synthetic biology - uzhffffffff-b34e-2810-ffff-ffff9f... · 2016. 5. 11. · synthetic biology for...
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SYNTHETIC BIOLOGY
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SYNTHETIC BIOLOGY FOR DE NOVO
ASSEMBLY OF MEMORY DEVICES –
GLOSSARY
(Non-)volatile memory – synthetic memory circuits (do not) need
active cellular processes to maintain their state
volatile memory > e.g. transcription-based
non-volatile m. > recombination-based
volatile memories are bistable
stochastic switching between states should
be rare
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LOGIC GATES
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GENETICALLY ENCODED OPERATORS
AND REGULATORS IN VITRO
In vitro memory circuits are typically
composed of interlocking negative and
positive feedback loops that form a
bistable core
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GENETICALLY ENCODED OPERATORS
AND REGULATORS IN VIVO
RECOMBINATION-BASED
TRANSCRIPTIONAL FEEDBACK LOOPS
PROTEIN
PHOSPHORYLATION
RNA EDITING
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APPLICATIONS OF SYNTHETIC
BIOLOGY
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REPROGRAMMING THE
MICROBIOME
PAPER #1 – KOTULA JW ET AL., PNAS 2014
«PROGRAMMABLE BACTERIA DETECT AND RECORD AN
ENVIRONMENTAL SIGNAL IN THE MAMMALIAN GUT”
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MEMORY ANALYSIS OF E. COLI
- Modify E. coli with memory capabilities , transfer into
mice, record events during antibiotic exposure
- Prerequisites
- inital «nonmemory» state should be highly stable, only
failing as a result of mutation
- «memory» state should also be highly stable
- Chromosomal integration instead of plasmids to minimize
loss of genetic information
- Integrated elements should not impose high fitness burden
on the host
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TRIGGER/REPORTER SYSTEM WITH A
TOGGLE SWITCH
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cl/Cro//tetP-Cro SYSTEM FROM
BACTERIOPHAGE LAMBDA
- repressor state of induction-deficient cI ind- lysogen only fails due to mutation
- little burden on bacterial host since only 100-200 monomers of cl ind- are
present per cell, and, if activated < 1000 molecules of Cro
- Tn10 tetracycline repressor is particularly sensitive to anhydrotetracycline
(ATC) >> 100 ng/mL ATC will cause full derepression of the promoter without
inhibiting growth of tetracycline-sensitive E.coli
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SELECTING THE OPTIMAL MUTANT
ATC
42°C
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ATC EXPOSURE LEADS TO CI>CRO
SWITCH FOR >5D IN VITRO
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DETECTION OF ATC IN THE
MAMMALIAN GUT
- PAS132: mutated clone chosen for analysis
- Contains a mutation in rspL, conferring resistance to
Streptomycin
- Colonization of mice with E. coliPAS132
- Female Balb/c mice received 107 bacteria per animal via
oral gavage while receiving streptomycin in the drinking
water (+/- ATC)
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ATC-INDUCED LASTING CI>CRO
SWITCHES IN VIVO
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ATC-INDUCED LASTING
CI>CRO SWITCHES IN VIVO
IN AN UNCHARACTERIZED
(E.COLI 16S RIBOSOME
CONTAINING)
COLIFORM BACTERIUM
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CONCLUSIONS
- Stable (& bistable) expression system in 2 different E.coli (one as yet uncharacterized) strains to record exposure to antibiotic treatment
- Lasting recordings over hours to days (and multiple cell cycles) with no significant fitness burden and no observed «spontaneous», stochastic switching of states
- <> FLP based recombination systems are non-volatile and may be leaky due to constitutive low expression of FLP recombinase
- Stable cI/Cro system could be completely transformed into E. coli and may further have stabilizing function on the transcripts
- May lead to design of efficient, probiotic bacteria for diagnostics or treatment
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PAPER #2 – GOLDMAN N ET AL., NATURE 2014
“TOWARDS PRACTICAL, HIGH-CAPACITY, LOW-
MAINTENANCE INFORMATION STORAGE IN
SYNTHESIZED DNA”
All 154 of Shakespeare’s sonetts
(ASCII «.txt»)
Watson&Crick, Nature 1953
(«watsoncrick.pdf»)
«EMBL-EBI.jpg» 26s, «ihaveadream.mp3» Huffmann code (ASCII «.txt»)
>> 757’051 bytes
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- 4-fold redundancy per DNA fragment
- Reverse complements of each fragment to minimize data loss
- total of 153,335 strings of DNA, each comprising 117 nt (100 nt data + 17 nt barcoding)
- No homopolymers
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• Oligo library synthesis
• ~1.2 x 107 copies per fragment
DNA synthesis
Lyophilize DNA
USA > UK > GER
• Resuspension
• Amplification
• Purification
EMBL
Sequencing on
Illumina HighSeq
In «paired-end» mode
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79.6 x 106 read-pairs of 104 bases in length
full-length reconstruction in silico
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TROUBLESHOOTING
2x25bp could not be assigned > manual filling through neighbor comparison >
«100% correctly aligned sequences»
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CONCLUSIONS & OUTLOOK –
ENCODING LARGER DATA VOLUMES
Indexing DNA
grows
logarithmically to
the encodable data,
while total amount
of synthetized DNA
for coding grows
sub-linearly
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CONCLUSIONS & OUTLOOK –
READ ERRORS AS A FUNCTION OF
COVERAGE
- Error is zero when ≥2% of the reads are used - taken from the originally obtained 79.6 × 106 read-pairs of all but “watsoncrick.pdf”
- Manual correction of «watsoncrick.pdf» leads to a minimal error rate of 0.0036%
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CONCLUSIONS & OUTLOOK –
COST EFFECTIVENESS
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CONCLUSIONS & OUTLOOK –
COST EFFECTIVENESS
- Costs can further be decreased by lower sequencing
coverage (now mean sequencing coverage ~ 1’308x)
~ 80 PB (80x1000 TB) of data as of writing of
Paper, growth of ~15 PB per year
- 10% is maintained on disk, CASTOR
migrates regularly between tapes
- Access to old data decreases considerably
after 2-3 years after aquisition
- Current costs ~ 12’400$ per MB for coding
and ~220$ per MB for decoding
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CONCLUSIONS
& OUTLOOK
- With current synthesis & reading
costs as well as current coding
scheme, DNA-based storage may
be cost-effective for archives of
several megabytes within a ~600-
5’000 yr period
- If costs decrease by 1 log, ~50-
500 yr period
- If costs decrease by 2 logs, < 50
yrs
- ~2.2 PB of information per gram
DNA
- long-term storage seems to be
evolutionary proven
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PAPER #3 – YANG ET AL., NATURE METHODS 2014
“PERMANENT GENETIC MEMORY WITH >1-BYTE
CAPACITY”
LSTP
«large serine-type phage»
integrases
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finding LSTP integrases
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CHARACTERIZATION OF MEMORY
SWITCHES AND ORTHOGONALITY
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CREATING A MEMORY ARRAY WITH 11 ORTHOGONAL
INTEGRASES FOR RECORDING OF 2^11 = 2048
DIFFERENT STATES (> 1 BYTE)
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LOGIC GATES
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LOGIC AND-GATE
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CIRCUITS OF MULTIPLE
RECOMBINASES