sRNAs in Artificial Gene Circuits and Bioremediation Applications
Lee F. 3,6, Hsiao T. 2,6, Borchardt E. 2,6, Musselman M. 1,6, Chang A. 2,6, Cooper R. 2,6, Waltersdorf S. 2,6, Entler M. 1,6
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Manaster J. , Evans C. , Jin Y.S. , Rao C.V.
Department of Bioengineering1, School of Molecular and Cellular Biology2, Department of Biochemistry3, Chemical and Biomolecular Engineering4, Food Science Human Nutrition5, University of Illinois at Urbana-Champaign6
Targeted inhibition and activation through sRNA Biobricks
We are the UIUC-Illinois iGEM Bioware team. Our team consists
of eight undergraduates in Molecular and Cellular Biology,
Bioengineering and Biochemistry at the University of Illinois at
Urbana-Champaign. This is our third year participating in the
iGEM competition, and we are excited to present the research we
have worked on full-time throughout this past year.
Goal
The Illinois iGEM team has worked to create a set of endogenous
and artificial sRNA regulator Biobricks to be used in cellular
stress responses and which contribute to bistability in artificial
gene circuits. A bacterial decoder and a bacterial metal detection
system of arsenic and gold demonstrates the capacity of sRNA
regulation in artificial gene circuits.
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ompA
2.) Public Outreach:
With our survey results in mind, we prepared several
presentations that we used to introduce synthetic biology to our
community.
gadX
TARGET SEQUENCE::GFP FUSION
Implementation of logic gate through sRNA Biobricks
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Input Input
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IPTG Atc
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FLUORESCENCE
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Metal Remediation System
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0 mM [IPTG], 1 mM [IPTG], 0 mM [IPTG], 1 mM [IPTG],
0 mM [atc]
0 mM [atc]
1 mM [atc]
1 mM [atc]
This cellular system allows cells to withstand high concentrations of
arsenic and gold while allowing the cells to bind the metals on their
surface. sRNAs regulate the expression of efflux pumps.
STATEMENT
A AND NOT B
atc AND NOT IPTG
A = Input
D = Output
Biobricks
PgolS Promoter
GolS Sensor
Comparison between riboregulators and sRNAs
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Target Gene Expression
1-Hour after Regulatory
RNA Induction
0 mM [IPTG]
hfq+
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1mM [IPTG]
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Riboregulator
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sRNA
www.PosterPresentations.com
1.) Survey:
Background: 11 questions designed to gage our audience’s awareness
and preconceived notions about synthetic biology.
Results: What are the first three words that come to mind when
you hear the words “synthetic biology?” Confusing, Complicated,
Bacteria, Genetics.
What kind of science and/or synthetic biology do you
see in pop culture? GATTACA, Jurassic Park, iRobot
All Biobricks are RFC10 compatible
SRNA
D4
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III. The bioremediation portion of the project is designed to
remove ionic arsenic and gold from solution. Arsenic was
chosen due to its detrimental health effects, and gold was
chosen due to its market value and potential in nanoparticle
fabrication.
TEMPLATE DESIGN © 2008
Biobricks
sRNA Target
MicA OmpA
MicF OmpF
GadY GadX
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FLUORESCENCE
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1mM [IPTG] hfq+
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2-4 Binary Decoder Truth Table
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Human Practices Project
Goal:
To better connect with our community and to spread the word about
synthetic biology. This was to both correct any misconceptions that
might be out there as well as to reinforce accurate ideas.
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Previously, the majority of engineered, genetic regulation
within bacteria has been achieved through the use of
transcriptional regulators. However, the recent explosion of
interest from the emerging field of RNA regulation provides
new insights into the dynamic nature of genetic regulation.
Small non-coding RNAs (sRNAs) comprise the chief
regulatory mechanism for eliciting primary responses to
environmental stresses. Acting in conjunction with proteins
such as hfq (RNA chaperone), sRNAs provide a costeffective, specific and rapid response that is essential in
targeting gene transcripts for regulation.
II. A decoder is a logic construct that allows for the deprocessing of coded information. According to permutations
of the possible combination of binary inputs, the decoder
activates a specific and unique location. The intended inputs
of our 2-4 binary decoder are the presence or absence of
either IPTG (gratuitous inducer of the Lac operon) and atc
(gratuitous inducer of the Tet operon).
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I.
A2
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0 mM [IPTG] hfq+
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Background
A1
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Performance of Bacterial Decoder based on sRNA Biobricks
180000
FLUORESCENCE
Illinois iGEM
Riboregulator
Key 1: BBa_J01008
Lock 1: Bba_J01010
GolB Binder
Ges Efflux
ParsR Promoter
ArsR* Sensor
ArsB* Efflux
*Submission in Progress
Gold
Arsenic
- VS -
After cells have collected the metal, they can be washed to purify their
specified metal and then recycled for a subsequent collection.
Events:
 Engineering Open House
 Biology Open House
 Science Olympiad National Tournament
 G.A.M.E.S. Camp
 Quad Day
 Surrounded By Science TV Program
Conclusion
Summary of Accomplishments:
 Designed and constructed 31 Biobricks for the Bacterial
Decoder and Au-As Bioremediation systems
 Constructed a Bacterial Decoder that uses sRNA logic
gates to regulate the expression of four fluorescent
proteins
 Engaged the immediate community in a practical and
extensive human practices project
Future Work
 Continue development of Bacterial Decoder 2.0, a
completely sRNA regulated gene circuit
 Construct Au-As Bioremediation system
 Keep informing the public about Synthetic Biology and
making advances in Human Practices
Acknowledgements