a synthetic biology approach to microbial fuel cell...
TRANSCRIPT
A Synthetic Biology Approach
to Microbial Fuel Cell
Development
Where’s Rolla?
Missouri S&T Energy Initiatives
iGEM Team at Missouri S&T
Small, but growingRaising awarenessStudent organization
Student design team
Presentations
iGEMers: Mining one gene at a time.
Geobacter sulfurreducens as model organism
Primary goal: optimize the electron shuffle
E. coli as a chassis to substitute for Geobacter Genome already contains key proteins in pathway
Lacks outer membrane cytochromes
Prelude
Fig. 2 – Modified from Macmillan Publishers Ltd., Derek Lovely: Bugjuice: Harvesting energy
from microorganisms. This shows the theoretically proposed pathway for electrons to the
extracellular space of Geobacter sulfurreducens. Note OmcB, OmcE, OmcS, OmcT
T
Dr. Derek Lovely: Research with Geobacter
e- transport without pilin gene
Dr. Alfred Spormann: Research with Shewanella
Cloned omcB-like gene into E. coli
Generated current
Prior Scientific Findings
Microbial Fuel Cell: polycarbonate casing, copper wire,
carbon felt (coated with E-TEK ELAT GDE), Nafion 117,
butyl rubber
Our Microbial Fuel Cell
Fig. 1 – Microbial Fuel
Cell constructed by
members of iGEM and
ChemE Car
http://www.hart-isee.com/uploads/images/Vehicles/How-a-fuel-cell-works.jpg
Fuel Cell Model
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1.2
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Vo
ltag
e
Time (Hrs)
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0 50 100 150 200 250P
ow
er
(mW
/m2)
Cu
rren
t (m
A/m
2)
Time (Hours) Current (mA/m2)
Power (mW/m2)
Courtesy of
Varun Paul,
Graduate
Student at
Missouri S&T
“haloalkaliphilic bacteria”
Fuel Cell Power Generation Model
omcB gene omcE gene
Geobacter
sulfurreducens
chromosome
omcT geneomcS gene
pPCR2.1 pPCR2.1 pPCR2.1 pPCR2.1
•PCR amplification
•EcoRI digest of pPCR2.1
pomcB plasmid•TOPO TA cloning
pomcT plasmidpomcS plasmidpomcE plasmid
Bba_J61100
plasmid•SpeI/PstI digest of Bba_J61100
plasmid
Bba_J61100
plasmid
Bba_J61100
plasmid
Bba_J61100
plasmid
omcB gene omcE gene omcS gene omcT gene
•XbaI/PstI digest of pomc
pomcB RBS plasmid pomcE RBS plasmid pomcS RBS plasmid pomcT RBS plasmid•Ligation
omcE RBS gene omcT RBS gene
•XbaI/PstI digest of E and T
•SpeI/PstI digest B and S
•Ligation
pomcBE RBS plasmid pomcST RBS plasmid
•XbaI/PstI digest of pomcST
omcST RBS gene
pomcBEST RBS plasmid
•SpeI/PstI digest pomcBE
•Ligation
Insertion of omc genes from G. sulfurreducens into E. coli
1
2
3
4
5
6
7
Parts Added to Registry
omcB: BBa_K269000
omcE: BBa_K269001
omcS: BBa_K269002
omcT: BBa_K269003
Cloned into pCR2.1/ All sequenced
Objective is to clone all 4 genes with Ribosome binding sites into one plasmid
All parts meet safety requirements
Geobacter is a biosafety level 1 organism
PCR cloning of omc gene (step 1)
Geobacter
sulfurreducens
chromosome
PCR Amplification
Xb
aI
Sp
eI
Eco
RI
Pst
I
omc gene
PCR
Product
+
pPCR2.1
Eco
RI
Eco
RI
AmpR
Xb
aI
Sp
eI
Eco
RI
Pst
I
omc gene
pomc plasmid
AmpR
Eco
RI
Eco
RI
Xb
aI
Sp
eI
Eco
RI
Pst
I
omc gene
pomc plasmid
AmpRX
baI
Sp
eI
Eco
RI
Pst
I
RBS
RBS part
AmpR
SpeI/PstI digest
XbaI/PstI digest
Bba_J61100
Xb
aI
Sp
eI
Eco
RI
Pst
I
RBS
RBS part
AmpR
Xb
aI
Sp
eI
Pst
I
omc gene
Eco
RI
Eco
RI
Bba_J61100
Adding RBS (step 2)
+
LigationX
baI
Sp
eI
Eco
RI
Pst
IomcB gene
pomc RBS plasmid
AmpR
RBS
Xb
aI
Sp
eI
Eco
R
I Pst
IRBS
RBS part
AmpR
Xb
aI
Sp
eI
Pst
I
omcB geneBba_J61100
Adding RBS (step 3 – repeated for each omc gene)
Xb
aI
Sp
eI
Eco
RI
Pst
IomcE gene
pomcE RBS plasmid
AmpR
RBS
Xb
aI
Sp
eI
Eco
RI
Pst
IomcB gene
pomcB RBS plasmid
AmpR
RBS
SpeI/PstI digest
XbaI/PstI digest
Xb
aI
Sp
eI
Eco
RI
Pst
IomcB gene
pomcE RBS plasmid
AmpR
RBSX
baI
Sp
eI
Pst
I
omcE geneRBS
Combining 2 genes (step 4)
+
Ligation
Xb
aI
Sp
eI
Eco
RI
Pst
IomcE gene
pomcBE RBS plasmid
AmpR
RBSomcB geneRBS
Xb
aI
Sp
eI
Eco
RI
Pst
IomcB gene
pomcE RBS plasmid
AmpR
RBS
Xb
aI
Sp
eI
Pst
I
omcE geneRBS
Combining 2 genes (step 4 continued)
Combining 4 genes (step 5)
Xb
aI
Sp
eI
Eco
RI
Pst
IomcE gene
pomcBE RBS plasmid
AmpR
RBSomcB geneRBS
Xb
aI
Sp
eI
Eco
RI
Pst
IomcT gene
pomcST RBS plasmid
AmpR
RBSomcS geneRBS
SpeI/PstI digest
XbaI/PstI digest
Xb
aI
Sp
eI
Pst
I
RBS omcS gene RBS omcT gene
Xb
aI
Sp
eI
Eco
RI
Pst
IomcE gene
pomcBE RBS plasmid
AmpR
RBSomcB geneRBS
+
Ligation
Xb
aI
Sp
eI
Eco
RI
Pst
I
pomcBEST RBS plasmid
AmpR
Xb
aI
Sp
eI
Eco
RI
Pst
IomcE gene
pomcBE RBS plasmid
AmpR
RBSomcB geneRBS
Xb
aI
Sp
eI
Pst
I
RBS omcS gene RBS omcT gene
omcE geneRBSomcB geneRBSRBS omcS gene RBS
omcT gene
Combining 4 genes (step 5 continued)
^(plasmid)
+
Ligation
Xb
aI
Sp
eI
Eco
RI
Pst
I
pomcBEST RBS plasmid
AmpR
Xb
aI
Sp
eI
Eco
RI
Pst
IomcE gene
pomcBE RBS plasmid
AmpR
RBSomcB geneRBS
Xb
aI
Sp
eI
Pst
I
RBS omcS gene RBS omcT gene
omcE geneRBSomcB geneRBSRBS omcS gene RBS
omcT gene
Combining 4 genes (step 5 continued)
^(plasmid)
Challenges
Anaerobic nature of Geobacter
Solubility of Nafion membrane
Host vector has an extra PstI site complicating removal of omc genes for ligation into RBS vector
Ligation/transformation inefficiency
Future Work
Testing the system and modeling
Determine redox potential for each cytochrome
Electron transport based on redox potential for each new cytochrome and existing electron transfer proteins in E. coli
Possible coupling of Shewanella
oneidensis or Geobacter
sulfurreducens with our new
E. coli strain to improve efficiency
Fig. 3 – S. oneidensis reducing metal. Image via
Asylum Research: M. El-Naggar, USC and Y. Gorby, J.
Craig Venter Institute
GAANN Fellows
Doctoral Research and
Training
in
Alternative Energy
Technologies
@
U.S. Citizen or U.S. Permanent Resident
Doctoral Student under the Program Faculty Advisorship
GPA of at least 3.5/4.0 [transcripts to be submitted]
GRE test scores in the 85th percentile
3 Letters of Recommendation
Research Field in Alternative Energy
Planning a Career in Teaching and Research
Eligibility Requirements [Minimum]
Financial
Free Application for Federal Student Aid (FAFSA) to be filled out
by the recipient. Fellowship stipend up to $30,000/yr with tuition and
fees fully paid for.
A limited number and highly competitive
GAANN Program Team in Alternative Energy
Program Director: Dr. Sunggyu “KB” Lee
Technical Co-Directors: Dr. John W. Sheffield
Dr. Fatih Dogan
Program Facilitator: Dr. David B. Henthorn
Program Faculty Co-PI: Dr. Daniel Forciniti
Dr. Scott E. Grasman
Dr. Kimberly Henthorn
Dr. Kakkattukuzhy M. Issac
Dr. Douglas K. Ludlow
Dr. Dave Westenberg
Dr. Yangchuan Xing
Program Secretary: Ms. Melody Mueller
Interdisciplinary Graduate Education Program in Alternative Energy (IGEAE)
For further information, please contact Dr. Dave Henthorn, [email protected].
The project members would like to thank Dr. Dave Westenberg and Dr. Katie Shannon from Missouri University of Science and Technology for their guidance and support as well as Mr. Fred Kielhorn for his generous gifts. We would also like to give thanks to the following as funding sources for our research and participation in the iGEM competition at Massachusetts Institute of Technology:
Missouri S&T Opportunities for Undergraduate Research Experience Program
Missouri S&T Department of Biological Sciences
Missouri S&T Department of Chemical and Biological Engineering
Missouri S&T Energy Research and Development Center
Missouri S&T Materials Research Center
Missouri S&T cDNA Resource Center
MidSci Scientific
Monsanto Corporation
Dr. Chang-Soo Kim
Dr. Susan Childers, Dr. Derek Lovely, Dr. B. Treu, & Ron Haas
Special Thanks
[1] Bug juice: harvesting electricity with microorganisms, Derek R. Lovely
[2] Possible Nonconductive Role of Geobacter sulfurreducens PilusNanowires in Biofilm Formation, Reguera et al.
[3] Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens, Holmes et al.
[4] International Genetically Engineered Machines Competition (iGEM).
http://parts.mit.edu/igem07/index.php/Main_Page
References
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