signals. Cell3 processes these signals and provides an output. Cell1 and Cell2 communicate with Cell3 via conjugation. Cell1 and Cell2 will be R-type conjugating cells, Cell3 will be an F-type cell. The phenomona associated with the cell types can be better explained by the conjugation folks. 

 	Cell1 will provide a ribosome containing a Lock_::promoter/rbs::LacI::terminator.

Cell2 will provide a ribosome containing a promoter/rbs::key::terminator. Cell3 will contain a ribosome with LacI-inversion-site::RFP::terminator

If Cell3 receives both Cell1 and Cell2 plasmids, output of Cell3-rfp will desist. Initial experiments should integrate a tag into successful transmission in order to assay and show hotness. Apparently, conjugation rates are fairly low (10-30%, with enough time), so conjugating 2 plasmids could see a 99% loss of signal. In order to decrease the apparent signal loss, it might be useful to tag cell-death onto cells that don’t receive plasmids. This is a tough riddle though, and for another time.

dlk 07/13/06
Addressable communication could be used to build graphical models. In the computer science sense, graphical models represent some decision making algorithm with 'nodes' and 'edges'. Bayesian networks are the canonical example of a graphical model, and a special case of a Bayesian network is the artificial neural network (ANN), which has achieved wide success in handwriting and voice recognition. If we can use addressable communication between cells to build an artificial neural network then we have the ability to use a lawn of bacterial cells to compute very non-trivial algorithms. An artificial neural network running in a lawn of cells would have very different characteristics than one implemented on a computer, including being probabilistic and massively parallel.

Constructing an artificial neural network using addressable communication between bacteria cells 

   Each node in the network is labeled {1...n} and has a corresponding lock {1...n}
   Each node in the network accepts input in the form of its key {1...n}
   Each node in the networks sends its output with a different lock

A simple type of ANN is organized into hierarchal layers with all data flowing in one direction. (A feed forward network) In this case, a node in layer one would be able to produce the locks which are opened by layer 2, layer 2 produces the locks opened by layer 3, and so on. In a 'recurrent' ANN, each node would be able to produce the locks of any other node in the network. Some internal logic in the cell would be used to determine when input levels are satisfied and what output to produce.

With two lock key pairs, it may be possible to compute the logic NAND function using an ANN framework. 

   1) The logic function NAND (Not AND, or the opposite of AND) is 'logically sufficient' meaning you can string together only NAND gates to compute _any_ logic function. (This is also true of NOR gates)
   2) ANN's can be used to compute NAND
   3) Computation of NAND using ANNs is different from designing the NAND logic into the cell 
   4) If you can show NAND computation, you have in principal shown general purpose computation
   5) If you can build ANNs, you can build extremely powerful computational machinery
   6) You can start talking about building general graphical models, which can do even more powerful computation
   7) The probabilistic and massively parallel natural of a bacteria ANN might make possible computations which are infeasible on digital computers.
   8) You're introducing both data representation (state) and computation into biologically designed networks 
   9) The learning algorithms for this would be awesome
   10) a lot of this can be described with math

How to build a NAND gate with addressable communication

We need to be able to build specific networks, determine the network topology, create a liquid culture mix of all the cell types, and grow. Expose to stimulus and the lawn should compute a function.

We need some way of determining HI and LOW from BACKGROUND 

   Use a common signal and measure it's intensity
       threshold somewhere above ambient to be LOW
       HI is somewhere above low
   Use two signals, take the strongest
       Chemical 1 is hi, chemical 2 is low, strongest concentration
   Either way, the signal should be capable of decay faster than diffusion
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Berk2006-LogicGatesTeam&oldid=55045"