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.

Synthetic Biology

Synthetic Biology

1. The design and fabrication of biological componentsand systems that do not already exist in the naturalworld

2. The re-design and fabrication of existing biologicalsystems.

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

Nature Biotechnology - 24, 952 - 953 (2006) doi:10.1038/nbt0806-952 Blueprint of an oil-eating bacteriumVíctor de Lorenzo

Oil Eating Microorganisms

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?

1. 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

Mycoplasma Mycoides (Donor) Mycoplasma Capricolum (Recipient)

Key Phases for Successful Transplantation

1. Isolation of intact donor genome

2. Preparation of recipient cells

3. Installation of isolated genome into recipient cells

tetM & lacZ Mycoides cells grown in 10ug/ml tetracycline

Centrifuged, resuspended

incubated, mixed with LMP agarose

100 ul x 20

Plugs solidified @ 4oC

Lysed, Proteinase K,Wash (4x)

DNAPFGE

Check for intact circular DNA

Donor Genomic DNA Preparation

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

DNAMelted @ 65oC

Incubated overnight w/ β-agarase I

DNA

~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

Incubate 37oC, pH 6.2 Washed, resuspended in CaCl

Held on ice

capricolum (recipient)

mycoides (Donor)

DNA

400ul SP4- medium

Incubated 30min RT

DNA DNA

10ug transfer RNAPEG fusion Buffer

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. mycoides2. Created chemically competent M. capricolum

recipient cells3. Isolated putatively transformed colonies4. 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

TopProbe w/ mycoides specific antibody (anti-VchL)

Result: Bound M. Mycoides donor genome and transplants Did not bind capricolum colonies

BottomProbe 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 confidencescores that indicated they were derivedfrom M. capricolum genes, each case proved tobe an artifact of either sequencing errors or geneboundary annotation errors (table S2).

2000ng DNA = 1.6 x 1013 genomes

Optimization of Genome Transplantation

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

Prepared as recipient cells

Incubation w/ fusion buffer

Plated on SP4 agar plates w/ tetracycline & chloramphenicol

Only colonies in 5% PEG grew30X increase when CaCl2 added

tetMCAT

Successful fusion

Preparation of Recipient Cells

Incubate 37oC, pH 6.2 Washed, resuspended in CaCl

Held on ice

Capricolum (recipient)

Mycoides (Donor)

DNA

400ul SP4- medium

Incubated 30min RT

DNA DNA

10ug transfer RNA*PEG fusion Buffer

Concluding Remarks1. 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

Http://parts.mit.edu/registry/index.php/Part_Types

Registry of Standard Biological Parts

•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.

Synthetic Chromosome – Venter Institute

http://www.guardian.co.uk/science/2007/oct/06/genetics.climatechange