C RISPR -C AS : O UR M ETHODS Circular ¡Polymerase ¡Extension ¡ Modified ¡QuikChange™ ¡ Cloning ¡(Quan ¡ et ¡al. , ¡2011) mutagenesis ¡(Liu ¡ et. ¡al , ¡2008) – Liga(on ¡and ¡restric(on-‑free – Liga(on ¡and ¡phosphoryla(on-‑free – Inexpensive ¡(~$2 ¡in ¡enzymes ¡per ¡ – High ¡PCR ¡efficiency ¡for ¡easy ¡ reac(on) ¡ transforma(on Liu, ¡H., ¡& ¡Naismith, ¡J. ¡H. ¡(2008). ¡An ¡efficient ¡one-‑step ¡site-‑directed ¡dele;on, ¡inser;on, ¡single ¡and ¡mul;ple-‑site ¡plasmid ¡ mutagenesis ¡protocol. ¡ BMC ¡biotechnology , ¡ 8 (1), ¡91. Quan, ¡J., ¡& ¡Tian, ¡J. ¡(2011). ¡Circular ¡polymerase ¡extension ¡cloning ¡for ¡high-‑throughput ¡cloning ¡of ¡complex ¡and ¡combinatorial ¡ DNA ¡libraries. ¡ Nature ¡protocols , ¡ 6 (2), ¡242-‑251.
C RISPR -C AS : O UR M ETHODS - CPEC •PCR-addition of overlapping regions (bb pref/suff) to insert •PCR-linearization of vector •Insert and vector extend (via Polymerase action, using each other as template Circular plasmid contains the integrated insert, including our standard prefix and suffix Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. doi:10.1371/journal.pone.0006441
S YNTHETIC B IO -C OMMUNICATION B IO W IRES 1. A TOMIC C RISPR -C AS 2. I NTERCELLULAR 3. T IME D E -E XTINCTION 4. S PACE
D E -E XTINCTION : T HE R OAD M AP Origin of Life • Building Blocks • Amino Acid Evolution Proof of Concept • Long Term Evolution • Bioinformatic Confirmation Application • CasA Recognition • Specific Mechanism
D E -E XTINCTION : P ROCESS • Protein Sequence Data • PFAM Database • Phylogenetic Tree • Geneious + PHYML • Substitution Model • ProtTest • Sequence Reconstruction • PAML + Lazarus
O RIGIN OF L IFE : A MINO A CIDS Background • HisC codes for histidine • CysE codes for cysteine Goals Histidine • Understand their evolution Cysteine • Chicken and egg question Process • Predict ancestral genes • Synthesize • Test basic function
O RIGIN OF L IFE : C HICKEN OR E GG ? Circles indicate protein interaction sites Green • Single Conserved His/Cys Site indicates • Molecular Clock > 120,000 conserved Years regions • Total Distance in Billions
O RIGIN OF L IFE : C YS E R ESULTS 0.23 0.18 0.18 OD OD 0.13 0.13 0.08 0.08 0 10000 20000 30000 40000 50000 60000 70000 0 10000 20000 30000 40000 50000 60000 70000 Time (secs) Time (secs) Vmax Points = 21 Vmax Points = 21 Well H1 H2 H3 H4 H5 H6 Well E1 E2 E3 E4 E5 E6 Vmax 0.085 0.078 0.059 0.067 0.049 0.073 Vmax 0.159 0.104 0.085 0.078 0.082 0.090 R^2 0.770 0.850 0.905 0.883 0.891 0.906 R^2 0.557 0.769 0.727 0.629 0.659 0.776 Positive Control ( Trento 2012) Ancestral Gene Negative Control Positive Control Ancestral Test
O RIGIN OF L IFE : H IS C R ESULTS 0.28 0.23 0.23 0.18 OD OD 0.18 0.13 0.13 0.08 0.08 0 10000 20000 30000 40000 50000 60000 70000 0 10000 20000 30000 40000 50000 60000 70000 Time (secs) Time (secs) Vmax Points = 21 Vmax Points = 21 Well F1 F2 F3 F4 F5 F6 Well C1 C2 C3 C4 C5 C6 Vmax 0.142 0.164 0.107 0.105 0.115 0.117 Vmax 0.138 0.117 0.102 0.132 0.107 0.130 R^2 0.762 0.949 0.965 0.744 0.933 0.937 R^2 0.811 0.862 0.828 0.847 0.871 0.904 Positive Control Ancestral Gene Negative Control Ancestral Test
P ROOF OF C ONCEPT : L ENSKI E XPERIMENT The Experiment • E coli Evolution • Started in 1988 • 50,000 generations Our Goals • Ancestral Reconstruction • Whole Genome • Proof of Concept • Modeling Evolution
P ROOF OF C ONCEPT : L ENSKI E XPERIMENT Results • Lazarus ~90% • Consensus ~99% Implications • Timeline too short? • Problems with algorithm? • Not enough strains?
A PPLICATION : C RISPR Target: CasA • CASCADE Complex • Mechanism • Protein Modeling Testing • Test functionality - purified proteins • Compare to modern sequences
D E -E XTINCTION : B IO B RICKS 7 BioBricks: Ancestral HisC Modern HisC 3 characterized AroE Ancestral CysE 4 currently being tested Ancestral CasA Modern CasA CasBCDE
F UTURE A IMS Proof of Concept Application • Alternative Programs • Biochemical Assay • Functional Assay
D E -E XTINCTION : E THICS
D E -E XTINCTION : A CCOMPLISHMENTS ✓ Predicted Ancestral Genes ✓ Successfully Tested Ancestral Gene Function ✓ Modeled Ancestral Proteins ✓ Collaborated with Dr. Rich Lenski to Model and Tested Reconstruction Methods ✓ Submitted 7 BioBricks!
S YNTHETIC B IO -C OMMUNICATION B IO W IRES 1. A TOMIC C RISPR -C AS 2. I NTERCELLULAR 3. T IME D E -E XTINCTION E U :CROPIS 4. S PACE
E U :CROPIS Euglena Combined Regenerative Organic Food Production A German satellite In mission scheduled to Space launch in 2016
P OWER C ELL Goal Develop a universal, sustainable energy source to power biological tools Application Feed the biological tools that will transform raw materials into fuel, food, drugs, and other products useful to settlers
E U :CROPIS Brown-Stanford iGEM team pioneers PowerCell for in Launch! We create a situ resource Activate first chromogenic utilization on Mars gravity level biosensor for and ground PowerCell in B. controls. subtilis 2011 2016 2013
EuCROPIS Microfluidic Chip
Anabaena Bacillus subtilis (Brown-Stanford 2011) (Stanford-Brown 2013)
P ROGRESS We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A) Used oligos to construct gene promoters Assembled plasmid containing: sucrose metabolism promoter, a ribosome binding site, and a red chromoprotein Synthesized the equivalent construct for sporulation Transformed into B. subtilis using an integration vector
3A Assembly of Promoter and RBS Ribosome binding sacY promoter destination plasmid: pSB1K3 site S X S X S X P E E P P E Amp Kan Chlor Cut with Cut with Cut with E and P E and S X and P S P S X E X E P Kan
3A Assembly of Promoter and RBS Ribosome binding sacY promoter destination plasmid: pSB1K3 site S X S X S X P E E P P E Amp Kan Chlor Cut with Cut with Cut with E and P E and S X and P S P S X E X E P Kan Ligate Ligate M X E S S Grow on Amp E P Kan plate Kan X P Chlor
3A Assembly of Promoter and RBS Ribosome binding sacY promoter destination plasmid: pSB1K3 site S X S X S X P E E P P E Amp Kan Chlor Cut with Cut with Cut with E and P E and S X and P S P S X E X E P Kan Ligate Ligate M X E S S Grow on Amp E P Kan plate Kan X P Chlor Cut with E and P E X S P
Cloning Strategy Schematic chromoprotein promoter and RBS destination plasmid S P E X X S X S P P E E Cut with Cut with Cut with E and S X and P E and P S X S P E X P E Ligate Ligate M X S E P
Cloning Strategy Schematic chromoprotein promoter and RBS destination plasmid S P E X X S X S P P E E Cut with Cut with Cut with E and S X and P E and P S X S P E X P E Ligate Ligate M X S E P Cut with E and P S P E X PCR E X BamHI S P HindIII
Cloning Strategy Schematic chromoprotein promoter and RBS destination plasmid S P E X X S X S P P E E Cut with Cut with Cut with E and S X and P E and P S X S P E X P E Ligate Ligate M X S E P Cut with E and P S P E X PCR integration vector E X BamHI S P amyE’ HindIII BamHI HindIII ‘amyE Cut with BamHI and HindIII and ligate BamHI amyE’ amyE’ BamHI Homologous Bacillus recombination genome HindIII HindIII E E y y m m a a ‘ ‘
R ESULTS Bacillus with sucrose metabolism promoter + RFP (Phase Contrast)
R ESULTS Bacillus with sucrose metabolism promoter + RFP (Fluorescence)
R ESULTS B. subtilis with sucrose metabolism Bacillus with sucrose metabolism promoter + fluorescent promoter + RFP chromoprotein eForRed (Fluorescence) (DIC overlaid with fluorescence)
F UTURE A IMS Fall 2013: Isolate colonies containing our construct and characterize promoters Winter 2013: Helium balloon test 2014: Biocompatibility tests with microfluics chip 2015: Prepare for mission 2016: EuCROPIS Satellite Mission
E U :CROPIS - A CCOMPLISHMENTS Constructed and synthesized prototype chromogenic biosensors to detect sucrose induction and sporulation 5 BioBricks: SacY promoter Contributed on project to validate PowerCell SacY + RBS Spo0A promoter on the EuCROPIS satellite mission and send synthetic biology SacY + RBS + eforRed SacY + RBS + RFP into outer space
O UTREACH
C ALIFORNIA A CADEMY OF S CIENCES
C ALIFORNIA A CADEMY OF S CIENCES
C ALIFORNIA A CADEMY OF S CIENCES
B IO E NGINEERING B OOTCAMP
B AY A REA /NYC M AKER F AIRES
C OLLABORATED WITH O THER T EAMS
S OCIAL M EDIA P RESENCE
I GEM M EMES !
H UMAN P RACTICES An economic analysis on improving the productivity of and lowering the costs for iGEM teams by employing DNA synthesis
B IO W IRES : 2 B RICKS SUMMARY C RISPR -C AS : 3 B RICKS D E -E XTINCTION : 7 B RICKS E U CROPIS: 5 B RICKS P RESENTED TO C ENTER I GEM M EMES D IRECTOR , NASA A MES B IO E B OOTCAMP T WO M AKERS F AIRES (SF C ALIFORNIA A CADEMY AND NY) OF S CIENCES B AY A REA AND N EW E CONOMIC A NALYSIS E NGLAND M EETUPS OF I GEM W ORKFLOW D E -E XTINCTION E THICS
A CKNOWLEDGMENTS Dr. Jimmy Xu (Brown): Lab space and advice on conductivity testing, Dr. Meyya Meyyapan (NASA): Advice for nanoscale analysis, Yifan Zhang (Brown): Performing mass spectrometry tests, Kyeong Kyu Kim (SKKU): Performing crystallization, Mark Capece (Stanford): Performing NMR, Dr. Corey Liu (Stanford): NMR support, Dr. Vittor Pinhiero (MRC): Project advice, Dr. Akira Ono (Kanagawa University): Project advice, Dr. Vesna Mitrovic (Brown): NMR support, Dr. David Bikard for Cas9 genomic clones, Coli Genomic Stock Center for bacterial strains, Dr. Judith Bender, Brown University for E. coli strains and plasmids, Dr. Michael Bagdasarian, Michigan State University for RP4 E. coli strains., Dr. David Relman, Stanford VA Hospital for pertussis gDNA, Dr. Rich Lenski (and lab), Dr. Nathan Wolfe, Dr. Joe Thornton, Daniel R. Zeigler, Ph.D., BGSC Director and the Bacillus Genetic Stock Center for Bacillus integration vectors, Lilah Rahn-Lee for advice on choosing B. subtilis genes, Elwood Agasid for explaining the EuCROPIS mission from an engineering perspective, Dr. Orr Yarkoni, BioE Bootcamp, Brown University, UTRA and Science Center, DNA 2.0, Biomatters Ltd. for free licences to its Geneious software, Jane Berry at EMD Millipore, NASA Ames Research Center, Rhode Island Space Grant Consortium, Stanford University VPUE and Provost, T eri Hanks, UC Davis 2013 iGEM team, Jason Hu, Dr. Rocco Mancinelli, Dr. Michael Lin, Pete Worden, Director NASA Ames, Jesica Navarrete, Dr. Ivan Lima, Joseph Michael “Mike” Grace, Diana Gentry, Cyprian Verseux, Kendrick Wang, Dr. Kosuke Fujishima, Ryan Kent, Simon Wong and Dr. Robert Siegel for the photos of The Stanford Dish A special thank you to our faculty advisors, Dr. Lynn Rothschild, Dr. Joseph Shih, and Dr. Gary Wessel, for all their help and support this summer. We wish you could be here with us today!
Stanford-Brown 2013 Supplemental Material
BioWires
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