WINFREE, Erik  CalTech BIOCHEM CIRCUITS in Cell-From Systems 21 Mar 2013

CalTech Prof; CS  and CNS and Bioengr  at Caltech

DNA and Natl Algos DNA Group:

intro by  R  Dass

Motivation:

personal level:  why do science?
what is our place in the world?
(was in high school)
what struck me:  one could really understand  a computer from beginning to end... everything
and  program it...

and cells are also little programs...  looked the same to me

DNA to RNA  to protein... both info systems

tranformation of a concept to embodiment in the real world:
happens with computer programs... and 
same thing with  CELLS/ LIFE:

program  concepts  become real in cells:  
how does it happen?

The story

Stan Leibler  hosted him at  Princeton
(and Michael Owitz:  made a ring oscillator in E Coli)
1999  I want to make a genetic regulatory  oscillaor in E coli!  
(he stayed around for an extra year to do this  (Michale Owitz did)

but  Erik  thout...(in 1999)  get rid of the cell and just do it in a test tube...
struggled, struggled for  many, may years  and finally publishe dit in 2011
(Michael did it in a cell in 2001)

My arguments to Michael:

there is so much abt cells that we don't understand.  Attempts to build circuits in cells
bumps up against the unknow.
(the unknown is good in bio  (perhaps) cuz its the springboard to new discos)

Even the simplest bio org is too complex for a complete understand...

wht we need is a series of ever more complex biochem systems
building gradually from a single molec
(liek we do with transistors and logic)

once reliable,  systemcatic building blocks are found,  we shud be able to create and explore expon more
compelx systems.

Even tho  it took me and  a few students ten years to build the oscillator,
its worth it... cuz we are learning how to put info into a molecule...

Questions about Information-based Chem

what kinds of decn task or control raks can be carried out by chemistry and what cannot?

What are the most efficient ways to do it?

Do you need more biochem complexity to carry out more complex tasks?

What is the simplest basis for progrmmable circuitry?

Can we study  "toy cases" to help build a rigorous fnd for design and analys of biochem circuits?

*****
Next....  I will present a  TOY CASE that will hep build a basis:  like LEGO BLOCKS...

CASE 1:  The genetic regulatory cicuit  architecture  (like Michael Elowitz's  ring oscillator)

How simple can it be?

over 5000 genes in many E coli strains...

can we do without cells?

can you make  a system will all the compoents but without the cell
(but with the DNA,  transcription factors, etc)

T7  RNA polymerase
E coli  RNase H and R
add in DNA with difft sequences
have something to make a signal and to shut it down...

see Kim,  Hopfield, Winfree  neural net comp  by in vitro transcip circuits in NIPS 2004
and  5 other papers

Kim, White, Winfree,  Construciton of an in vitro bistable circuit  etc...

Jongmin Kim  was his  v first grad student  (great)

Erik's advisor was John Hopfield

we need RNA itslef as a gtranscrip factor to turn genes on and off...

have short DNA  maye  100 to 150  bases...

he shows an in vitro  transcrip switch...

shows a small amt of DNA with an incomplete  promoter  that takes an activator olognucleotide

imcomplete proters transcibe poorly;
promoter swith nicks transcribe well

want a  switch like behavior... how to get there?

threshold  behavior by sequestration
ie  some threshold concentration of the transc  factor is required to achieve the step funciton..

so,  something soaks up the transcrip factor until there is an excess that turns the system off...

direct math connex  btwn  neural networkd vs transcrip cicuits

RNA levels are analog to the firing levels of neurons  and syanpses a re analog to
these transcrip switches...

can make  comelx attractor states...
can do winner take all selection...

lots of things u can do int heory...

What we learned...

most of teh machinery of the cell is NOT nec. for logical control of behavior in principles.

the same set of molecs can perform vastly difft functions...

Kristin White  (an undergrad)  helped on this...

Example switch seq  T7 promoter

need toehold of 8 nucleotides  (at front)

then 27  bases to turn this switch off  then the T7 promoter then the output the a hairpin at the end...

;modular   design... a working switch that you can modify...

characgerizing switches...

ON  has a fluorphore  that is quenched

OFF is unquenched  (and glows)

create some molecs that were either ON or OFF

produces  RNAP  goes to RNase H which produces  A2...

he shows kinetics of  fluorescence...  (sigmoid curves)

can get some nice curvies...

concentraitons in nanoMoles

switch one turns on switch 2...

does gel expts....  can match the model...

but (some species are being produced that are NOT part of the model)

Model...   a dozen or more  Michaelis menton eqns...

Table 1  model params...

he then shows a mutually inhbitory circuit...

Sw12  (produces 1 but regul by 2)  and
Sw21  (produces 2 but regul by 1)

he shows a ring oscillator  (like a hpfield network) that
shoudl botain bistable behavior...

can get bistabel behavior   (it is cell free, so can tune it and change concetnrations)

can systematically change  concentraitons to see if it is  stable for one species being high or
theother species beinghigh vs bistable in the middle...

But his expeeriment is difft fromteh model  (perhaps cuz of time constants for RNAs...
cuz  of  an excess of certian RNA species)

What we learned

in vitro transcrip circuitry can be systematicaly designed and buitla dnit works!

mechais mdoels can be useful for designs
keys tsem params can be easily varied..

BUT...
beware of unintednde dsecondary struc
beware of promiscuous  RNA...  etc.

new undergrad  Pakpoom  Sub......  long name  2012  paper...

inverter and repeater switch designs

then Jongmin built an oscil  based on this...

two switch negative feedback oscilaltor...

depends on Hill exponent...
looka t null clines for the 2 swtiches...

T21  in Texas Red  and T12 in Tamra  (difft fluoros)

Sw12 produces rA1 which  control Sw21  tha tprocues  rA2...

gel characterizaiton...   get junk species that accumulate...

oscillator  did work  DESPITE  accum of junk  (that it had to overcome...

beare of oversimplified models and intuitions!

need system taht is robust  (eg to waste species)

PNAS  2011  big paper
Franco,  Friedrichs, King, Jungmann  et al  ...

coupling principels

oscill behavior is a func of  switch characgeristics...

DNA  produces RNA  (but say its absored by L, a load)  the load will damp the oscill

so, systemis sensit  to amt of load...

can get around that (by controlling the load)

use  a SNA tweezer...

the load is an DNA tweezer...

opeare the tweezer with RNA transcript...

can take out the load  using tweezers...

incr amts of load shuts down the oscillation...

*****

now  some unpub  work...

collab with Fritz Simmel in Germany...

small volume effects...

the smaller system , the more noise we wud expect due to (shot effects) small number effects..

100 micro liter system...

show  100 micron scale bar...

microemuslion  with every droplet that has  an scillator in it...

each driplet oscilaltors do high frame rate photogr...

r =  7.5 micorns

he shows a movie...

oscillators in water in oil emulsion...

these are 20 to 30  micron droplets... osc like xmas  tree  lights!!

can look at behavior as a func of size of droplets

small droplet  (less than 5 mics)  more stochasticity...

bigger: less variablity...

some oscillate for over 10 hours!

can see some that are sustained, some damped and some strongly damped...

thot  that this would be  explaiend by  (gilespie style simulatiosn)

but stoch reaciton kinetics do not explain it...

but partitioning effects do  (better)

perhaps  two droplets do not  get the same amt of stuff when born...  yes...

eg  if  droplet 1  gets more  RNase H thandropelt 2  (that is their lowest conc species)

Yes... partitioningdistribs are wide...

big summary:  borader picture

biology,  computers, and chemistry

but if u can program chemistry  then perhaps u can do  synthetic biology...