Berk2006-LogicGatesTeam

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wrb 08/16/06

• note: 8.14's tecan was of J23039(5). this time around i grew J23039(6). It showed much stronger Luxation over J23039(5), by fluorescense
• I also tried to grow T9002 in LuxGFP media, but that didn't work!
• I also tried to grow I13601, and I13601 with IPTG, since TG1 cells are Lac blahblah. but i didn't really know how much iptg to add, even though i told Chris I'd be able to figure out. =( oops. one more try tomorrow morning!

wrb 08/14/06, part 2

• progress: it's taking longer to do everything than i thought.
• saw 7x increase in fluorescence between J23039 and J23039+T9002.
• sequence data says constructs worked. sequence data is not perfect though. The parts made it in but the parts are not necassarily wwhat they should be / what we thought they were.
• growing up more stuff. Putting T9002 in a heavily LuxI'd media (via pAC-LuxGFP) that we trust as well as one we don't (J23039). growing at 30C because Chris suspects Lux system is not super stable at 37C.
• Also growing the J23039 at 37C to test that hteory
• also continuing the growth of this morning's growth, in hopes that diffusion "just takes a little longer." doubtful.
• preparing for Wed. meeting to explain system and plan
• preparing for move to boston.

dlk 08/15/06

• Progress: It’s taking longer to build a simulation of conjugation than I thought it would. A key difficulty is the effect of ‘clumping’. I feel like, at least in solution, the clumping plays an important role, but so far my attempts to build a good model for clumping conjugation are not working. I may just do a uniform mixture model of conjugation and come back to the clumping question once everything else is worked out.
• To build a real neural network, we need some concept of inhibition or ‘negative weights’ in addition to the backprop error mechanism which ‘zeros’ weights that are contributing to the error. My first proposal for this, (see side figure) is to split each ‘node’ into two cell types, one which represents the positive weight (and passes forward the gent marker) and one which represents the negative weight, (and passes forward the CCD)
  Single signal
  
  Or two signals
  
• This greatly complicates both the Hebbian re-enforcement and the backpropogation of error. I don't yet have a solution, but I think the solution will be to have each lock be unique and coorespond to a 'return' address as well as the 'destination' address.
• The feedback mechanisms make sense in the context of a single layer and a multi-layer.
  Single layer
  
  Multi-layer feedback
  
• If I can figure out the backwards addressing, then this feedback mechanism will work for both Hebbian learning and backprop. The backwards addressing is the key design issue.
• As can be seen in the multi-layer feedback, it is important that the next stage of the feedback is sent before the cell death occurs. I assume this can be done by placing the CCD on another lock with is opened by the first lock. (The first lock opens letting both the next layer feedback and the key for CCD activate)
• I think the best way to visualize the information flow will be using a dynamic graph.
  

I'm using Ali Cemgil's dynamic belief net graphing package for matlab, which I'll turn into a movie, with each time step of the simulation being a frame in the animation. So the final output will be a network graph that evolves through time providing a visual representation of the information processing function that the cell culture is computing. I don't think open wetware will let me post MPEG files, so I'll stick them on one of my servers and link them here, but I'm not going to be generating those till I figure out the backprop addressing issue and the conjugation clumping issue.

• Will is awsome for being in lab at 2am. mad props.

wrb 08/14/06

• started more overnights, this time @ 30C: pAC-LUXGFP in LB, T9002 + J23039, I13601 + J23039, J23039, and T9002 all in LBcarb. juice went in at 2am.

dlk 08/12/06 conjugation clumping [[1]]

wrb 08/10/06

• still no flourescence.
• transformed J23040 miniprep that showe positive results in digest on 08/07/06. started overnights.
• PCR colony with the 6 J23039 parts grown (K009, G00101). a few of them showed the activity. I have the cells left over from their overnight in the freezer.

...j23039 and j23040 are pretty much done, it looks like.

• time to spec. out parts into conjugation
• time to spec. out parts with locks and keys
• time to make parts work in solution (not just parts, systems...)

dlk 08/09/06

• Talked with chris and john about how to make the simulations useful
• Use a simpler system, Key1 with gent marker -> cell (lock1, that is locking key2) -> lock2-tra -> regulate back to the cell with a cell death command
• Cells that receive the gent marker live
• Cells that produce the wrong key output receive the cell death command
• two forms of regulation, upregulate with the gen marker if the lock key pair matches, down regulate with the cell death command if the output is incorrect
• allows for a back prop style learning, but not really scalable, works for small scale proof of concept.
• pretty pictures will be required

wrb 08/08/06

• still nothing growing green.
• transferred .5 mL of each J23039 culture into fresh LBamp with .5mL T9002. (5mL total cultures)
• if they don't show green soon, the rest of the J23039 cultures will be miniprepped and pcr'd tonight for sanity. maybe T9002 is bogus?

wrb 08/08/06

• started overnights @ 9.30 am. growing in LBamp: J23039, J23040, J23039+T9002, J23039+I13601, J23039+I13601+J23040
• tubes i expected to glow green did not. everything is still white. leaving these overnights in, in hopes that small molecule diffusion leading to gene expression just takes a long time.
• picked 6 more J23039 colonies to grow.

wrb 08/07/06

• PCR on some more colonies from the construction, using KB009/G00101 oligos.
• gel digest on miniprep from last week's overnights. cut eco/pst
• i am optomistic j23039 construct worked, though not all checks were perfect. the j23040 pcr did not come out, but one of the test digests showed correct results. construct completed?

wrb 08/06/06

• trying to test constructs J23039 and J23040. J23040 is much bigger (almost 800 bp's) than either of the first parts, so, running a gel of the miniprep.
• J23039 is harder to test: one of the parts in J23039, R0040, contributed only ~70 unique bp's to the construct. Could grow J23039 up and do a LuxI assay? but i don't know how to do that. Starting overnights of J23039 and T9002 cells in the same vial. should cause fluoresense if everything is working. if it doesn't work, too many problems would be possible for the issue to be instantly resolvable.

wrb 08/04/06

• miniprepped overnights that had been overnighting for almost 40 hours. I think that generall it's not great practice to do that.
• gave part numbers to the constructs. see J23039 and J23040.

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
• I no longer think the first simulation of T9002 given on 07/31/06 is correct
• the below shows simulation of r0040, B0034, C0062 producing luxR as levels of tetracycline drop

• 

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