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DNA by zhouwenjuan


									Synthetic Biology
              Synthetic Biology

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

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

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
            Why use these bacteria?
Mycoplasma Mycoides (Donor)              Mycoplasma Capricolum (Recipient)

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
    - 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
  Donor Genomic DNA Preparation
                                                    100 ul x 20

                         Centrifuged, resuspended

                         incubated, mixed with
                         LMP agarose
tetM & lacZ Mycoides
cells grown in 10ug/ml                                                               Plugs
tetracycline                                                                                         Lysed, Proteinase K,
                                                                                     solidified      Wash (4x)
                                                                                     @ 4oC

                                                                   PFGE                            DNA
                                                                  Check for intact
                                                                  circular 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

                    •   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
             Preparation of Recipient Cells

                                                                Washed, resuspended in CaCl
                         Incubate 37oC, pH 6.2

                                                                     Held on ice

capricolum (recipient)
                                                                                              PEG        10ug transfer RNA

                                                                     Incubated 30min RT

      DNA                                            DNA                                           DNA
 mycoides (Donor)
                                                 400ul SP4- medium

      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

          •    Transplants contain multiple copies of IS1296
               insertion sequence
          •    59% (44 of 75) were same as donor DNA blot

          •    Banding differences due to IS1296 transposition

          •    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
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
Colony Hybridization
         Probe w/ mycoides specific antibody (anti-VchL)

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

         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       Incubation
     recipient cells   w/ fusion                                        tetM
                       buffer                                          CAT

                                                             Only colonies in 5% PEG grew
                                                             30X increase when CaCl2 added
      Plated on SP4 agar plates w/
      tetracycline & chloramphenicol
             Preparation of Recipient Cells

                                                            Washed, resuspended in CaCl
                     Incubate 37oC, pH 6.2

                                                                 Held on ice

Capricolum (recipient)
                                                                                          *PEG      10ug transfer RNA

                                                                 Incubated 30min RT

      DNA                                        DNA                                          DNA
 Mycoides (Donor)
                                             400ul SP4- medium
                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
Registry of Standard Biological Parts
       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.


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