NASA Ames, Stanford University, Brown University ...2012.igem.org/files/poster/Stanford-Brown.pdf · Microbes can be used for mining and recycling due to their sensitivity and affinity

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