Penn State iGEM 20112011.igem.org/files/presentation/Penn_State.pdfcI + RBS P RM O R3 O R2 O R1 P R...
Transcript of Penn State iGEM 20112011.igem.org/files/presentation/Penn_State.pdfcI + RBS P RM O R3 O R2 O R1 P R...
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Penn State
iGEM 2011 Bacterial Dosimeter
Presenters: Alex Bina, Jamie Colletta,
Elyse Merkel, Jim Rose, &
Lauren Rossi, Vishal Saini
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http://www.google.com/imgres?q=fukushima&um=1&hl=en&sa=N&rls=com.microsoft:en-us:IE-SearchBox&rlz=1I7DMUS&biw=1280&bih=819&tbm=isch&tbnid=a86-Nu7197jNtM:&imgrefurl=http://kasamaproject.org/2011/08/06/from-hiroshima-to-fukushima-remembrance-and-crisis/&docid=Sz3QKcQ-x-KZyM&w=535&h=338&ei=M4WPTtuqEqXl0QHN5LVR&zoom=1&iact=rc&dur=219&page=2&tbnh=136&tbnw=191&start=15&ndsp=20&ved=1t:429,r:4,s:15&tx=89&ty=60
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Project: “Bacterial Dosimeter”
Goal: Detecting Radiation through DNA
damage
Genetic Circuit:
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Project: “Bacterial Dosimeter ”
Activator: RecA
Sensor
Reporter
Additional Work
Dosimeter Device Design
NYC_wetware Collaboration
Human Practices
Conclusions
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Activator: RecA RecA is a protein found naturally in E. coli
Functions:
Repairs DNA damage through recombination
Cleaves Lambda phage cI repressor
Goal: To use RecA to activate our genetic system through its proteolytic activity.
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Activator: RecA
Challenges:
Common laboratory strains (DH10β) contain
inactivated RecA mutation: “RecA1”
Not compatible with standard Biobrick
assembly methods
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Activator: RecA
Our question:
How to prevent
recombination and loss of
cloned genes, while
maintaining proteolytic
activity of RecA?
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Activator: RecA
Solutions
Cyan = RecA activation
Green = Arg 243
Magenta = Lys 286
W = Wild type RecA
M = Mutated RecA
Red = BioBrick restriction sites
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Activator: RecA Test circuit was developed to determine if
the mutations were successful
Mutations successful: RecA cleaves the cI
repressor
Mutations fail: homologous double
terminators will recombine
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Sensor: λ Phage Switch
• Design based on the lambda phage lysogenic vs. lytic
switch
• Based on design by Penn State 2007 iGEM team
• Switch is activated in presence of DNA damage
Circuit under normal conditions:
cI cI cI cI
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cI + RBS PR PRM OR3 OR2 OR1 RBS + Cro RBS + TEV B0015
Sensor: λ Phage Switch
cI Cro TEV
cI cI cI
RecA with ssDNA
cI
cI
cI
cI
DNA Damage Due to Radiation
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Sensor: λ Phage Switch
Attached three different strength
ribosome binding sites to the cI repressor
gene to vary the translation rate
Higher RBS strength on cI repressor higher
threshold of radiation required to activate
the switch
cI RBS
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Reporter Project
Original Part:
BBa_K316007
Part utilizes reporter
enzyme immobilized by
fusion to GFP
Cloned and created a
catalog of the individual
fusion parts included in
the original form of the
part created by the
Imperial College London
2010 iGEM team
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Reporter
Reporter System
Characteristics:
1. Fast-acting
2. Color Pigment
Output
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E GFP Cleave Site Fusion Linker
Fusion Protein Variations
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E GFP Cleave Site Fusion Linker
E Cleave Site Fusion Linker GFP
E Fusion Linker Cleave Site GFP
Fusion Protein Variations
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Reporter Project
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Xyle Mutation
Original Xyle gene sequence contains two
NgoMIV sites and one AgeI site
Not compatible with Assembly 25 methods
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Xyle Mutation • Synonymous mutations were created at each site
Green = Xyle; Yellow = Assembly 25 Scar; Blue = Linker;
Red = Suffix restriction sites
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• Initial repression model of the operating region
promoters.
• Model was too simple, and more work needed to be
done.
Modeling
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• The new model takes into account
binding of Cro and RNAP to the Operating
Region.
• This graph shows the concentration of Cro
and CI proteins over time after the system
is activated.
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Or Data
We measured the Pr promoter on the operating region of
the sensor part by using RFP.
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Dosimeter Design
Design goal: portable, durable, and reliable
Reusability: camera and film pairing.
Hollow case made out of a hard plastic camera
Tray will be for one-time use film
After use the tray will be developed in catechol, etc. to show the different levels of radiation present.
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Collaboration with NYC-
Wetware Control
TS TR
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Human Practices
(Re) Designing Life is a video designed to
explain synthetic biology in an
understandable way for a general
audience
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Video: Audience Demographic: the Elderly Untapped audience
Active voters
Presentation and Survey
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Video: Survey Results
Agree = 8 (50%)
Neutral = 6 (38%)
Strongly Disagree = 2 (12%)
I Have a Better Understanding of Synthetic Biology as a Result of Watching this Video
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Improvements to the Video
More mature tone
Less abstract analogies explaining
synthetic biology techniques
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Parts Submitted
Parts Submitted to Registry BBa_K648000 mCherry with terminator
BBa_K648005 Short Fusion Protein Linker
BBa_K648006 Long 10AA Fusion Protein Linker with Standard 25 Prefix/Suffix
BBa_K648008 TEV protease cleaveage site with Standard 25 Prefix/Suffix
BBa_K648008 RecA Cleavage Site with Standard 25 Prefix/Suffix
BBa_K648011 Standard 25-Ready Xyle Reporter
BBa_K648013 GFP with Standard 25 Prefix/Suffix
BBa_K648028 Cro, Lamda Repressor which activates the lytic cycle
BBa_K648029 OR, operating region for Lambda switch
BBa_K648102 RecA (mutation in the amino acid Lys 286)
BBa_K648101 RecA
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Where we are now:
RecA :
First addition of RecA part to registry
Introduced all five mutations into one
plasmid
Assembled complete testing construct
Unable to test mutated RecA plasmid with
test construct due to time constraints
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Where we are now:
Sensor:
Characterized Pr promoter of the operating
region
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Where we are now:
Reporter:
Cloned each individual subunit.
Assembled intermediates.
Unable to characterize all expected
reporter systems due to time constraints
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Labwork Timeline:
Mutated Xyle
Successfully
Cloned
RecA
First Assembly
Cloned
final
reporter
subunit:
GusA
RecA
Test
Circuit
Completed
RecA
mutations Characterized
Or Part
Test RecA
Mutant
Combinations
Characterize
Complete
Reporter
Variations
Sensor
Testing:
Gamma
Facility
Jamboree
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We would like to thank our
advisors Dr. Tom Richard,
Dr. Howard Salis, and our
mentor Mike Speer.
Penn State 2011
Additionally we would like
to extend our gratitude to
all of our sponsors!