the effect of pdms based microdevice channel width on plasmid dna transformation efficiency in e....
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PAPER
University of California, Berkeley, College o
Effect of PDMS-based Microdevice Channel Width on Plasmid DNA
Transformation Efficiency
Albert Peng,a Simrunn Girn,
a Regine Labog,
Submitted 9th December 2010
The effect of PDMS-based microdevice channel width on GFP plasmid transformation efficiency 5
in E. coli was studied in this project. Four different device designs consisting of 50 µm, 100 µm,
250 µm, and 500 µm channel widths were used in conjunction with s
soft lithography fabrication techniques to create PDMS microdevices. Multiple chemical
transformation trials using optimal macroscale heat shock parameters
devices, and data was collected from agar plate cultures and subsequently analyzed by the ImageJ 10
software package. Although we have successfully demonstrated chemical transformation in a
microscale environment, our data suggests that variability in transformation efficiency introduced
by experimental error is large enough such that any potential influence channel width may have on
transformation efficiency is masked.
Introduction 15
Plasmid DNA transformation is a key molecular biology
concept of introducing new functionality to existing bacteria
strains by importing desired DNA molecules into cells. DNA
transformation in E. coli is generally accomplished by
chemical and electrical means, and various studies have been 20
performed to maximize transformation efficiency for both
methods. While there are advantages and disadvantages to
both techniques, chemical transformation is cheaper and more
accessible than electroporation and is the mai
study. 25
Although heat shock chemical transformation is widely
used and accepted,6 it is relatively unclear how it functions.
During chemical transformation, it is theorized that
in a ����� and E. coli solution envelop the cell membrane
thus producing a net positive charge on the surface, attracting 30
the negatively charged plasmid DNA.1,3,4 A heat shock step
then opens pores on the cell surface and facilitates passage
through the cell membrane due to the close proximity of the
plasmid DNA to the cell. An ice incubation step is thought to
reduce the thermal motion of the DNA and allow further 35
binding to the cell membrane.1 Finally a warm
incubation in rich LB media allows the cells to recover from
the previous disturbances to cellular processes and promotes
survivability of the culture. In addition, this incubation step
could allow further uptake of plasmid into the cell as sort of a 40
second heat shock step.1
Traditional transformation optimization stud
always been done at macroscale.5 Applications such as
genomic and cDNA library construction typically require
transformation with low DNA copy, so it is necessary to find 45
parameters that maximize transformation efficiency.
transformation has been shown to be possible at microscale,
the influence of channel width on transformation efficiency
has never been studied. Since the exact mechanism of plasmid
DNA uptake in E. coli during chemical transformation is 50
unknown, it is important to study all the possible parameters
University of Calfornia, Berkeley
of Engineering 2010
based Microdevice Channel Width on Plasmid DNA
Efficiency in E. coli
Regine Labog,a and Yiqing Zhao
a
based microdevice channel width on GFP plasmid transformation efficiency
was studied in this project. Four different device designs consisting of 50 µm, 100 µm,
250 µm, and 500 µm channel widths were used in conjunction with standard photolithography and
soft lithography fabrication techniques to create PDMS microdevices. Multiple chemical
transformation trials using optimal macroscale heat shock parameters1 were performed with these
ate cultures and subsequently analyzed by the ImageJ
software package. Although we have successfully demonstrated chemical transformation in a
microscale environment, our data suggests that variability in transformation efficiency introduced
l error is large enough such that any potential influence channel width may have on
Plasmid DNA transformation is a key molecular biology
concept of introducing new functionality to existing bacteria
strains by importing desired DNA molecules into cells. DNA
is generally accomplished by
ans, and various studies have been
performed to maximize transformation efficiency for both
methods. While there are advantages and disadvantages to
both techniques, chemical transformation is cheaper and more
accessible than electroporation and is the main focus of this
Although heat shock chemical transformation is widely
it is relatively unclear how it functions.
During chemical transformation, it is theorized that ���� ions
solution envelop the cell membrane
thus producing a net positive charge on the surface, attracting
A heat shock step
then opens pores on the cell surface and facilitates passage
close proximity of the
plasmid DNA to the cell. An ice incubation step is thought to
reduce the thermal motion of the DNA and allow further
Finally a warm 37�
incubation in rich LB media allows the cells to recover from
previous disturbances to cellular processes and promotes
survivability of the culture. In addition, this incubation step
could allow further uptake of plasmid into the cell as sort of a
Traditional transformation optimization studies have almost
Applications such as
genomic and cDNA library construction typically require
transformation with low DNA copy, so it is necessary to find
parameters that maximize transformation efficiency.1 While
tion has been shown to be possible at microscale,2
the influence of channel width on transformation efficiency
has never been studied. Since the exact mechanism of plasmid
during chemical transformation is
study all the possible parameters
that may affect transformation efficiency, such as the channel
size in which transformation occurs. For our study a standard 60
chemical transformation procedure is used with chemically
competent E. coli cells and GFP. While
are specific to the strain of E. coli and GFP plasmid used in
these experiments, the results gathered could be useful for
future work with other strains of bacteria and plasmids.65
Materials and Methods 60
Device Fabrication
The design of our 50 µm, 100 µm, 250 µm, and 500 µm 75
channel width devices was drawn using AutoCAD and sent to
an external manufacturer to produce mylar masks for
photolithography (Figure 1). When designing our device we
needed to incorporate three functions: an inlet fo
GFP loading, a heat shock chamber with the required channel 80
dimensions, and an outlet to collect the pool. S
chosen for the transformation chamber to maximize volume
while maintaining the designated channel widths. In addition,
the devices were designed to have identical volumes of 3.4 µL
each in order to make channel width the only varying factor 85
between devices. This resulted in fewer S
channel width devices compared with the smaller channel
width devices.
Fig. 1 A 50 µm channel width device design
University of Calfornia, Berkeley | BioE 121L
Bioengineering | 1
based Microdevice Channel Width on Plasmid DNA
that may affect transformation efficiency, such as the channel
size in which transformation occurs. For our study a standard
chemical transformation procedure is used with chemically
cells and GFP. While the results we obtain
and GFP plasmid used in
these experiments, the results gathered could be useful for
future work with other strains of bacteria and plasmids.
our 50 µm, 100 µm, 250 µm, and 500 µm
channel width devices was drawn using AutoCAD and sent to
an external manufacturer to produce mylar masks for
photolithography (Figure 1). When designing our device we
needed to incorporate three functions: an inlet for E. coli and
GFP loading, a heat shock chamber with the required channel
dimensions, and an outlet to collect the pool. S-curves were
chosen for the transformation chamber to maximize volume
while maintaining the designated channel widths. In addition,
e devices were designed to have identical volumes of 3.4 µL
each in order to make channel width the only varying factor
between devices. This resulted in fewer S-curves for the larger
channel width devices compared with the smaller channel
A 50 µm channel width device design
2 | Bioengineering
Fig. 3 Experimental procedure used for transformation
A standard contact photolithography procedure with
negative SU-8 2035 photoresist was then done using the
previously created mylar mask and a 4” silicon wafer. Contact
photolithography was used to keep production costs low while 5
maintaining high resolution of features. Spin coating
parameters were chosen to create a single final photore
height of 50 µm for all devices (Figure 2), and the appropriate
UV exposure times were chosen to accommodate the contact
aligner measured UV intensity, which is variable with the age 10
and quality of the UV bulb. After the necessary heat,
developing and cleaning treatments, the wafer is then placed
into a vacuum chamber for silanizing. Silanizing the wafer
allows cured PDMS to be more readily removable from the
surface of the wafer and is essential for soft lithography.15
Fig. 2 Close-up channel dimensions of a 50 µm device
The silanized wafer with all the device features was then
used as a mold for PDMS soft lithography. A 10:1 ratio of
base to curing agent was weighed out and thoroughly mixed,
resulting in a 50 g base: 5 g curing agent mixture. This 20
solution was degassed by vacuum and then poured over the
clean wafer, and allowed to cure overnight on a
plate. Once cured, the PDMS layer was carefully peeled off
the wafer. Individual devices were cut out from the PDMS
sheet and 1 mm holes were punched at the inlet and outlet. 25
Devices and microscope glass slides were then both tape
cleaned and chemically cleaned by acetone, IPA and DI water,
and subjected to UVO treatment to modify the surface
chemistry to facilitate bonding. The PDMS devices and sli
were then bonded together to produce a final useable 30
microdevice.
Experimental Procedure
After our devices were created, we began running chemical
transformation trials (Figure 3). 20 µL of chemically
University of California, Berkeley, College
Experimental procedure used for transformation
A standard contact photolithography procedure with
was then done using the
previously created mylar mask and a 4” silicon wafer. Contact
photolithography was used to keep production costs low while
maintaining high resolution of features. Spin coating
parameters were chosen to create a single final photoresist
, and the appropriate
UV exposure times were chosen to accommodate the contact
aligner measured UV intensity, which is variable with the age
and quality of the UV bulb. After the necessary heat,
cleaning treatments, the wafer is then placed
into a vacuum chamber for silanizing. Silanizing the wafer
allows cured PDMS to be more readily removable from the
surface of the wafer and is essential for soft lithography.
s of a 50 µm device
The silanized wafer with all the device features was then
used as a mold for PDMS soft lithography. A 10:1 ratio of
base to curing agent was weighed out and thoroughly mixed,
resulting in a 50 g base: 5 g curing agent mixture. This
ution was degassed by vacuum and then poured over the
clean wafer, and allowed to cure overnight on a 55� hot
plate. Once cured, the PDMS layer was carefully peeled off
the wafer. Individual devices were cut out from the PDMS
hed at the inlet and outlet.
Devices and microscope glass slides were then both tape
cleaned and chemically cleaned by acetone, IPA and DI water,
and subjected to UVO treatment to modify the surface
chemistry to facilitate bonding. The PDMS devices and slides
were then bonded together to produce a final useable
After our devices were created, we began running chemical
. 20 µL of chemically
competent E. coli was thawed on ice for 30 minutes50
which 2 µL GFP plasmid obtained through miniprep was
added and mixed by gentle tapping. After incubating on ice
for 30 minutes, 5 µL of this solution was vacuum loaded into
each device. Vacuum loading was done on ice until the entire
device was loaded. The devices were then placed on a hot 55
plate set at 42� for 30 seconds as monitored by a
thermocouple, and then placed on ice for 2 minutes. A syringe
was placed at the inlet of each device and used air pressure to
evacuate the device of bacteria, and pool was collected at the
outlet and incubated in 50 µL LB-Amp media for 1 hour. The 60
appropriate dilutions were made and the culture was plated on
agar-Amp plates and allowed to grow overnight. Pictures were
taken the following day and colonies were counte
ImageJ software.
Results and Discussion
Prior to performing any transformation experiments, we
attempted to vacuum load our devices with
that vacuum loading is a viable technique to introduce
solutions into microdevices. A picture using phase contrast 65
microscopy was taken demonstrating successful vacuum
loading (Figure 4). A total of 28 devices were then
successfully used in transformation runs and had enough
colonies on their corresponding plates to be counted.
Transformation efficiency is determined quantitatively as the 70
total colony count on each plate, with higher counts equating
to higher transformation efficiencies.
Fig. 4 Phase microscopy image of E. coli loaded in a 50 µm device
The first set of experiments we tried to perform included: 3 65
x 50 µm, 3 x 100 µm, 3 x 250 µm, and 3 x 500 µm channel
e of Engineering 2010
was thawed on ice for 30 minutes, after
which 2 µL GFP plasmid obtained through miniprep was
added and mixed by gentle tapping. After incubating on ice
for 30 minutes, 5 µL of this solution was vacuum loaded into
each device. Vacuum loading was done on ice until the entire
ded. The devices were then placed on a hot
for 30 seconds as monitored by a
thermocouple, and then placed on ice for 2 minutes. A syringe
was placed at the inlet of each device and used air pressure to
pool was collected at the
Amp media for 1 hour. The
appropriate dilutions were made and the culture was plated on
Amp plates and allowed to grow overnight. Pictures were
taken the following day and colonies were counted by the
Prior to performing any transformation experiments, we
attempted to vacuum load our devices with E. coli to show
that vacuum loading is a viable technique to introduce
solutions into microdevices. A picture using phase contrast
microscopy was taken demonstrating successful vacuum
). A total of 28 devices were then
ormation runs and had enough
colonies on their corresponding plates to be counted.
Transformation efficiency is determined quantitatively as the
total colony count on each plate, with higher counts equating
loaded in a 50 µm device
The first set of experiments we tried to perform included: 3
x 50 µm, 3 x 100 µm, 3 x 250 µm, and 3 x 500 µm channel
University of California, Berkeley, College of Engineering 2010 Bioengineering | 3
width devices. We wanted to do three runs of each channel
width in order to average the data from all three and generate
more reliable results. Out of these runs only: 1 x 50 µm, 2 x
100 µm, 2 x 250 µm, and 2 x 500 µm devices were able to
generate any measurable data (Figure 5). Some devices were 5
not able to load completely in a reasonable amount of time
and had to be discarded. In addition, our initial batch of 50
µm channel width devices were not bonded very well to the
glass slides, and popped off when we attempted to use air
pressure to empty the device of E. coli. 10
Fig. 5 Colony count data gathered from the first set of transformations
The data generated using these devices shows that colony
count decreases as channel width increases, since the 100 µm
devices had an average of 900 colonies while the 250 µm and
500 µm devices had an average of 800 and 600 colonies, 15
respectively. This suggests that smaller channel widths
coincide with higher transformation efficiency. However, due
to the low number of successful trials for each device, we
decided to do more transformations in order to confirm our
findings. 20
For the second set of transformation runs we wanted to see
if there was a legitimate difference in transformation
efficiency between smaller and larger channel widths. Since
our data from the first set of runs was relatively sparse due to
experimental error, we decided that we should only focus on 25
two channel widths and make sure that we believe our results.
We ran trials with 4 x 100 µm and 4 x 250 µm devices in the
same fashion as the first set of runs and gathered the colony
data (Figure 6). The data shows that the average colony
number from the 100 µm and 200 µm devices are 1000 and 30
1200 respectively, which is in direct contradiction of the trend
observed in the first set of runs. This new data suggests that
there is relatively little difference between the transformation
efficiency of the 100 µm and 200 µm channel width devices.
Judging from the extreme variability of the individual trials in 35
the second run (1500 colonies in trial 1 and 400 colonies in
trial 4 of the 250 µm set), it appeared that our experimental
methods were still unable to generate consistent results.
Fig. 6 Colony count data gathered from the second set of 40
transformations
In a last attempt to obtain coherent data, we performed a
third and final set of transformations. For these trials we used:
4 x 50 µm, 4 x 100 µm, 4 x 250 µm, and 4 x 500 µm channel
width devices. The 50 µm and 500 µm channel widths 45
performed the best at an average of 250 and 300 colonies
respectively, while the 100 µm and 250 µm channel widths
had 100 and 180 colonies each (Figure 7). Unfortunately this
data still does not agree with our previous runs, and we must
end this project with inconclusive results. 50
Fig. 7 Colony count data gathered from the third set of transformations
Different dilution factors were used for each run prior to
plating, so the colony counts between runs are very different
in our data. However, only the relative difference in colony
counts between individual devices within runs matters, and 55
from the three sets of runs that we performed, there was no
clear trend indicating the effect of channel width on
transformation efficiency. One reason for this could be due to
experimental error. The transformation has been shown to be
very robust even at a 10x dilution factor across all device 60
widths, indicating that slight errors in experimental procedure
such as inexact transfer volumes can result in high variability
4 | Bioengineering University of California, Berkeley, College of Engineering 2010
in colony counts. For example trial 1 of the 50 µm device in
run 2 had 600 colonies while trial 2 of the same device in the
same run had only 100 colonies, even though they both
experienced a 10x dilution before plating. Any effect that
channel width may have had on these colony counts would 5
have been masked by the extreme variability introduced by
experimental error.
Conclusion
Colony count data collected from three separate runs of
multiple transformation trials did not reveal a clear trend 10
between microdevice channel width and transformation
efficiency. Transformation was robust amongst all devices
even at high dilution factors, suggesting that the effect of
channel width is small compared to the inherently high
transformation efficiency. Variability in colony counts 15
introduced due to experimental error also contributed to the
inability to generate consistent data. Due to limitations in our
original device design and time constraints we must end this
project with inconclusive results. Future work can be done to
improve both device design and the experimental procedure 20
by performing everything on-chip, to minimize compounding
errors due to inexact off-chip activities such as E. coli
evacuation from the device, dilution factors, and inconsistent
plating technique.
References 25
a College of Engineering, Bioengineering Department, University of
California, Berkeley,CA, 94704, USA.
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