SUMMARY
Stanford-Brown 2012Advisors:
Lynn RothschildNASA Ames, Stanford University, Brown University
Joseph ShihStanford University Gary WesselBrown University
Debha Amatya
Bryce Bajar Gabriel Ben-Dor Julia Borden Benjamin Geilich Jason Hu Chris Jackson
SYNTHETIC ASTROBIOLOGYTHE TRANSIT OF
HUMAN PRACTICESMeeting with New York Times
science journalist Carl Zimmer
Tabling at the San Francisco and New York Maker Faires, helping kids do DNA extractions.
Organizing a Tranist of Venus viewing at Stanford University
Meeting other local iGEM teams at UCSF. Brainstorming, collaborating, and playing pick-up soccer.
Being featured in Wired Magazine’s blog, Wired Science.
Collaborating on the “Science in Action” exhibit with the California Academy of Sciences.
BIOMININGMicrobes can be used for mining and
recycling due to their sensitivity and
affinity for metal ions. We want to bring
these advantages up to space.
We’re creating a chimeric flagellar system
with metal-binding sites that allows the
easy extraction and collection of minerals.
Vishesh Jain Bella Okiddy Rashmi Sharma Aaditya Shidham Kendrick Wang Michelle Yu
THE GENE CONSTRUCT
N-TERMINUS
FLAGELLA
In our biomining project, we ran
into hurdles surrounding patent
law of genes and pathways.
This served as a launchboard
for our human practices project:
a guide to biopatents for future
iGEM teams, and a review of the
ethical discussions surrounding the
patenting of genetic information.
OUTREACH
SOURCES
ACKNOWLEDGEMENTSThe team would like to thank Jim Head and his team, DNA 2.0, Geneious, the California Academy of Sciences, the Michael Z. Lin lab, Pete Worden,
Carl Zimmer, Linda Kahl, Gary Lee, Kevin Jackson, and David Grinspoon. We’d also like to thank the Rhode Island Space Grant, the Stanford Vice
Provost for Undergraduate Education, Stanford Bioengineering REU (Research Experience for Undergraduates), and Brown UTRA (Undergraduate
Teaching and Research Award) for their sponsorships We would also like to thank the many members of the Rothschild lab who gave much needed
advice and guidance over the summer: Andre Burnier, Jesse Palmer, Kosuke Fujishima, Chris Venter, Jesica Navarete, Diana Gentry, Mike Grace, Ivan
Paulino Lima, and Rocco Mancinelli.
FLIC codes for flagellin, a monomer that assembles into flagella.
We can mount a metal-binding site
Molly A. Bergman,Lisa A. Cummings,Robert C. Alaniz,Laura Mayeda,Ivana Fellnerova,Brad T. Cookson. CD4+-T-Cell Responses Generated during Murine Salmonella enterica Serovar Typhimurium Infection Are Directed towards Multiple Epitopes within the Natural Antigen FliC. 2005 Infect Immun. 73(11): 7226–7235E.Coli FliC gene: http://biocyc.org/ECOLI/sequence-rc?type=GENE&object=EG10321Kouichi Kuroda and Mitsuyoshi Ueda. Molecular design of the microbial cell surface toward the recovery of metal ions. Current Opinion in Biotechnology 2011, 22:427–433Benita Westerlund-Wikstrom, Jarna Tanskanen, Ritva Virkola, Jorg Hacker, Martin Lindberg, Mikael Skurnik and Timo K.Korhonen. Functional expression of adhesive peptides on flagellin (Section - Purification of Chimeric Flagella) Protein Engineering vol.10 no.11 pp.1319-1326, 1997Chiaramello A.E., Zyskind, J.W.: Coupling of DNA replication to growth rate in Escherichia coli: A possible role for guanosine tetraphosphate. J. Bacteriology 1990, 172:2013-2019.Quiñones A., Wandt G., Kleinstauber S., Messer W.: DnaA protein stimulates polA gene expression in Escherichia coli. Molecular Microbiology 1997, 23: 1193-1202.Sun L., Jacobson B., Dien B., Srienc F., Fuchs J.: Cell Cycle Regulation of the Escherichia coli nrd Operon: Requirement for a cis-Acting Upstream AT-Rich Sequence. J. Bacteriology 1994, 176: 2415-2426.Sun L., Fuchs J.: Escherichia coli Ribonucleotide Reductase Expression is Cell Cycle Regulated. Molecular Biology of the Cell 1992, 3:1095-1105.Messer W.: The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication 2002, 26:355-374.Ferullo D.J., Cooper D.L., Moore H.R., Lovett S.T.: Cell cycle synchronization of E. coli using the stringent response, with fluorescence labeling assays for DNA content and replication. Methods 2009, 48:8-13.Grinspoon, David Harry. Venus Revealed: A New Look below the Clouds of Our Mysterious Twin Planet. Reading, MA: Addison-Wesley Pub., 1997.Chattopadhyay, M. K. (2006). Mechanism of bacterial adaptation to low temperature. J. Biosci., 31 (1), 157-165.Riley, M., Staley, J. T., Danchin, A., Wang, T. Z., Brettin, T. S., Hauser, L. J., Land, M. L., Thompson, L. S. (2008). Genomics of an extreme psychrophile, Psychromonas ingrahamii. BMC Genomics, 9(210), 1-19.Calahan, D., Dunham, M., DeSevo, C., Koshland, D.E. (2011). Genetic analysis of desiccation tolerance in Saccharomyces cerevisiae. Genetics, 189: 507-519.Bonaterra, A., Camps, J., Montesinos, E. (2005). Osmotically induced trehalose and glycine betaine accumulation improves tolerance to desiccation, survival and efficacy of the postharvest biocontrol agent Pantoea agglomerans EPS125. FEMS Microbiol. Lett., 250: 1-8.Cayley, S., Lewis, B. A., Record Jr., T. (1992). Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12. J. Bacteriol. 174(5): 1586-1595.
C-TERMINUS
MULTIPLE CLONING SITE
Multiple cloning sites allow us to design and insert sequences.
We’ve succefully inserted the copper binding sites HypB1, HypB2, and HTTC.
First we cut out the sequence in the native Flic for the disposable
region.
EXCISED DNA
FLIC GENE
on flagella by inserting it into the flagellin sequence.
• IntroducedSYNTHETIC BIOLOGYasaTOOLforASTROBIOLOGY
• IsolatedpartsthatIMPROVE RESISTANCEtoBASIC CONDITIONSandDESICCATIONIne.colI
•developedtwoNOVELandEFFECTIVECELL-CYCLE DEPENDENT PROMOTERSforuseasREMOTE BIOSENSORS
HELL CELLSpace is filled with extremes, and
we’re here to prepare prospective
microbial astronauts for their hellish
commute.
To custom engineer extremophiles
for space exploration, we took cues
from natural adaptations against the
elements.
This not only broadens the scope of
applications of synthetic biology, but
also tests the limits of life, terrestrial
and extraterrestrial.
MODEL MECHANISM GENES
RA
DIA
TIO
N
Deinococcus radiodurans
Hymenobacter
Manganese transport
Superoxide dismutases
DNA repair mechanisms
Sod Cu/ZnSod MnDpsGDpsMPMntHRecA
CO
LDPsychromonas ingrahamii
Glycine betaine pathway
betA/B
DES
ICCA
TIO
N Saccharomyces cervisiae
Trehalose biosynthesis pathway
Glycine betaine pathway
betA/BotsA/B
BA
SICIT
Y Escherichia coli Buffers sdaB
RESULTS
PEr
cEn
t Su
rv
ivA
L
GRAPH 3: Percent
survival of transformed
bacteria after
varying exposure to
UV light
OD
60
0
timE (SEcOnDS)
GRAPH 4: Optical density of transformed bacteria in pH
9.5 solution over time
RADIATION
BASICITY
GRAPH 5: Percent
survival of transformed
bacteria under
dessicative conditions
DESICCATION
PEr
cEn
t Su
rv
ivA
L
timE (SEcOnDS)
Transformed bacteria survived comparably or better than negative control. The best performing biobrick, dps MP, binds to DNA to prevent radiation damage.
All transformed bacteria performed better than negative control. recA is involved in DNA repair, and sdaB produces biological buffers.
betAB and otsAB, which produce osmoprotectants, performed two orders of magnitude better than negative control.
1.00E-08
1.00E-06
1.00E-04
1.00E-02
1.00E+00
Negative Control
mntH
bet
ots
1.00E-05
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E+00
0 5 10 15 20 25 30 35 40
Control
mntH
recA
sod Cu/Zn
dps MP
0
0.4
0.8
1.2
1.6
0 4 8 12 16 20 24
recA negative control sdaB dps
timE uv ExPOSurE (SEcOnDS)
VENUS LIFE
We’re studying bioaerosols by
coupling cell cycle promoters and
fluorescent proteins to monitor
aerosolized bacteria.
This will provide insight into the
feasibility of life in Venusian clouds,
as proposed by Carl Sagan in 1967.
These constructs can also provide
possible remote sensors here on
Earth.
POLAP nrdPplasmid
POLAP DnAPinrDP OPErOn FOr ribOnucLEOtiDE rEDuctASE
0
1.0
2.0
3.0
4.0
5.0
6.0
6040200
GRAPH 1: nrdP-promoted fluorescence suggests cell cycle dependence
FLu
Or
ESc
Enc
E/c
ELL
DEn
Sity
(x1
05)
timE (minutES)OD and fluorescence were taken from a cell-
synchronized culture. The peaks show that
fluorescence increases with fission, and thus
the nrd promoter is cell cycle dependent.
GRAPH 2: polAP-promoted fluorescence suggests DNA
replication dependence
2.0
2.5
3.0
3.5
6050403020100
FLu
Or
ESc
Enc
E/c
ELL
DEn
Sity
(x1
05)
timE (minutES)OD and fluorescence were taken from a cell-
synchronized culture. The peaks are more
periodic than the nrd-promoted fluorescence.
We suspect these peaks to correlate with DNA
replication.
KEY:trial 1trial 2average
KEY:trial 1trial 2average
MICROSCOPY CONFIRMS PROMOTER ACTIVITY FOR NRDP AND POLAP
ISOLATE PROMOTERS WITH PCR
PrOmOtEr rEGiOnS OF intErESt:
• ImprovedpartBBa _ K133038fromslovenIa2008BySTANDARDIZING LIGATION IntoflagellaandENGINEEREDthee.colIFLAGELLUMtoEXTRACT METALSInsItu
• MODELEDBacterIalgrowthIntheVENUSIAN ATMOSPHERE
•wroteGUIDEStoBIOETHICSandGENE PATENT LAWforsmoothernavIgatIonofthemoralandlegalaspectsofsynthetIcBIology
(iGEM) Maker Faire Poster Julia.indd 1 10/12/12 3:59 PM
