Berk2006-LogicGatesTeam

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dlk 08/04/06

• first attempt at handling pair wise interactions appears to work
• it looks pretty fast
• none of the units are correct, so all quantities are unitless, unclear what they should be
• on the units subject, I think this might be a PoPS-ish simulation
• the below shows simulation of r0040, B0034, C0062 producing luxR as levels of tetracycline drop

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dlk 07/31/06

• i've got a simulation of T9002 working
• everything is modeled probalisticly
• takes into account diffusion and protein half life, doesn't take into account secondary structure or pairwise cytoplasm reactions
• time to talk to chris about how that fancy light sensor machine works to see if we can do a real world v simulation comparison in the time domain
• time to write up what I did in detail

  

wrb 07/31/06'

• plates had lots of little colonies on 'em. starting overnights in LBcarb, and the r0040+f1610 in LBamp/kan, since the construct SHOULD be on PSB1AK3. cultures went in at 11:30pm
• actually, no LBcard in site. started r0040+f1610 in LBkam for giggles.
• ought to put these constructs into the registry and get them their own part names.

wrb 07/30/06

• gel purify
• ligation: choosing insert and vector with the BglI cut site is kind of wierd...Bgl is more or less in the middle of the plasmid. chose the xba, bgl cut pieces to be "vector"
• transformation (heat shock) abd plated onto carb plates.
• plates went into 37deg. @ 5.30pm .

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...(and the gel came out ok)
• 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.
• tomorrow: gel purify, ligate, transform
• monday morning: look at plates, run testdigest

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