Synthetic Bio- Communication 1 S YNTHETIC B IO -C OMMUNICATION 1. A - - PowerPoint PPT Presentation

Synthetic Bio- Communication

Stanford-Brown 2013

1

SYNTHETIC BIO-COMMUNICATION

• 1. ATOMIC
• 3. TIME
• 2. INTERCELLULAR
• 4. SPACE

• 1. ATOMIC

BIOWIRES

SYNTHETIC BIO-COMMUNICATION

• 2. INTERCELLULAR
• 3. TIME
• 4. SPACE

• 1. ATOMIC
• 2. INTERCELLULAR

BIOWIRES CRISPR-CAS

SYNTHETIC BIO-COMMUNICATION

• 3. TIME
• 4. SPACE

• 3. TIME

CRISPR-CAS

DE-EXTINCTION

SYNTHETIC BIO-COMMUNICATION

• 2. INTERCELLULAR
• 4. SPACE

BIOWIRES 1. ATOMIC

• 3. TIME

CRISPR-CAS

DE-EXTINCTION

SYNTHETIC BIO-COMMUNICATION

• 2. INTERCELLULAR
• 1. ATOMIC
• 4. SPACE

EU:CROPIS BIOWIRES

SYNTHETIC BIO-COMMUNICATION

• 4. SPACE

EU:CROPIS

DE-EXTINCTION 3. TIME

CRISPR-CAS 2. INTERCELLULAR BIOWIRES 1. ATOMIC

Every year, technology companies spend billions on R&D Much of this research depends

• n advancements in nanoscale

manufacturing

BIOWIRES

Wires too Large! Fabrication Expensive Infinite Resistance

• Miniaturization necessary
• Increasing #transistors per

chip

• Thinner wire is better!
• Lithography machines highly

expensive

• Time-intensive

e-­‑ e-­‑

$ $ $

PROBLEMS WITH CURRENT TECHNOLOGY

BIOWIRES

Access Inhibition

• Access costs and

techniques make nanotechnology inaccessible to most

• This science is not shared

Wires too Large! Fabrication Expensive Infinite Resistance

• Miniaturization necessary
• Increasing #transistors per

chip

• Thinner wire is better!
• Lithography machines highly

expensive

• Time-intensive

e-­‑ e-­‑

$ $ $

PROBLEMS WITH CURRENT TECHNOLOGY

BIOWIRES

Access Inhibition

• Access costs and

techniques make nanotechnology inaccessible to most

• This science is not shared

W e ’ v e S

• l

v e d A l l T h r e e !

Wires too Large! Fabrication Expensive Access Inhibition

• 1-atom thick silver wire in

DNA coat

• DNA can self assemble, self-

replicate and self-catalyze

• DNA platform makes

nanodevice design available to all investigators

Smallest Ever Self-Assembling SynBio: DNA cheap and reliable

BIOWIRES:OUR SOLUTIONS

Canonical DNA A C G T T A

• Governed by H-bonding
• Purine(G)+ pyrimidine(C)
• Not conductive

Non-bonding CC Mismatch A C C T T A

• C-C mismatch: two

pyrimidines

• Space not filled
• No bond formed

Ag+ binding CC Mismatch A C C T T A

• Silver ion (Ag+) fills gap
• Binds to N3 sites on C
• Our hypothesis:

enhanced conductivity

BIOWIRES

MECHANISM

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Future: DNA Origami

• 32bp10CC (10 ions)
• DNA duplex
• Fully characterized
• 50bp24CC (24 ions)
• DNA and RNA hairpin
• Coils: EM wave

generation

• Sheets: microchip

template

• Bundles: signal

transduction BBa_K1218026: “Star”

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BBa_K1218022: Hairpin

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BRICKS AND DEVICES

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

IS SILVER BOUND?

Evidence of Silver Binding:

Thermal Denaturation: Absorbance Change Bba_K1218026 cannot anneal without silver; Melting shows silver is present

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

IS SILVER BOUND?

Thermal Melts PAGE FRET Phen Green

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

IS SILVER BOUND?

Thermal Melts PAGE FRET Phen Green

WHAT IS THE MOLAR RATIO (AG:CC)?

Evidence of 1:1 Ag:CC Molarity:

Gel Electrophoresis: PAGE analysis Molar Ratio Increase 0:1 1:10 3:10 1:2 1:1 3:2 2:1 3:1 4:1

Molar Ratio Increase Duplex formation 0:1 1:10 3:10 1:2 1:1 3:2 2:1 3:1 4:1 Bba_K1218026+cannot+anneal+without+silver+ As 1:1 molar equivalence is reached, a duplex is formed

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

IS SILVER BOUND?

Thermal Melts PAGE FRET Phen Green

WHAT IS THE MOLAR RATIO (AG:CC)?

PAGE FRET ESI-MS (Brown)

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

IS SILVER BOUND?

Thermal Melts PAGE FRET Phen Green

WHAT IS THE MOLAR RATIO (AG:CC)?

PAGE FRET ESI-MS (Brown)

WHAT IS THE STRUCTURE?

Structural Analysis

Ion Distribution: TEM + Negative Staining Molar ¡Ra/o ¡Increase

Dark areas = low electron density DNA Light areas = high electron density regions like Ag+

BBa_K1218026: Silver ion pattern 1&2 bp apart– TEM confirms structure

Structural Analysis

Ion Distribution: TEM + Negative Staining

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

IS SILVER BOUND?

Thermal Melts PAGE FRET Phen Green

WHAT IS THE MOLAR RATIO (AG:CC)?

PAGE FRET ESI-MS (Brown)

WHAT IS THE STRUCTURE?

TEM COSY

• H NMR (Stanford)

X-ray Crystallography (SKKU collaboration)

Structural Analysis

Bond Site: COSY-H NMR Molar ¡Ra/o ¡Increase

Overlay(shows(shi.(in(H52H6(cross(peak(with(Ag+,(indica=ng(N3(bond(site( Shi.(when(silver(added(

BIOWIRES

SO WHAT CAN WE (AND OTHER TEAMS) BUILD?

Bba_K1218026: Duplex Bba_K1218022: DNA/RNA DNA Origami

IS SILVER BOUND?

Thermal Melts PAGE FRET Phen Green

WHAT IS THE MOLAR RATIO (AG:CC)?

PAGE FRET ESI-MS (Brown)

WHAT IS THE STRUCTURE?

TEM COSY

• H NMR (Stanford)

X-ray Crystallography (SKKU collaboration)

IS IT CONDUCTIVE?

Spin-coupling NMR (Brown) DC Chip Analysis

BIOWIRES: ACCOMPLISHMENTS

✓ Showed uptake of silver by DNA via thermal

denaturation

✓ Demonstrated near 1:1 molar equivalence

between mismatches and silver ions via PAGE

✓ Visually confirmed paired ion distribution via

TEM

✓ Confirmed bond site using COSY-H NMR

BBa_K1218022 BBa_K1218026

We are the first team to develop a synbio platform for quasi-1D nanowire assembly from DNA!

• 1. ATOMIC
• 2. INTERCELLULAR

BIOWIRES CRISPR-CAS

SYNTHETIC BIO-COMMUNICATION

• 3. TIME
• 4. SPACE

Hey ¡bro

What’s ¡up?

CRISPR-CAS:

INTERCELLULAR COMMUNICATION

“If ¡we ¡are ¡not ¡careful, ¡we ¡will ¡soon ¡ be ¡in ¡a ¡post-­‑an<bio<c ¡era”

• ­‑ ¡Dr. ¡Tom ¡Frieden, ¡Director ¡of ¡the ¡Center ¡for ¡Disease ¡Control

Prokaryotic immune system that targets invading DNA

WHAT IS CRISPR-CAS?

CRISPR-CAS

Targets virulence genes

CRISPR-CAS

CRISPR-CAS

Donor bacteria can conjugate system to recipient bacteria

CRISPR-CAS

CRISPR-CAS

s

CRISPR-CAS

s

Recipient bacteria expresses CRISPR-Cas system

CRISPR-CAS

CRISPR-CAS

CRISPR-CAS

CRISPR-­‑Cas ¡on ¡a ¡ Popula<on ¡Level

CRISPR-CAS

✓ Obtained RP4-containing strains ✓ Midiprepped RP4 plasmid ✓ Transformed RP4 into electrocompetent DH5α ✓ Demonstrated successful conjugation of RP4 cells with cells harboring RFP plasmid

CRISPR-CAS:

DEMONSTRATING CONJUGATION

BBa_K1218011 BBa_K1218014 BBa_K1218000

CRISPR-CAS:

SUBMITTED BRICKS

Minimal CRISPR Array BsaI sites for easy reprogramming pCas9 Degrades target DNA dCas9-ω fusion Activates or represses target genes

• 3. TIME

CRISPR-CAS

DE-EXTINCTION

SYNTHETIC BIO-COMMUNICATION

• 2. INTERCELLULAR
• 4. SPACE

BIOWIRES 1. ATOMIC

Origin of Life

• Building Blocks
• Amino Acid Evolution

Application

• CasA Recognition
• Specific Mechanism

Proof of Concept

• Long Term Evolution
• Bioinformatic Confirmation

DE-EXTINCTION:THE ROADMAP

Protein Sequence Data

• PFAM Database

Phylogenetic Tree

• Geneious + PHYML

Substitution Model

• ProtTest

Sequence Reconstruction

• PAML + Lazarus

43

DE-EXTINCTION:PROCESS

Background

• HisC codes for histidine
• CysE codes for cysteine

Goals

• Understand their evolution
• Self-containment?

Process

• Predict ancestral genes
• Synthesize
• Test basic function

Histidine Cysteine

ORIGIN OF LIFE:AMINO ACIDS

• Conserved His/Cys Sites
• Molecular Clock > 120,000 Years
• Total Distance in Billions

ORIGIN OF LIFE:CHICKEN OR EGG?

Circles ¡indicate ¡protein ¡ac<ve ¡sites Highlighted ¡ regions ¡are ¡ conserved ¡ sites

Time (secs) OD

10000 20000 30000 40000 50000 60000 70000 0.08 0.13 0.18 Well E1 E2 E3 E4 E5 E6 Vmax 0.085 0.078 0.059 0.067 0.049 0.073 R^2 0.557 0.769 0.727 0.629 0.659 0.776 Vmax Points = 21

Time (secs) OD

10000 20000 30000 40000 50000 60000 70000 0.08 0.13 0.18 0.23 Well H1 H2 H3 H4 H5 H6 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 Vmax Points = 21

Positive Control (Trento 2012) Ancestral Gene

ORIGIN OF LIFE:CYSE RESULTS

Positive Control Ancestral Gene

Time (secs) OD

10000 20000 30000 40000 50000 60000 70000 0.08 0.13 0.18 0.23 Well C1 C2 C3 C4 C5 C6 Vmax 0.138 0.117 0.102 0.132 0.107 0.130 R^2 0.811 0.862 0.828 0.847 0.871 0.904 Vmax Points = 21

Time (secs) OD

10000 20000 30000 40000 50000 60000 70000 0.08 0.13 0.18 0.23 0.28 Well F1 F2 F3 F4 F5 F6 Vmax 0.142 0.164 0.107 0.105 0.115 0.117 R^2 0.762 0.949 0.965 0.744 0.933 0.937 Vmax Points = 21

ORIGIN OF LIFE:HISC RESULTS

Target: CasA

• CASCADE Complex
• Mechanism
• Protein Modeling

Testing

• Test functionality -

purified proteins

• Compare to modern

sequences

APPLICATION:CRISPR

Target: CasA

• CASCADE Complex
• Mechanism
• Protein Modeling

Testing

• Test functionality -

purified proteins

• Compare to modern

sequences

APPLICATION:CRISPR

Target: CasA

• CASCADE Complex
• Mechanism
• Protein Modeling

Testing

• Test functionality -

purified proteins

• Compare to modern

sequences

APPLICATION:CRISPR

Target: CasA

• CASCADE Complex
• Mechanism
• Protein Modeling

Testing

• Test functionality -

purified proteins

• Compare to modern

sequences

APPLICATION:CRISPR

Target: CasA

• CASCADE Complex
• Mechanism
• Protein Modeling

Testing

• Test functionality -

purified proteins

• Compare to modern

sequences

APPLICATION:CRISPR

DOES THE MODEL WORK?

• E. coli

A- A+ A-1 A+6

Thanks to Rich Lenski (MSU) we can validate the model

DOES THE MODEL WORK? LENSKI DATA

The Experiment

• E coli evolution
• 25 years
• 12 strains
• 40,000 generations

Our Goals

• Ancestral Reconstruction
• Whole Genome
• Proof of Concept
• Modeling Evolution

Results

• Lazarus ~90%
• Consensus ~99%

Implications

• Timeline too short?
• Problems with model?
• Not enough strains?

PROOF OF CONCEPT: LENSKI DATA

Application

• Biochemical Assay
• Functional Assay

Proof of Concept

• Alternative Models: ML, Bayes,

MP

• Selective Pressure

FUTURE AIMS

DE-EXTINCTION: BIOBRICKS

7 BioBricks: 3 characterized 4 currently being tested

Ancestral CasA CasBCDE Modern CasA Modern HisC Ancestral HisC Ancestral CysE AroE

DE-EXTINCTION:

ETHICS

✓ 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!

DE-EXTINCTION:ACCOMPLISHMENTS

• 3. TIME

CRISPR-CAS

DE-EXTINCTION

SYNTHETIC BIO-COMMUNICATION

• 2. INTERCELLULAR
• 1. ATOMIC
• 4. SPACE

EU:CROPIS BIOWIRES

Euglena Combined Regenerative Organic Food Production In Space

A German satellite mission scheduled to launch in 2016

EU:CROPIS

Brown-Stanford iGEM team pioneers PowerCell for in situ resource utilization on Mars

2011 2016

EU:CROPIS

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

POWERCELL

Brown-Stanford 2011

2011 2016

We create a chromogenic biosensor for PowerCell in B. subtilis

2013

EU:CROPIS

2011

2015

Load microfluidics chip into satellite

EU:CROPIS

2011 2016

Launch! Activate first gravity level and ground controls.

EU:CROPIS

Eu:CROPIS Microfluidics Chip

Anabaena

(Brown-Stanford 2011)

Bacillus subtilis

(Stanford-Brown 2013)

Anabaena

(Brown-Stanford 2011)

Bacillus subtilis

(Stanford-Brown 2013)

Anabaena

(Brown-Stanford 2011)

Bacillus subtilis

(Stanford-Brown 2013)

PROGRESS

We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A)

PROGRESS

We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A)

Used oligos to construct gene promoters

PROGRESS

We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A)

Used oligos to construct gene promoters Assembled 2 plasmids containing: sucrose metabolism promoter, a ribosome binding site, and a red chromoprotein/RFP

PROGRESS

We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A)

Used oligos to construct gene promoters Assembled 2 plasmids containing: sucrose metabolism promoter, a ribosome binding site, and a red chromoprotein/RFP Synthesized the equivalent construct for sporulation

PROGRESS

We identified genes associated with regulation of sucrose metabolism (sacY) sporulation (spo0A)

Used oligos to construct gene promoters Assembled 2 plasmids containing: sucrose metabolism promoter, a ribosome binding site, and a red chromoprotein/RFP Synthesized the equivalent construct for sporulation Transformed into B. subtilis using an integration vector

PROGRESS

RESULTS

Bacillus with sucrose metabolism promoter + RFP (Phase Contrast)

Bacillus with sucrose inducer promoter + RFP (Fluorescence)

• B. subtilis with sucrose inducer

promoter + fluorescent chromoprotein eForRed (Fluorescence)

RESULTS

FUTURE AIMS

Fall 2013: Isolate colonies containing our construct and characterize promoters

FUTURE AIMS

Fall 2013: Isolate colonies containing our construct and characterize promoters Winter 2013: Helium balloon test

FUTURE AIMS

Fall 2013: Isolate colonies containing our construct and characterize promoters 2014: Biocompatibility tests with microfluidics chip Winter 2013: Helium balloon test

FUTURE AIMS

2015: Prepare for mission Fall 2013: Isolate colonies containing our construct and characterize promoters 2014: Biocompatibility tests with microfluidics chip Winter 2013: Helium balloon test

FUTURE AIMS

2015: Prepare for mission 2016: EuCROPIS Satellite Mission Fall 2013: Isolate colonies containing our construct and characterize promoters 2014: Biocompatibility tests with microfluidics chip Winter 2013: Helium balloon test

FUTURE AIMS

EU:CROPIS - ACCOMPLISHMENTS

Constructed and synthesized prototype prototype chromogenic biosensors to detect sucrose induction and sporulation 5 BioBricks:

BBa_K1218001 BBa_K1218023 BBa_K1218021 BBa_K1218025 BBa_K1218020

Contributed on project to validate PowerCell

• n the EuCROPIS

satellite mission and send synthetic biology into outer space

EU:CROPIS - ACCOMPLISHMENTS

Constructed and synthesized prototype prototype chromogenic biosensors to detect sucrose induction and sporulation 5 BioBricks:

BBa_K1218001 BBa_K1218023 BBa_K1218021 BBa_K1218025 BBa_K1218020

OUTREACH

CALIFORNIA ACADEMY OF SCIENCES

CALIFORNIA ACADEMY OF SCIENCES

BIOENGINEERING BOOTCAMP

BAY AREA/NYC MAKER FAIRES

COLLABORATED WITH OTHER TEAMS

SOCIAL MEDIA PRESENCE

77

IGEM MEMES!

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HUMAN PRACTICES

An economic analysis on improving the productivity

• f and

lowering the costs for iGEM teams by employing DNA synthesis

SUMMARY

BIOWIRES: 2 BRICKS CRISPR-CAS: 3 BRICKS DE-EXTINCTION: 7 BRICKS EUCROPIS: 5 BRICKS PRESENTED TO CENTER DIRECTOR, NASA AMES TWO MAKERS FAIRES (SF

AND NY)

BAY AREA AND NEW ENGLAND MEETUPS

IGEM MEMES

BIOE BOOTCAMP CALIFORNIA ACADEMY

OF SCIENCES

ECONOMIC ANALYSIS

OF IGEM WORKFLOW

DE-EXTINCTION ETHICS

ACKNOWLEDGMENTS

• 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, Teri 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.

Supplementary Slides

Stanford-Brown 2013

83

Evidence of Silver Binding:

Thermal Denaturation: FRET fluorescence

Evidence ¡of ¡Silver ¡Binding:

Fluorescent ¡Metal-­‑Binding: Phen ¡Green ¡quenching

Efficiency/Molar Analysis

ESI-MS (data being analyzed)

• 12e Palindrome

1. T esting #ions incorporated 2. T esting effect of molar ratio Control Sequence

Bond Geometry

COSY-H NMR Left: data from T

• rigoe

(2012): expect to see shift

• n H5,

indicating N3 bonding site for Ag ion; Below: preliminary data with no silver

Structural Analysis

Ion Distribution: TEM + Negative Staining Molar ¡Ra/o ¡Increase Dark ¡areas ¡= ¡low ¡electron ¡density ¡DNA Light ¡ ¡areas ¡= ¡high ¡electron ¡density ¡regions ¡like ¡Ag+

Structural Analysis

Ion Distribution: TEM + Negative Staining Image ¡is ¡threshold ¡filtered ¡in ¡ImageJ ¡(NIH)

Structural Analysis

Ion Distribution: TEM + Negative Staining Par/cle ¡ ¡shapes ¡extracted ¡based ¡on ¡size ¡data

Structural ¡Analysis

Ion Distribution: TEM + Negative Staining Molar ¡Ra/o ¡Increase Dark ¡areas ¡= ¡low ¡electron ¡density ¡DNA Light ¡ ¡areas ¡= ¡high ¡electron ¡density ¡regions ¡like ¡Ag+ 124/649 ¡par/cles ¡are ¡paired ¡at ¡the ¡1bp ¡(3.4A) ¡level ¡in ¡one ¡test

Ion Distribution: TEM + Negative Staining

Structural Analysis

De-Extinction

FAQ: PFAM

• The Pfam database contains

protein families (hence the name) with annotations and sequence alignments.

• We began our process using

these alignments.

FAQ: GENEIOUS + PHYML

• Geneious offers a range of

capabilities as well as extensions for specific tasks.

• PhyML constructs trees using

maximum likelihood (a parametric method).

• Maximum parsimony

computes the tree with the fewest changes.

• Maximum likelihood

computes the most likely changes.

FAQ: PROTTEST

• Takes the sequence and tree

data and uses an approach based on PhyML. It compares the data to several empirical substitution models (WAG, JTT, etc.)

• Empirical models use

parameters estimated from real data. They can be more accurate than mechanistic models.

FAQ: PAML + LAZARUS

• PAML stands for Phylogenetic

Analysis by Maximum

• Likelihood. It is a package of

programs for evolutionary computation.

• Lazarus is an interface that

uses PAML on large datasets and find the ancestral sequence with the maximum posterior probability.

FAQ: DIFFERENT MODELS?

• Maximum Likelihood (ML)

maximizes likelihood at the “tips” of te tree

• Parsimony minimizes the

number of ancestral state changes

• Bayesian includes uncertainty

about the phylogenetic tree in its calculations

FAQ: ALTERNATIVE PROGRAMS?

• RASP (Reconstruct Ancestral

State in Phylogenies)

• Bayesian or Parsimony
• Phyrex
• Parsimony
• FastML
• Web-based Maximum

Likelihood

• MrBayes
• Bayesian

FAQ: CHOOSING AN OUTGROUP

EuCROPIS

SPACE MICROBIOLOGY

Stresses from space:

• 1. Weightlessness
• 2. Radiation
• 3. Other: vacuum, thermal, UV, orbital debris

How space affects biology:

• Solar radiation, UVB, UVC are hazardous to nucleic acids and proteins
• Freezing
• Desiccation
• Reduced lag phase and increased final cell population numbers
• May affect genetics: some genes seem to be gravity-dependent
• Increased microbial virulence
• Decreased effectiveness of antibiotics
• Effects of cosmic radiation

Why can’t we simulate microgravity?

• Some discrepancy between theory and data
• Some ways to simulate, like clinostat or rotating wall vessel but do not fully

reproduce the effects of space

Space Microbiology (Hornek, Klaus, Mancinelli 2010)

BACILLUS

• 1. Physiology
• Spores have protective envelope, a dehydrated protoplast
• 2. Flight tested
• Bacillus spores have been flown in space (Apollo 16, Spacelab 1, LDEF, etc)
• Exposed to space vacuum, radiation and cosmic rays, etc
• Can survive several years if protected from high intensity solar UV-radiation
• Space vacuum does cause some mutagenesis though
• 3. Record-setting organism
• After nearly six years in space, still 1-2% of spores are viable, even without protection

from dehydration caused by the space vacuum

• Bacillus found at higher elevation in atmosphere than any other organism (41km)

*Apollo 16 and 17: only time microorganisms went outside earth’s magnetic field Responses of B subtilis spore to space environment: results from experiments in space (Gerda Hornek 1996) Space Microbiology (Hornek, Klaus, Mancinelli 2010)

PowerCell

TIMELINE

E X sacY promoter P S E X P S destination plasmid: pSB1K3 Ribosome binding site Cut with E and S Cut with X and P Cut with E and P X S P E P E X P S E X S P E X S Ligate Ligate Cut with E and P M Grow on Kan plate E X P S Kan Chlor Amp Kan

3A Assembly of Promoter and RBS

Kan X P Chlor E S Amp

Text

E X S P E X promoter and RBS P S E X P S destination plasmid chromoprotein Cut with E and S Cut with X and P Cut with E and P X S P E E X P S E X S P E X S Ligate Ligate Cut with E and P M Cut with BamHI and HindIII and ligate Homologous recombination

amyE’ ‘ a m y E

BamHI HindIII

amyE’ ‘ a m y E

BamHI HindIII Bacillus genome integration vector HindIII BamHI

amyE’ ‘amyE

PCR E X S P BamHI HindIII P

Cloning Strategy Schematic