Isolation of Microorganisms and Tagging with Marker Genes

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Isolation of Microorganisms and Tagging with
Marker Genes

• A Physiological Study
• by Ingvor Irene Zetterlund

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Isolation of Microorganisms and Tagging with
Marker Genes

• Aim to study a particular microorganism in a
  complex community by means of marker genes gfp
  lux.
• The gfp gene encodes for green fluorescent
  protein. GFP absorbs UV light fluorescing green
  light.
• The lux gene is a marker used for determination
  of cellular metabolic activity.

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Materials

• Bacteria- the isolate from the soil of a potted
  plant grown on ampicillin- the isolate from the
  soil of a potted plant grown on kanamycin- E.
  coli CC118 carrying the vector PUT gfp lux- E.
  coli DH5a as a control of the transformation-
  GFP tagged Arthrobacter chlorophenolicus A6G as a
  control microorganism.

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The soil of the Gardenia Used in the Project
Gardenia Before its Withering
Infected Gardenia
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Materials

• Reagents- LB medium- PBS- TAE buffer- 0,9
  NaCl- Nycodenz- Plates with ampicillin,
  cycloheximid, kanamycin, cycloheximid and
  kanamycin.
• Kits- QIAprep- Wizard DNA Clean-UP system.

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Methods Isolation of Bacteria

• The bacterial cells were isolated from soil on
  plates with LB medium and the antibiotic
  cycloheximid. Then them were grown in LB medium
  with cycloheximid.

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Methods Isolation of the Plasmid DNA

• E. coli CC118 carrying the vector PUT gfp lux
  plasmid was grown on the plates with LB medium
  and kanamycin.
• The vector plasmid DNA was isolated using QIAprep
  and purified by Wizard DNA Clean-UP system.
• For the vector plasmid isolation the cell lysis
  must be incomplete.
• The QIAprep plasmid purification is based on
  alkaline lysis of bacterial cells.
• Then DNA are absorbed onto silica-gel membran of
  a QIAprep spin column.

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Methods Agaros Gel Electrophoresis

• The quality of the plasmid DNA was analyzed by
  agaros gel electrophoresis after it was cut by
  restriction enzimes.

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Methods Transformation of the Bacterial Cells

• Transformation is a transferring in a cell of the
  external DNA, which incorporates into the
  recipient cell genome.
• Electrocompetent cells were prepared by growth
  overnight with shaking in- Isolate - LB medium
  with cycloheximid at RT. - E. coli DH5a - LB
  medium without antibiotics at 28oC.

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Transformation of the Bacterial Cells
Electroporation

• Competent cells were prepared by electroporation.
  
• The bacterial cells and the purified plasmid were
  placed into an electroporation cuvette and
  exposed to a strong electric field.
• The cell membrane becomes more permeable to DNA
  after this procedure.
• The transformed cells were grown in LB medium
  with ampicillin (IA) and LB medium with kanamycin
  (IK) overnight with shaking.

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Reintroduction to Soil

• Four microcosms were started with- The isolate
  grown on ampicillin- The isolate grown on
  kanamycin- GFP tagged Arthrobacter
  chlorophenolicus A6G as a control
  microorganism- Water as a control microcosm.
• The physiological condition of the bacterial
  cells was analyzed by flow cytometry
  (FACS-Calibur) and colony forming units CFU.

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Methods Flow Cytometry

• The flow cytometry allows analyzing hundreds of
  the bacterial cells per second when they pass a
  laser beam.
• The tagged cells can be distinguished by their
  fluorescence intensity.
• The number of the tagged cells was counted by the
  formula

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Methods Nycodenz

• The bacteria cells were separated from soil by
  equilibrium density centrifugation in continuous
  Nycodenz gradients.

Aquatic phase
Soil mixture
Bacterial cells
Nycodenz
Heavy soil particals
Before centrifuging
After centrifuging
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Results

• Quantity of the isolates and A6G cells are shown
  in relation to the background fluorescence from
  soil.
• IA isolate grown on ampicillin, IK isolate
  grown on kanamycin
• The results from the FACS were analized by Excel
  and the graphs were approximated. By Chi-test
  their new patterns were confirmed.

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Results The Isolate Grown on Ampicillin

• Have demonstrated a faint capability of the
  reintroduction to soil.
• During the first week the number of the tagged
  cells increased modestly.
• The following four days the numbers were
  decreasing, and it became under the level of the
  background fluorescence from soil.
• The number of CFU was four log units less than
  the number of the tagged cells.

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Results The Isolate Grown on Kanamycin

• The number of the tagged cells of the isolate
  grown on kanamycin was increasing during nine
  days and trebles itself.
• The following five days it was decreasing but
  remained higher than the background fluorescence
  from soil.
• The quantity of the CFU was four log units less.
  It was the highest in the beginning and it was
  decreasing all the time.

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Results A6G

• A6G showed a good capability of being introduced
  to soil.
• The number of the tagged cells were increasing
  during the experiment. It achieved the top after
  14 days.
• The number of CFU was four log units less than
  the number of the tagged cells.
• CFU was highest after a week, then it sank.

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Results

• The isolate grown on ampicillin could not be
  reintroduced to soil.
• The isolate grown on kanamycin have demonstrated
  a certain degree of capability of the
  reintroduction to soil.
• A6G showed a good capability of being
  reintroduced to soil.

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Discussion

• The soil community made it difficult for the
  transformed bacteria and A6G to establish
  themselves.
• Unfavourable for the bacterial cells
  environment- competition with other
  microorganisms,- predators, got many of them
  dead or possibly entered dormancy.

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Factors Influenced the Results

• The plasmid DNA was not purified excellently.-
  During electroporation a lot of short circuits
  were appeared.- Too high voltage could kill most
  of the bacterial cells.- Few bacteria were
  transformed and reintroduced to soil.- That was
  more difficult for a lower amount of the cells to
  establish themselves in the soil.
• The beads were counted by means of Burkners
  chamber inexactly, sometimes rubbish on the
  objective of the microscope was taken as beads.
• The flow cytometry was done at different time
  after the experiment was prepared. The beads are
  sensitive to light and the long waiting for the
  FACS could influence the results.
• There was lack of spreader in the lab, and I
  spread bacterial cells on the plates with one
  handmade. It was dropping with ethanol onto the
  medium, which could kill the bacteria.
  Consequently a fewer amount of the CFU could grow
  on the plates.
• The bacteria were observed during two weeks. A
  longer experiment could have other results.