Transcript Isolation of Microorganisms and Tagging with Marker Genes

Isolation of Microorganisms and
Tagging with Marker Genes
A Physiological Study
by Ingvor Irene Zetterlund
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.
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 DH5α as a control of the
transformation;
- GFP tagged Arthrobacter chlorophenolicus
A6G as a control microorganism.
The soil of the Gardenia Used in the
Project
Gardenia Before its Withering
Infected Gardenia
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.
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.
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.
Methods: Agaros Gel Electrophoresis
 The quality of the plasmid DNA was analyzed
by agaros gel electrophoresis after it was cut
by restriction enzimes.
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 DH5α - LB medium without antibiotics
at 28oC.
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.
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 (FACSCalibur) and colony forming units CFU.
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:
Number
cells

Events
Events
cells
beads
 Bead
conc
 0 ,9  10
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
Results
Monitoring of Quantity of the Isolates and A6G
Cells
4
3,00
3
2
1
0
-1
1
2
3
4
5
-2
Observations
Isolat&Amp
Isolat&Kanam
6
Number of Cells * 10^8
Number of the Cells x 10^8
Number of the Isolates and A6G Cells
2,50
2,00
1,50
IA
1,00
IK
0,50
A6G
0,00
-0,5020-feb
24-feb
27-feb
01-mar
03-mar
05-mar
-1,00
A6G
Date
 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.
Results: The Isolate Grown on Ampicillin
 Have demonstrated a faint
3,0
6
2,5
5
2,0
4
1,5
3
1,0
2
0,5
1
0,0
20-feb 24-feb 27-feb 01-mar 03-mar 05-mar
Number of the 0,975
cells, *10^8
CFU * 10^4
1,223
2,164
2,004
0,843
2,424
0,5
5,5
0,125
0,02
0,015
Number of the cells, *10^8
CFU * 10^4
0
CFU * 10^4
The Isolate Grown on Ampicillin
Number of the Cells * 10^8
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.
Results: The Isolate Grown on Kanamycin
 The number of the tagged
The Isolate Grown on Kanamycin
5,0
1,2
4,5
1,0
4,0
3,5
0,8
3,0
0,6
2,5
2,0
0,4
1,5
1,0
0,2
0,5
0,0
20-feb 24-feb 27-feb 01-mar 03-mar 05-mar
Number of the 1,023
cells, *10^8
CFU, *10^4
1,624
3,322
4,731
1,524
3,553
1
0,5
0,4
0,125
0,015
Number of the cells, *10^8
0,0
CFU, *10^4
CFU * 10^4
Number of the Cells * 10^8
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.
Results: A6G
 A6G showed a good
A6G
6
1,4
5
1,2
0,8
3
0,6
2
0,4
1
0
Number of the cells,
*10^8
CFU, *10^5
1,0
4
0,2
20-feb 24-feb 27-feb 01-mar 03-mar 05-mar
1,09
1,015
2,139
1,678
0,35
1,25
0,1605 0,2075 0,5725
Number of the cells, *10^8
1,163
5,414
CFU, *10^5
0,0
CFU * 10^5
Number of the Cells * 10^8
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.
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.
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.
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 Burkner’s 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.