sRNAs in Artificial Gene Circuits and Bioremediation...
Transcript of sRNAs in Artificial Gene Circuits and Bioremediation...
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sRNAs in Artificial Gene Circuits and Bioremediation Applications
University of Illinois Urbana-Champaign 2010 iGEM
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
TEAM ADVISORS
The University of Illinois iGEMBioware Team
HUMAN PRACTICES
GOAL
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
COURTNEY FUENTES-EVANSYONG SU JIN Ph.D.JOANNE MANASTERCHRISTOPHER RAO Ph.D.
HUMAN PRACTICES
TEAM ADVISORS GOAL
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
The 2010 UIUC iGEM project has two components
Heavy metal bioremediation
Modular sRNA logic gates
HUMAN PRACTICES
TEAM ADVISORS GOAL
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
Our goal is to develop a sRNA-mediated artificial gene circuit and to ultimately implement it into a heavy metal bioremediation system.
HUMAN PRACTICES
TEAM ADVISORS GOAL
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
Project Part I: Goal•Our goal is to construct a bacterial decoder based on sRNA
•A = INPUT
•D = OUTPUT
HUMAN PRACTICES
A1 A2 D1 D2 D3 D4
0 0 1 0 0 0
0 1 0 1 0 0
1 0 0 0 1 0
1 1 0 0 0 1
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
Approach: Small RNAs
•Small single stranded fragment of RNA
•Used as responders to cellular stress in bacteria
•Targets specific sequences on mRNA
•Can activate or repress target gene transcripts
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
Motivation: Small RNAs
•As engineered biological designs grow in complexity, multiple levels of genetic regulation will be required.
•Most applications in synthetic biology involve a system’s novel response to its external environment.
•The Parts Registry is still beginning to explore these areas
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
HUMAN PRACTICES
sRNA Activate Target Gene Expression
sRNA Repress Target Gene Expression
GFP gadXGadY
Biobricks
GadY
Biobricks
MicA
MicFompA GFPMicA
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
HUMAN PRACTICES
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
ompF ompA gadX
FLU
OR
ESC
ENC
E
TARGET SEQUENCE::GFP FUSION
0 mM [IPTG] hfq+
1mM [IPTG] hfq+
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
sRNAs can be used to make simple logic gates
HUMAN PRACTICES
GFPompA gadX
micA
gadY
IPTG (B) atc (A) Expected Output
0 0 0
0 1 1
1 0 0
1 1 0
STATEMENTA AND NOT B
atc AND NOT IPTG
Plac
Ptet
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
HUMAN PRACTICES
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
0 mM [IPTG], 0 mM [atc]
1 mM [IPTG], 0 mM [atc]
0 mM [IPTG], 1 mM [atc]
1 mM [IPTG], 1 mM [atc]
Flu
ore
sce
nce
IPTG atc Expected Output
Observed Output
0 0 0 0.35
0 1 1 1.00
1 0 0 0.11
1 1 0 0.27
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
sRNAs can be used in a Bacterial Decoder Function
HUMAN PRACTICES
020000400006000080000
100000120000140000160000180000200000
0 mM [IPTG], 0 mM [atc]
1 mM [IPTG], 0 mM [atc]
0 mM [IPTG], 1 mM [atc]
1 mM [IPTG], 1 mM [atc]
Flu
ore
sce
nce
Characterization of K091101 (lac-tet fusion promoter)
IPTG atcExpected Output
GFP EYFP ECFP mCherry
0 0 1 0 0 0
1 0 0 1 0 0
0 1 0 0 1 0
1 1 0 0 0 1
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
HUMAN PRACTICES
IPTG atcExpected Output
Observed Output
0 0 1 0 0 0 1.00 0.01 0.00 0.01
1 0 0 1 0 0 0.10 1.00 0.00 0.00
0 1 0 0 1 0 0.09 0.43 1.00 0.01
1 1 0 0 0 1 0.03 0.39 0.10 1.00
Performance of A Bacterial Decoder
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
DECODERsRNA BIOBRICKS LOGIC GATESGOAL
HUMAN PRACTICES
Part I: Summary•Submission of 7 basic sRNA Biobrick
parts
•Successful construction of a bacterial
decoder using sRNA regulation.
•Only 3 functional sRNAs
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OVERVIEW RNA BIOREMEDIATION CONCLUSIONHUMAN PRACTICES
BIOBRICKSARSENIC CONSTRUCTGOLD
Project Part II: Gold and Arsenic Bioremediation System
Our goal is to develop a sRNA mediated artificial gene circuit and to ultimately implement it into a heavy metal bioremediation system.
MODELING
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Project Part II: Goal is to remove metals from water
Arsenic needs to be removed from drinking water
OVERVIEW RNA BIOREMEDIATION CONCLUSIONHUMAN PRACTICES
ARSENICGOLD BIOBRICKSMODELINGCONSTRUCT
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Project Part II: Bacteria are inexpensive and effective solution
OVERVIEW RNA BIOREMEDIATION CONCLUSION
ARSENICGOLD
HUMAN PRACTICES
BIOBRICKSMODELINGCONSTRUCT
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ARSENICGOLD BIOBRICKSMODELINGCONSTRUCT
OVERVIEW RNA BIOREMEDIATION CONCLUSIONHUMAN PRACTICES
The Bioremediation system uses metal-binding proteins that attach to the cell’s outer surface and collect free-floating metal ions.
Transmembraneprotein (LamB)
Metal Binding protein (GolB or ArsR)
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OVERVIEW RNA BIOREMEDIATION CONCLUSIONHUMAN PRACTICES
ARSENICGOLD BIOBRICKSMODELINGCONSTRUCT
sRNA logic gates regulate efflux pumps. Arsenic and gold ions leave cell through efflux pumps, which keeps the cell alive
sRNA
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ARSENICGOLD
Bioremediation Small RNA Construct
HUMAN PRACTICES
Sensor
Constitutive
Promoter
Inducible
Promoter
GadY
Constitutive
Promoter
Efflux pump GadX
Efflux pump GadX
GadY
GadY
BIOBRICKSMODELINGCONSTRUCT
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ARSENICGOLD
HUMAN PRACTICES
MODELINGCONSTRUCT BIOBRICKS
Parts involved in bioremediation
Gold Arsenic
Sensor GolS ArsR
Efflux GesABC ArsB
Binder GolB-LamB ArsR-LamB
Promoter PGolS PArsRInducible
Promoter
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ARSENICGOLD
Bioremediation Schematic:
EffluxSensor
Binder
Constitutive
Promoter
Constitutive
Promoter
Inducible
Promoter
HUMAN PRACTICES
BIOBRICKSMODELINGCONSTRUCT
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ARSENICGOLD
Final Construct
HUMAN PRACTICES
BIOBRICKSMODELINGCONSTRUCT
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ARSENICGOLD
HUMAN PRACTICES
MODELINGCONSTRUCT BIOBRICKS
Strong Arsenic Removal Based on
Cell Concentration and Affinity for
Arsenic
The projected Arsenic
collection for our
system is well over
50ppb, particularly at
cell concentrations
over 7mg/mL.
100
90
80
70
60
50
40
30
20
10
0
Wild type E. coli
with up-regulated
ArsR.
Our projected
Bioremediation
System
Regular. Coli
0 1 2 3 4 5 6 7 8
9 10 Dry Weight of Cells (mg/mL)
Ars
enic
Colle
ction (
ppb)
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ARSENICGOLD
HUMAN PRACTICES
MODELINGCONSTRUCT BIOBRICKS
Biobricks
PArsR Promoter
ArsR Sensor
ArsR-LamB
Binder
ArsB-GadX
Efflux
GadY(sRNA)
ArsBstabilizer
Biobricks in Au-As Bioremediation System
Biobricks
PGolS Promoter
GolS Sensor
GolB-LamB
Binder
Ges-GadX
Efflux
GadY(sRNA)
Gesstabilizer
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
RESULTSGOAL MEDIASURVEY OUTREACH
Overview
Goal: better connect with our community to spread the word about synthetic biology.
Customize presentation: survey
Meet in a variety of settings: public presentations
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
RESULTSGOAL MEDIASURVEY OUTREACH
The Survey
•Short Survey on public conceptions and beliefs about synthetic biology
•Distribution Method: online
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
RESULTSGOAL SURVEY OUTREACH
Survey Population Characteristics
Location N umber of Responses
Illinois 65
Indiana 1
Missouri 1
Texas 1
Wisconsin 1
India 1
Taiwan 1
Vietnam 1
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
RESULTSGOAL SURVEY OUTREACH
Survey Questions/Answers:Q: What are the first 3 words that come to mind when you hear the words “Synthetic Biology?”
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
RESULTSGOAL SURVEY OUTREACH
Survey Questions/Answers
Q: What kind of science and/or synthetic biology do you see in pop culture (music, movies, tv etc.)?
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
RESULTSGOAL SURVEY OUTREACH
• Engineering Open House
• Biology Open House
• Science Olympiad
National Tournament
• G.A.M.E.S. Camp
• Quad Day
•Surrounded By Science
Public Outreach
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ACKNOWLEDGEMENTSJUDGING
Continuing Work
•Continue human practices
•Integrate small RNA logic gates with bioremediation system
•Test the metal system
•Compare our collection system to existing systems
•Prepare for 2011 UIUC iGEM team!
FUTURE WORK
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ACKNOWLEDGEMENTSFUTURE WORK JUDGING
Medal Criteria
Bronze:
•Successfully complete and submit a Project Summary form. Create and share a Description of the team's project via the iGEM wiki
•Present a Poster and Talk at the iGEM Jamboree
•Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Parts
•Submit DNA for at least one new BioBrick Part or Device to the Registry of Parts
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ACKNOWLEDGEMENTSFUTURE WORK JUDGING
Medal Criteria
Silver:
•Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected.
•Characterize the operation of at least one new BioBrick Part or Device and enter this information on the Parts or Device page via the Registry of Parts
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ACKNOWLEDGEMENTSFUTURE WORK JUDGING
Medal Criteria
Gold:
•Help another iGEM team
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ACKNOWLEDGEMENTSFUTURE WORK JUDGING
HUMAN PRACTICES
We collaborated on various levels with a number of teams
•UIUC Software
•Project Advice:•Peking
•Purdue
•Surveys:•Warsaw
•METU Turkey Software
•Edinburgh The University of Illinois
Software Team
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ACKNOWLEDGEMENTSFUTURE WORK JUDGING
Medal Criteria
Gold:
•Help another iGEM team
•Characterize or improve an existing BioBrick Part or Device and enter this information back on the Registry.
•Completed a Human Practices project
HUMAN PRACTICES
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OVERVIEW RNA BIOREMEDIATION CONCLUSION
ACKNOWLEDGEMENTSFUTURE WORK JUDGING
HUMAN PRACTICES
Acknowledgements