Transcript Slide 1

Assessment of key parameters to achieve bacteria high transformability
in E. coli and A. tumefaciens
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Zach Regelin , Suzette Smedes , Kan Wang , Diane Luth , Ksenija Markovic , Helene Eckert , François Torney
1University
of Northern Iowa, 2 Melcher-Dallas Community School District, 3 Dept. of Agronomy, Iowa State University
June 13, 2006 – August 2, 2006
Project Description
Results and Discussion
Monitoring the Growth Curve
Growth Curve
Materials and Methods
The growth curves for different E. coli and A. tumefaciens
strains were determined by measuring optical densities
(ODs) and plating diluted culture samples. Growth curves
were determined for different inoculums and different media
(Figure 1).
Electrocompotent cells were prepared by a series of washing
and pelleting in 10% glycerol solution, then flash-frozen in
liquid nitrogen and stored at -80ºC. To genetically transform
the cells by electroporation, they were mixed with plasmids
and a 2.5 kV current was passed through the mix for 5.9 ms.
Cultures were plated on selective media to determine
transformation efficiencies.
Species
Strain
Inoculum
Medium
0.5
Dh5a Plate
0.4
ccdBR Plate
OD
Electroporation is a genetic transformation process in which a strong
electric field is used to introduce DNA into cells. Bacterial transformation
is important for molecular cloning, gene construct selection, and plasmid
production and storage. To optimize the electroporation process, several
key parameters were investigated, including: the strain of the bacteria, the
inoculum source, and the media type. Bacteria were grown under
different conditions and their growth was monitored. Bacteria grown to
different phases of growth under various conditions were used for
electroporation and transformation efficiencies were determined.
0.6
0.3
Dh5a glycerol
0.2
Using E. coli strains Dh5 and ccdBR, it was determined
that the strain, the media type, and the amount of
inoculum had a great effect on the growth but the source
of the inoculum had little effect. (data not shown)
ccdBR glycerol
0.1
0
0
50
100
150
Time (min)
Figure 2
For A. tumefaciens, the strain and the media type
had a great effect on the growth and the source of
the inoculum had a great effect for one strain’s
growth but little effect for the other’s (Figure 3).
Electroporation
Figure 3
E. coli in LB
E. coli in 2xTY
A. tumefaciens
in YEP
•CcdBR showed higher
efficiencies than Dh5.
•The higher OD cultures had
higher efficiencies.
•The media did not have a
significant effect.
Species
Strain
Inoculum
Medium
Figure 1
The standard deviations are
too large for these results to
be conclusive.
Figure 4
Acknowledgement
I would like to thank the National Science Foundation REU program for funding this project.