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Synthetic Biology
Synthetic Biology
1. The design and fabrication of biological components
and systems that do not already exist in the natural
world
2. The re-design and fabrication of existing biological
systems.
Synthetic Biology vs. Systems Biology
•
Systems biology studies complex biological systems as integrated wholes, using
tools of modeling, simulation, and comparison to experiment. The focus tends to
be on natural systems, often with some (at least long term) medical significance.
•
Synthetic biology studies how to build artificial biological systems for engineering
applications, using many of the same tools and experimental techniques. But the
work is fundamentally an engineering application of biological science, rather
than an attempt to do more science. The focus is often on ways of taking parts of
natural biological systems, characterizing and simplifying them, and using them as
a component of a highly unnatural, engineered, biological system.
http://syntheticbiology.org/FAQ.html
Oil Eating Microorganisms
Nature Biotechnology - 24, 952 - 953 (2006)
doi:10.1038/nbt0806-952 Blueprint of an oil-eating bacterium
Víctor de Lorenzo
MIT iGEM's Project: to sense and remove Hg ions from contaminated water.
Two Cell System
One cell will use the Mer promoter to sense
the presence of Mercury ions, then activate
the GFP fused downstream.
Second cell uses surface display mechanism
to exhibit a Mercury capturing peptide,
extracting the Mercury from the water.
Both cells also display polystyrene binding
peptides, and will thus be attached to a
polystyrene filter.
Genome Transplantation in Bacteria:
Changing One Species to Another
Carole Lartigue, John I. Glass,* Nina Alperovich, Rembert Pieper, Prashanth P. Parmar,
Clyde A. Hutchison III, Hamilton O. Smith, J. Craig Venter
Mycoplasma mycoides
Mycoplasma capricolum
?
Successful transplantation
-Clean change of one bacterial species into another
-No recombination between donor & recipient
chromosomes
Why use these bacteria?
Mycoplasma Mycoides (Donor)
1.
Mycoplasma Capricolum (Recipient)
Small Bacteria – Goat & Bovine Pathogens
Small Genome
No cell wall
2. Degree of Relatedness
- 76.4% of M. Mycoides genome could be mapped to M. Capricolum
genome
- Of 76.4% there was 91.5% nucleotide identity
3. Plasmids containing a M. mycoides LC origin of replication complex (ORC)
can be established in M. capricolum, whereas plasmids with an M.
capricolum ORC cannot be established in M. mycoides LC
Key Phases for Successful Transplantation
1. Isolation of intact donor genome
2. Preparation of recipient cells
3. Installation of isolated genome into recipient
cells
Donor Genomic DNA Preparation
100 ul x 20
Centrifuged, resuspended
incubated, mixed with
LMP agarose
tetM & lacZ Mycoides
cells grown in 10ug/ml
tetracycline
Plugs
solidified
@ 4oC
PFGE
Check for intact
circular DNA
Lysed, Proteinase K,
Wash (4x)
DNA
DNA
Intact DNA Remains in Plug
A. Intact Circular DNA stays in plug
- linear DNA, fragment, PRO, &
RNA migrate
B. Plasmid safe DNase digests
band but not plug
Silver Staining Indicates Naked DNA
A.
SDS-PAGE & silver stain of plugs
B.
Plugs boiled in SDS before or
after PFGE
C.
DNase I treatment
LC-MS/MS Analysis of Plugs
•
Background of non M.
Mycoides proteins run on
PFGE
•
No M. mycoides proteins
present in plugs not
exposed to PFGE
Liberation of DNA From Agarose Plug
Melted @ 65oC
DNA
DNA
Incubated overnight w/ β-agarase I
~10ug DNA (8 x 109 genomes)
Key Phases for Successful Transplantation
1. Isolation of intact donor genome
2.Preparation of recipient cells
3. Installation of isolated genome into recipient
cells
Preparation of Recipient Cells
Washed, resuspended in CaCl
Incubate 37oC, pH 6.2
Held on ice
capricolum (recipient)
PEG
fusion
Buffer
Incubated 30min RT
DNA
mycoides (Donor)
DNA
400ul SP4- medium
DNA
10ug transfer RNA
DNA
SP4 agar plates w/
tetracycline & Xgal
Genetic Analysis
• Displayed expected specific amplification
* IS1296, tetM, & lacZ may have recombined to
destroy arginine deiminase gene
Southern Blot Analysis
•
Donor, Recipient & putative transplants digested with
Hind III & run on 1% agarose gel
A.
•
•
Transplants contain multiple copies of IS1296
insertion sequence
59% (44 of 75) were same as donor DNA blot
•
Banding differences due to IS1296 transposition
B.
•
•
No probe hybridized with WT M. capricolum
92% clones were same as donor DNA blot
Transplant Genome Library Analysis
Whole Genome Libraries from 2 Transplant Clones
1300 random Sequence reads from each transplant
(1.09 million bp) all matched M. mycoides
*20 identical regions b/w 395 & 972 bp
Currently..
1. Isolated naked DNA from donor M. mycoides
2. Created chemically competent M. capricolum
recipient cells
3. Isolated putatively transformed colonies
4. Confirmed genotypic identity using PCR, southern
blot, & library screening
Successful Introduction of M. mycoides genome
into M. capricolum followed by subsequent loss
of capricolum genome during antibiotic
selection
Colony Hybridization
Top
Probe w/ mycoides specific antibody (anti-VchL)
Result: Bound M. Mycoides donor genome and transplants
Did not bind capricolum colonies
Bottom
Probe w/ carpricolum specific antibodies (anti-VmcE & VmcF)
Result: Bound WT capricolum colonies
Did not bind mycoides donor genome or transplants
Proteomic Analysis – 2DE & MALDI-MS
•
Mycoides & transplants identical
•
Significant differences (50%) in capricolum
•
Mascot Algorithm
-red = identical to both species
-blue = unique to M. mycoides
*there were nine protein spots with confidence
scores that indicated they were derived
from M. capricolum genes, each case proved to
be an artifact of either sequencing errors or gene
boundary annotation errors (table S2).
Optimization of Genome Transplantation
2000ng DNA = 1.6 x 1013 genomes
PEG Based Method – Capricolum Cells Fuse
• PEG fusion buffer ([Tris 20 mM, NaCl 500 mM, MgCl2 20 mM, polyethylene glycol 8000 (PEG;
USB Corporation no. 19959)10%]
tetM
CAT
Successful fusion
Prepared as
recipient cells
Incubation
w/ fusion
buffer
Plated on SP4 agar plates w/
tetracycline & chloramphenicol
tetM
CAT
Only colonies in 5% PEG grew
30X increase when CaCl2 added
Preparation of Recipient Cells
Washed, resuspended in CaCl
Incubate 37oC, pH 6.2
Held on ice
Capricolum (recipient)
*PEG
fusion
Buffer
Incubated 30min RT
DNA
Mycoides (Donor)
DNA
400ul SP4- medium
DNA
10ug transfer RNA
Concluding Remarks
1.
Transplant occurred but mechanism of transplant is still unclear
- No demonstration of mosaicism
2.
Other methods for transplantation
- cation & detergent mediated transfection, electroporation, &
compaction of genome methods all unsuccessful
3.
Transplants performed without detergent & proteinase K
treatment were unsuccessful
- Improbability of finding naturally occurring free-floating, intact
naked genomes limits this transplantation phenomenon to the
laboratory
Registry of Standard Biological Parts
Http://parts.mit.edu/registry/index.php/Part_Types
Synthetic Chromosome – Venter Institute
•Synthetically created a chromosome that is 381 genes long and contains 580,000 base pairs
•The DNA sequence is based on the bacterium Mycoplasma genitalium which the team pared down
to the bare essentials needed to support life, removing a fifth of its genetic make-up. The wholly
synthetically reconstructed chromosome, which the team have christened Mycoplasma
laboratorium, has been watermarked with inks for easy recognition.
•The new life form will depend for its ability to replicate itself and metabolise on the molecular
machinery of the cell into which it has been injected, and in that sense it will not be a wholly
synthetic life form.
http://www.guardian.co.uk/science/2007
/oct/06/genetics.climatechange