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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