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 (FACS-Calibur) 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:

109,0 concbeads

cellscells Bead

Events

EventsNumber

Methods: Nycodenz

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

Soil mixture

Nycodenz

Aquatic phase

Bacterial cells

Heavy soil particals

Before centrifuging After centrifuging

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.

Monitoring of Quantity of the Isolates and A6G Cells

-1,00

-0,50

0,00

0,50

1,00

1,50

2,00

2,50

3,00

20-feb 24-feb 27-feb 01-mar 03-mar 05-mar

Date

Num

ber

of C

ells

* 1

0^8

IA

IK

A6G

Number of the Isolates and A6G Cells

-2

-1

0

1

2

3

4

1 2 3 4 5 6

Observations

Num

ber

of th

e C

ells

x 1

0^8

Isolat&Amp Isolat&Kanam A6G

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.

The Isolate Grown on Ampicillin

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Nu

mb

er o

f th

e C

ells

* 1

0^8

0

1

2

3

4

5

6

CF

U *

10^

4

Number of the cells, *10 8̂ CFU * 10^4

Number of thecells, *10 8̂

0,975 1,223 2,164 2,004 0,843 2,424

CFU * 10^4 0,5 5,5 0,125 0,02 0,015

20-feb 24-feb 27-feb 01-mar 03-mar 05-mar

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.

The Isolate Grown on Kanamycin

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

Nu

mb

er o

f the

Cel

ls *

10^

8

0,0

0,2

0,4

0,6

0,8

1,0

1,2

CF

U *

10^

4

Number of the cells, *10 8̂ CFU, *10^4

Number of thecells, *10 8̂

1,023 1,624 3,322 4,731 1,524 3,553

CFU, *10^4 1 0,5 0,4 0,125 0,015

20-feb 24-feb 27-feb 01-mar 03-mar 05-mar

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.

A6G

0

1

2

3

4

5

6

Nu

mb

er o

f th

e C

ells

* 1

0^8

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

CF

U *

10^

5

Number of the cells, *10 8̂ CFU, *10^5

Number of the cells,*10 8̂

1,09 1,015 2,139 1,678 1,163 5,414

CFU, *10^5 0,35 1,25 0,1605 0,2075 0,5725

20-feb 24-feb 27-feb 01-mar 03-mar 05-mar

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.