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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 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.
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)
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....
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
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