Biohacking: Theory and

Practice

Ron Shigeta PhD

Drew Waight PhD

@Biocurious

Nov 2011Illus David Goodsell http://mgl.scripps.edu/people/goodsell/illustration/public

BioPunks and BioHackers

“Biopunks are the visionaries and biotech wizards whose imaginations were set on fire by the knowledge that scientists had finally sequenced the human genome last year. Biopunks get off on creative genetic engineering, RNA research, cloning, and protein synthesis. Biopunks hack genomic data, lining up human genomes next to mouse genomes to find out what the two species have in common and what they don't.”

-Annalee Newitz Aug 8, 2001

“Your software is the abstract information pattern behind your genetic code, but your actual wetware is the physical DNA in a cell. A sperm cell is wetware with a tail, but it’s no good without an ovum’s wetware. A fertilized seed is self-contained wetware, and a plant cutting is wetware, too, as plants can reproduce as clones. ….I’ve come to understand that a body’s wetware is more than just its DNA. The autocatalytic system of biochemicals in each cell is a kind of wetware itself. The arrangement of a body’s cells—and the all-important tangling of the cortical neurons—is a higher-level kind of wetware as well.” -Rudy Rucker Aug 25, 2007

Wetware 101: Cellular Biology

Virus

• All life is a self replicating assemby of nucleic acids (DNA, RNA), amino acids (Protein), and lipids

• Eukaryotes (Animals, Plants, Fungi), are much larger than Prokaryotes (Bacteria, Archaea) and contain many more levels of complexity

Wetware 101: RNA and DNA

RNA DNA

• Chemically very similar, both are comprised of 4 bases and a phosphate backbone. DNA adopts a double helix and the majority is inaccessible due to tight supercoiling. DNA appears to serve an exclusively informational purpose

• RNA is normally found as a single stranded entity, is short lived and can adopt conformations which perform mechanistic and enzymatic functions. It is hypothesized that RNA is the original life molecule.

Wetware 101: Proteins• Amino acids are the building blocks of proteins. Peptide chains (proteins) fold to form secondary, tertiary and quaternary structures with different properties arising from the various side chains. The function of a protein is intrinsic to the 3 dimensional structure.

• With the exception of functional RNAs, proteins are responsible for all of the enzymatic, mechanistic and structural necessities for maintaining organisms.

Wetware 101: Central Dogma

RNA → ProteinCodon Usage Table

“Classical” Central Dogma

Wetware 101: Central Dogma

Wetware 101: Central Dogma

Wetware tools: PlasmidsA plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. Plasmids are double-stranded and, in many cases, circular. In the bacterial world, plasmids serve as mobile genetic elements which can move between individual organisms or even across species.

The use of plasmids are standard tools in modern microbiology and genetics. They are easy to manipulate and purify, can confer antibiotic resistance (or other selection markers)

The disadvantage to plasmid usage is that the organism will generally subvert, manipulate or kick out the plasmid after 100 generations or so

Polymerase Chain Reaction• The invention of PCR has made modern molecular biology possible. The reaction allows for the generation of high amounts of specific DNA genes or sequences (100s of ng/uL)

• By using PCR up to 2000bp of product can be amplified from a genomic source. Typically this DNA product is inserted either into a vector, or used to make recombinant chromosomal integrations.

• PCR uses primers (synthesized oligonucleotides ~40bp Tm=60C) specific to the flanking sequence of interest, dNTPs, template DNA, and a thermostable DNA polymerase (Taq, Pfu, Phusion)

• The reaction is thermocycled through melting, annealing and elongation steps.

Polymerase Chain Reaction

Restriction EnzymesA Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses.

Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially and are routinely used for DNA modification and manipulation in laboratories

Molecular CloningRestriction enzyme treated DNA fragments from PCR reactions or excised from existing plasmids can be used to insert into plasmid backbones. In this manner many different genes, promoters and other DNA elements can be shuffled around via “cut and paste”

Joining the sticky ends requires DNA ligase

BioBrick standard assembly

pGLO

Our Example system“Software” for the cell

bla (AmpR)GFP

AraC

ori

Img:wikipediahttp://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses24.html

pGLO

bla (Ampicillin Resistance)

GFP (Green Flourescent Protein)

ori

MCS (Multiple Cloning Sites)

AraC (Arabinose Repressor)

pGLO

bla (AmpR)GFP

AraC

ori

1

Arabinose Consumption

araBaraC

1) AraC both upregulates itself, and represses AraB AraA and AraD 2) Arabinose diffuses in3) AraC binds Arabinose, and 4) RNA polymerase may now transcribe araB/A/D

to mRNA and eventually produces protein

AraC

pBAD

AraC

AraC AraB

2 3 4

AraC

pC araA araD

AraA

AraD

http://userpages.umbc.edu/~lrowan1/arabinoseoperon.html

arabinose

1

pGLO a machine for detecting Arabinose

GFPAraC

1) Setup - Constitutive Expression of Sensor / Regulator(Endogenous or Exongenous)

1) Sensors receive signal2) Program logic turns on/off genes3) There is a readout of the signal

AraC

pBAD*

AraC

AraC

GFP2 3 4

Genetically Engineered Machines

• Osmolarity Sensor

• Day night smell alarm clock

• Cell photography

• Repair

• igem.org

• Partsregistry.org

iGEM 2011: MissouriOsmolarity Sensing - could detect excess sugar in a serum sampleendogenous EnvZ and OmpR make for a relatively simple machine

• EnvZ http://www.ncbi.nlm.nih.gov/gene/947272• OmpR http://www.uniprot.org/uniprot/P0AEJ4• http://ecoliwiki.net/colipedia/index.php/ompR:Expression

EnvZ

*OmpR

various

OmpR

various various

iGEM 2011: Missouri Endogenous EnvZ with three promotor sequences gives a non-linear response to OmpR binding…

• http://2011.igem.org/Team:Missouri_Miners/Project• Promotor sequence

http://partsregistry.org/Part:BBa_R0082

*OmpR

eFYP

*OmpR

eFYP

*OmpR

eFYP

*OmpR

*OmpR *OmpR

OneFYP

OneFYP

Off

EnvZ

Endogenous vs Exogenous Components

• Will cross talk with host mess up your logic?

• Does the host supply what your system needs to keep your machine running?

- pGLO has no endogenous 'logic' in E coli that does not have araC- Osmosis detector uses endogenous EnvZ/OmpR and signals in accord with what the bacteria feels

SBC 2004: AustinTowards bacterial photography through a partly endogenous pathway

• http://partsregistry.org/cgi/htdocs/SBC04/austin.cgi• Detailed Description of receptor:

http://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor#References

SBC 2004: AustinTowards bacterial photography through a partly endogenous pathway

• http://partsregistry.org/cgi/htdocs/SBC04/austin.cgi• Detailed Description of receptor:

http://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor#References

*OmpR

Cph8

BBa_R0082

BBa_I15010

LacZ Nfrag

BBa_E0033BBa_B0034

BBa_B0015

iGEM 2006: ChibaFresh scent by day, orange by nightWhat did Chiba Do?

• http://parts.mit.edu/wiki/index.php/Aromatic_Bacteria_:_Chiba_2006#parts

Activity - iGEM 2006: ChibaLets Guess What Chiba did

• http://parts.mit.edu/wiki/index.php/Aromatic_Bacteria_:_Chiba_2006#parts

iGEM 2010: NewCastleEngineering Bacillus subtilis to repair microcracks in cementSystem Architecture: one signal activates three different pathways relying on endogenous biosynthesis pathways

• http://2010.igem.org/files/poster/Newcastle.pdf

1

2

3

4

iGEM 2010: NewCastleQuorum Sensing with subtilin

• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207451/pdf/jbacter00088-0430.pdf• https://www.bio.cmu.edu/courses/03441/TermPapers/97TermPapers/lux/communication.html

iGEM 2010: NewCastleSwarming and Glue

BBa_K302012

LacI RepressorBBa_K302003

SacBBBa_K302005

Cons. prom.K143012

rbsK143021

RBS-cds junction G0000

sfp – surfactantK302010

rbsK143021

RBS-cds junction G0000

swrA – repair flagellaK302011

trans stopK302011/2

lacI

lacI

iGEM 2010: NewCastleMaking CaCO3 (cement)

(NH4)2CO3

http://www.enzyme-database.org/reaction/AminoAcid/Arg.html

iGEM 2010: NewCastleMaking CaCO3 (cement) BBa_K302015

LacI RepressorBBa_K302003

rbsK143021

Inhibitory RNA for ahrCFor arginine synthesisK302002

rbsK143021

rocF – Break down to UreaK302011

transcr stopK302011/2

lacIlacI

LacI RepressorBBa_K302003

Partsregistry.org/CatalogExploring the standards parts registry soon to be available at biocurious

• Promotors

– Constitutive (always on)

– Cell Signaling

– Metal Sensitive (iron copper lead)

– Phage Promotors (high intensity)

– USTC Logic promotors

• Protein Coding Sequences

• Ribosome Binding Sites

• Terminators

• Plasmids

Example: http://partsregistry.org/PBAD_Promoter_Family

Partsregistry.org/CatalogProtein Coding Sequences

– Reporters

• Fluorescent Proteins (GFP/YFP/CFP etc etc. Exogenous, cheap, independent)

• Luciferases – oxidizes luciferin to produce life (can be endogenous)

• LacZ – reaction with reagent chemicals to darken the cell

– Transcriptional Regulators (http://partsregistry.org/Protein_coding_sequences/Transcriptional_regulators)

• Positive Regulators (activator)

– luxR

• Negative Regulators (repressors)

– lacI

• Both (multiple)

– araC

– Selection Marker

• AmpR

• TetC

• KanR

Make our own PlasmidsRummaging through our parts box

• Transcriptional Regulators (inputs)

– araC (arabinose)

– EnvZ / ompR (osmolarity)

– Cph8 (light)

– (Quorum Sensing)

• Reporters (outputs)

– Carotene (orange color)

– Fluorescent proteins (nearly any color)

– LacZ (dark color)

– Swarming

– Scent Chemicals (citrus, pine, fruit)

• Promotors

– pBAD (arabinose)

– (ompR – osmotic pressure)

• Repressors

– pC (arabinose)

– LacI (lactose)

•Hardware:•Replication Origins (ORI)

•High Copy•Low Copy

•Selection markers• AmpR• KanR•CmfR•TetR

Finding your own Genes

• Parts registry http://partsregistry.org• NCBI http://ncbi.nih.gov

– Gene http://ncbi.nih.gov

– PubMed http://ncbi.nih.gov

• UniProt http:uniprot.org• KEGG http://kegg.org• ecoCYC http://biocyc.org

Exercise: Inserting a Gene into a Vector

You want to insert Bacteriorhodopsin, a light driven proton pump into an expression vector with tight regulation. You want to fuse this protein to GFP so you can locate it within the cell. The guy downstairs says he has the perfect vector and gives you an eppendorf tube with 5uL of the vector and tells you that its based off a pBAD backbone.

You get yourself some Halobacterium salinarium genomic DNA from ATCC and get to work....

Advanced Topics : Beyond iGEM

the SynBio Bleeding EdgeKeasling lab Arteminisin production in yeast. $ 0.25 dose from a fermentor

• http://www.nature.com/nature/journal/v440/n7086/full/nature04640.html• http://en.wikipedia.org/wiki/Artemisinin

From the SynBio Bleeding EdgeNeutraceuticals: Golden Rice and BioFuels

http://en.wikipedia.org/wiki/Golden_rice

Nature 438, 441-442 (24 November 2005) | doi:10.1038/nature04405

Advanced Topics: Beyond iGEM

doi:10.1016/j.jmb.2010.10.038

Advanced Topics: Beyond iGEM

A Tamsir et al. Nature 000, 1-4 (2010) doi:10.1038/nature09565

Construction of all 16 two-input Boolean logic gates.

Construction of an XOR gate by programming

communication between colonies on a plate.

Advanced Topics: Beyond iGEM

Science 21 October 2011: Vol. 334 no. 6054 pp. 366-369 DOI: 10.1126/science.1208144

Nature. 2000 Jan 20;403(6767):335-8.

Advanced Topics: Beyond iGEM

Nature Biotechnology 20, 1041 - 1044 (2002)

Nature Biotechnology (2011) doi:10.1038/nbt.2018

Advanced Topics: Beyond iGEM

Advanced Topics: Beyond iGEM

Biohacking In the Very Near Future....

Understanding the “machine language” of wetware is of paramount importance

What applications will be developed with the ability to program life forms from a computer (or smart phone)

Classical uses of Selection Different colors of GFP The Methuselah worm Seymour Benzer’s scent mutants in fruit flies

Other Resources

• EcoiWiki http://ecoliwiki.net/colipedia

• ATCC http://www.atcc.org/

• Carolina Biologicals

http://www.carolina.com/

• … see next week's bioinformatics meetup

http://www.meetup.com/BioCurious/events/40419392/

• BioCurious Project Google Doc

https://docs.google.com/document/d/1kPjdMKC85WBMy14aAH4fZWhfoGZybyZtItiEjczM6cc/edit?hl=en_US&pli=1

• rtshigeta@yahoo.com waight@msg.ucsf.edu