03+19+15

03/19/15  

Declare  your  major  J  Students  must  declare  a  major  during  or  before  the  spring  semester  of  their  second  year  or  will  not  be  permi?ed  to  register  for  the  fall  semester  of  their  junior  (or  third)  year.    If  you  have  not  declared  yet,  you  may  bring  the  following  to  class:    (1)  a  filled  out  copy  of  the  declara0on  of  major  form  (see  instrucGons  below)  (2)  an  unofficial  copy  of  your  transcript    I  will  collect  these  documents  and  make  sure  they  are  signed  and  filed  with  the  registrar  office.  The  procedure  is  outlined  at:    h?p://registrar.rice.edu/students/majors_minors/#declaring    You  can  ignore  the  advise  that  you  must  personally  deliver  the  signed  form  to  the  registrar  office:  if  you  get  these  form  to  me  now,  our  departmental  staff  will  file  them  with  the  registrar.      

Biosensor  

Biosensing  module  +  transducer  =>  output  

Whole  cell  sensing  system  

Example:  quorum  sensing  

Output  signals  

Output  signals  

Modular  structure  of  programmable  cells  

SyntheGc  Biosensors  

Biosensing  module  

•  Disease  biomarker/pollutant  that  interfaces  with  endogenous  regulatory  mechanism  

Transducers  

•  Use  bacterial  geneGc  circuitry  •  Rewire  exisGng  systems  to  perform  novel  funcGons  novel  and  generate  syntheGc  network  

•  Improve  sensiGvity  and  dynamic  range  of  output  

•  Integrate  mulGple  inputs  to  generate  desired  logic  gates      

INVERTER  

GENETIC  OSCILLATOR  

Construct  a  NAND  gate    (with  TFs)                OR  gate  (inducers)  

Construct  a  NOR  gate    (with  TFs)                            AND  gate  (inducers)  

AND  gate  

OR  gate  

AND  gate  (inducers)  

OR  gate  (inducers)  

AND  with  memory  

Feedback  loops  Response    Gme  Steady  state  output  levels  

ArGficial  control  of  gene  regulaGon?  

© 2005 Nature Publishing Group

Vol 438|24 November 2005

441

Engineering Escherichia coli to see lightThese smart bacteria ‘photograph’ a light pattern as a high-definition chemical image.

We have designed a bacterial system that isswitched between different states by red light.The system consists of a synthetic sensorkinase that allows a lawn of bacteria to func-tion as a biological film, such that the projec-tion of a pattern of light on to the bacteriaproduces a high-definition (about 100 mega-pixels per square inch), two-dimensionalchemical image. This spatial control of bac-terial gene expression could be used to ‘print’complex biological materials, for example, andto investigate signalling pathways through precise spatial and temporal control of theirphosphorylation steps.

Plants and some bacteria use a class of pro-tein photoreceptors known as phytochromesto control phototaxis, photosynthesis and theproduction of protective pigments1–3. Photo-receptors are not found in enterobacteria, suchas Escherichia coli, so we created a light sensorthat functions in E. coli by engineering a chimaera that uses a phytochrome from acyanobacterium.

A phytochrome is a two-component systemthat consists of a membrane-bound, extra-cellular sensor that responds to light and anintracellular response-regulator1. The response-regulators of most phytochromes do not haveDNA-binding domains and do not directlyregulate gene expression, so we fused a cyano-bacterial photoreceptor to an E. coli intracellularhistidine kinase domain (Fig. 1a, and see supplementary information). This design wasbased on the well studied E. coli EnvZ–OmpRtwo-component system, which normally reg-ulates porin expression in response to osmoticshock4. The EnvZ histidine kinase domain hasbeen used for the construction of functionalchimaeras5,6, and a plant phytochrome haspreviously been used to construct a two-hybrid gene expression system in yeast7.

To create the chimaera, we aligned membersof the phytochrome family with EnvZ andidentified potential functional crossover pointsbetween the Synechocystis phytochrome Cph1and EnvZ. (For methods, see supplementaryinformation.) The length and composition ofthe peptide that links a photoreceptor to itsresponse-regulator can affect signal transduc-tion5,6, and we therefore constructed a series ofchimaeras with variable linker lengths. Thevariants were transformed into a !EnvZ E. coli strain containing a chromosomal fusion between the OmpR-dependent ompCpromoter and the lacZ reporter4, which

enzymatically produces a black compound. The part of the photoreceptor that responds

to light, phycocyanobilin, is not naturallyproduced in E. coli. We therefore introducedtwo phycocyanobilin-biosynthesis genes (ho1 and pcyA) from Synechocystis that convert haem into phycocyanobilin8 (partsBBa_I15008, BBa_I15009; MIT Registry ofStandard Biological Parts) (Fig. 1a, inset).Individual Cph1–EnvZ chimaeras were thenactivated at 37 "C for 4 h with broad-spec-trum light and assayed for expression of the lacZ reporter. The chimaera Cph8(BBa_I15010) produced a particularly strongresponse to light (Fig. 1b).

For bacterial photography, we grew a lawnof bacteria on agar. The lacZ reporter was visu-alized by addition of S-gal (3,4-cyclohex-enoesculetin-#-D-galactopyranoside): LacZcatalyses the formation of a stable, insoluble,black precipitate from S-gal. Light repressedgene expression in the bacteria, giving a high-contrast replica of the applied image on

the biological film, in which light regionsappeared light and dark regions were dark(Fig. 1c, and see supplementary information).The lacZ activity showed a graded response toincreasing light intensity that was minimal inthe brightest light (Fig. 1d).

Our creation of a novel genetic circuit withan image-processing function demonstratesthe power and accessibility of the tool sets andmethods available in the nascent field of syn-thetic biology. The principle of programmedlight regulation should enable gene expressionto be spatially and temporally controlled inindividual cells and in populations, leading topotential application in bacterial microlithog-raphy, manufacture of biological materialcomposites and the study of multicellular signalling networks. Anselm Levskaya*, Aaron A. Chevalier†, JeffreyJ. Tabor†, Zachary Booth Simpson†, Laura A.Lavery†, Matthew Levy†, Eric A. Davidson†,Alexander Scouras†, Andrew D. Ellington†‡,Edward M. Marcotte†‡, Christopher A. Voigt*§||

BRIEF COMMUNICATIONS

P

pcyA

ho1

ompCpromoter

lacZ

Blackoutput

PCB

Haem

1,600

1,200

Mill

er u

nits

Out

put

800

400

0+Cph8

Position

– + –PCB

a b

c d

0.80

0.90

1.00

PCB

P

PCB

Figure 1 | Light imaging by engineered Escherichia coli. a, The chimaeric light receptor Cph8 containsthe photoreceptor from Cph1 (green) and the histidine kinase and response-regulator fromEnvZ–OmpR (orange); inset, conversion of haem to phycocyanobilin (PCB), which forms part of thephotoreceptor. Red light drives the sensor to a state in which autophosphorylation is inhibited (right),turning off gene expression. For details of genes, see text. b, Miller assay showing that Cph8 is active inthe dark (black bars) in the presence of PCB and inactive in the light (white bars). There is no light-dependent activity in the absence of Cph8 ($) and there is constitutive activity when only the histidinekinase domain of EnvZ is expressed (%), or when the PCB metabolic pathway is not included ($PCB).c, When an image is projected on to a bacterial lawn, the LacZ reporter is expressed only in the darkregions. d, Transfer function of the circuit. As the intensity of the light is increased by using a lightgradient projected from a 35-mm slide, the circuit output gives a graded response.

24.11 brief comms NEW 17/11/05 5:21 PM Page 441

Nature Publishing Group© 2005

© 2005 Nature Publishing Group

Vol 438|24 November 2005

441

Engineering Escherichia coli to see lightThese smart bacteria ‘photograph’ a light pattern as a high-definition chemical image.

We have designed a bacterial system that isswitched between different states by red light.The system consists of a synthetic sensorkinase that allows a lawn of bacteria to func-tion as a biological film, such that the projec-tion of a pattern of light on to the bacteriaproduces a high-definition (about 100 mega-pixels per square inch), two-dimensionalchemical image. This spatial control of bac-terial gene expression could be used to ‘print’complex biological materials, for example, andto investigate signalling pathways through precise spatial and temporal control of theirphosphorylation steps.

Plants and some bacteria use a class of pro-tein photoreceptors known as phytochromesto control phototaxis, photosynthesis and theproduction of protective pigments1–3. Photo-receptors are not found in enterobacteria, suchas Escherichia coli, so we created a light sensorthat functions in E. coli by engineering a chimaera that uses a phytochrome from acyanobacterium.

A phytochrome is a two-component systemthat consists of a membrane-bound, extra-cellular sensor that responds to light and anintracellular response-regulator1. The response-regulators of most phytochromes do not haveDNA-binding domains and do not directlyregulate gene expression, so we fused a cyano-bacterial photoreceptor to an E. coli intracellularhistidine kinase domain (Fig. 1a, and see supplementary information). This design wasbased on the well studied E. coli EnvZ–OmpRtwo-component system, which normally reg-ulates porin expression in response to osmoticshock4. The EnvZ histidine kinase domain hasbeen used for the construction of functionalchimaeras5,6, and a plant phytochrome haspreviously been used to construct a two-hybrid gene expression system in yeast7.

To create the chimaera, we aligned membersof the phytochrome family with EnvZ andidentified potential functional crossover pointsbetween the Synechocystis phytochrome Cph1and EnvZ. (For methods, see supplementaryinformation.) The length and composition ofthe peptide that links a photoreceptor to itsresponse-regulator can affect signal transduc-tion5,6, and we therefore constructed a series ofchimaeras with variable linker lengths. Thevariants were transformed into a !EnvZ E. coli strain containing a chromosomal fusion between the OmpR-dependent ompCpromoter and the lacZ reporter4, which

enzymatically produces a black compound. The part of the photoreceptor that responds

to light, phycocyanobilin, is not naturallyproduced in E. coli. We therefore introducedtwo phycocyanobilin-biosynthesis genes (ho1 and pcyA) from Synechocystis that convert haem into phycocyanobilin8 (partsBBa_I15008, BBa_I15009; MIT Registry ofStandard Biological Parts) (Fig. 1a, inset).Individual Cph1–EnvZ chimaeras were thenactivated at 37 "C for 4 h with broad-spec-trum light and assayed for expression of the lacZ reporter. The chimaera Cph8(BBa_I15010) produced a particularly strongresponse to light (Fig. 1b).

For bacterial photography, we grew a lawnof bacteria on agar. The lacZ reporter was visu-alized by addition of S-gal (3,4-cyclohex-enoesculetin-#-D-galactopyranoside): LacZcatalyses the formation of a stable, insoluble,black precipitate from S-gal. Light repressedgene expression in the bacteria, giving a high-contrast replica of the applied image on

the biological film, in which light regionsappeared light and dark regions were dark(Fig. 1c, and see supplementary information).The lacZ activity showed a graded response toincreasing light intensity that was minimal inthe brightest light (Fig. 1d).

Our creation of a novel genetic circuit withan image-processing function demonstratesthe power and accessibility of the tool sets andmethods available in the nascent field of syn-thetic biology. The principle of programmedlight regulation should enable gene expressionto be spatially and temporally controlled inindividual cells and in populations, leading topotential application in bacterial microlithog-raphy, manufacture of biological materialcomposites and the study of multicellular signalling networks. Anselm Levskaya*, Aaron A. Chevalier†, JeffreyJ. Tabor†, Zachary Booth Simpson†, Laura A.Lavery†, Matthew Levy†, Eric A. Davidson†,Alexander Scouras†, Andrew D. Ellington†‡,Edward M. Marcotte†‡, Christopher A. Voigt*§||

BRIEF COMMUNICATIONS

P

pcyA

ho1

ompCpromoter

lacZ

Blackoutput

PCB

Haem

1,600

1,200M

iller

uni

ts

Out

put

800

400

0+Cph8

Position

– + –PCB

a b

c d

0.80

0.90

1.00

PCB

P

PCB

Figure 1 | Light imaging by engineered Escherichia coli. a, The chimaeric light receptor Cph8 containsthe photoreceptor from Cph1 (green) and the histidine kinase and response-regulator fromEnvZ–OmpR (orange); inset, conversion of haem to phycocyanobilin (PCB), which forms part of thephotoreceptor. Red light drives the sensor to a state in which autophosphorylation is inhibited (right),turning off gene expression. For details of genes, see text. b, Miller assay showing that Cph8 is active inthe dark (black bars) in the presence of PCB and inactive in the light (white bars). There is no light-dependent activity in the absence of Cph8 ($) and there is constitutive activity when only the histidinekinase domain of EnvZ is expressed (%), or when the PCB metabolic pathway is not included ($PCB).c, When an image is projected on to a bacterial lawn, the LacZ reporter is expressed only in the darkregions. d, Transfer function of the circuit. As the intensity of the light is increased by using a lightgradient projected from a 35-mm slide, the circuit output gives a graded response.

24.11 brief comms NEW 17/11/05 5:21 PM Page 441

Nature Publishing Group© 2005

We  have  designed  a  bacterial  system  that  is  switched  between  different  states  by  red  light.  The  system  consists  of  a  syntheGc  sensor  kinase  that  allows  a  lawn  of  bacteria  to  funcGon  as  a  biological  film,  such  that  the  projecGon  of  a  pa?ern  of  light  on  to  the  bacteria  produces  a  high-­‐definiGon  (about  100  megapixels  per  square  inch),  two-­‐dimensional  chemical  image.  This  spaGal  control  of  bacterial  gene  expression  could  be  used  to  'print'  complex  biological  materials,  for  example,  and  to  invesGgate  signaling  pathways  through  precise  spaGal  and  temporal  control  of  their  phosphorylaGon  steps.   25