The document summarizes an experiment that studied the effect of microdevice channel width on plasmid DNA transformation efficiency in E. coli. Four PDMS microdevices with channel widths of 50 μm, 100 μm, 250 μm, and 500 μm were fabricated and used in chemical transformation trials. While transformation was successful, the data showed high variability and no clear relationship between channel width and transformation efficiency. Future work is needed to improve device design and experimental methods to better study the potential influence of channel width.

1. PAPER University of Calfornia, Berkeley | BioE 121L
Effect of PDMS-based Microdevice Channel Width on Plasmid DNA
based
Transformation Efficiency in E. coli
Albert Peng,a Simrunn Girn,a Regine Labog,a and Yiqing Zhaoa
Submitted 9th December 2010
5 The effect of PDMS-based microdevice channel width on GFP plasmid transformation efficiency
based
in E. coli was studied in this project. Four different device designs consisting of 50 µ m, 100 µ m,
250 µ m, and 500 µ m channel widths were used in conjunction with s standard photolithography and
soft lithography fabrication techniques to create PDMS microdevices. Multiple chemical
transformation trials using optimal macroscale heat shock parameters 1 were performed with these
10 devices, and data was collected from agar plate cultures and subsequently analyzed by the ImageJ
ate
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
l
transformation efficiency is masked.
that may affect transformation efficiency, such as the channel
15 Introduction 60 size in which transformation occurs. For our study a standard
chemical transformation procedure is used with chemically
Plasmid DNA transformation is a key molecular biology competent E. coli cells and GFP. While the results we obtain
concept of introducing new functionality to existing bacteria
are specific to the strain of E. coli and GFP plasmid used in
strains by importing desired DNA molecules into cells. DNA
these experiments, the results gathered could be useful for
transformation in E. coli is generally accomplished by
65 future work with other strains of bacteria and plasmids.
20 chemical and electrical means, and various studies have been
ans,
performed to maximize transformation efficiency for both
60 Materials and Methods
methods. While there are advantages and disadvantages to
both techniques, chemical transformation is cheaper and more Device Fabrication
accessible than electroporation and is the mai focus of this
main
75 The design of our 50 µ m, 100 µ m, 250 µ m, and 500 µ m
25 study.
channel width devices was drawn using AutoCAD and sent to
Although heat shock chemical transformation is widely an external manufacturer to produce mylar masks for
used and accepted,6 it is relatively unclear how it functions. photolithography (Figure 1). When designing our device we
During chemical transformation, it is theorized that ions needed to incorporate three functions: an inlet for E. coli and
fo
in a and E. coli solution envelop the cell membrane
80 GFP loading, a heat shock chamber with the required channel
30 thus producing a net positive charge on the surface, attracting
dimensions, and an outlet to collect the pool. S-curves were
S
the negatively charged plasmid DNA. 1,3,4 A heat shock step
chosen for the transformation chamber to maximize volume
then opens pores on the cell surface and facilitates passage while maintaining the designated channel widths. In addition,
through the cell membrane due to the close proximity of the the devices were designed to have identical volumes of 3.4 µ L
e
plasmid DNA to the cell. An ice incubation step is thought to 85 each in order to make channel width the only varying factor
35 reduce the thermal motion of the DNA and allow further
between devices. This resulted in fewer S-curves for the larger
S
binding to the cell membrane.1 Finally a warm
channel width devices compared with the smaller channel
incubation in rich LB media allows the cells to recover from
width devices.
the previous disturbances to cellular processes and promotes
survivability of the culture. In addition, this incubation step
40 could allow further uptake of plasmid into the cell as sort of a
second heat shock step. 1
Traditional transformation optimization stud have almost
studies
always been done at macroscale.5 Applications such as
genomic and cDNA library construction typically require
45 transformation with low DNA copy, so it is necessary to find
parameters that maximize transformation efficiency. 1 While
transformation has been shown to be possible at microscale,2
tion
the influence of channel width on transformation efficiency
has never been studied. Since the exact mechanism of plasmid
50 DNA uptake in E. coli during chemical transformation is Fig. 1 A 50 µm channel width device design
unknown, it is important to study all the possible parameters
University of California, Berkeley, College of Engineering 2010
o Bioengineering | 1

2. Fig. 3 Experimental procedure used for transformation
A standard contact photolithography procedure with 50 competent E. coli was thawed on ice for 30 minutes, after
minutes
negative SU-8 2035 photoresist was then done using the which 2 µ L GFP plasmid obtained through miniprep was
previously created mylar mask and a 4” silicon wafer. Contact added and mixed by gentle tapping. After incubating on ice
5 photolithography was used to keep production costs low while for 30 minutes, 5 µ L of this solution was vacuum loaded into
maintaining high resolution of features. Spin coating each device. Vacuum loading was done on ice until the entire
parameters were chosen to create a single final photorephotoresist 55 device was loaded. The devices were then placed on a hot
ded.
height of 50 µ m for all devices (Figure 2), and the appropriate
, plate set at for 30 seconds as monitored by a
UV exposure times were chosen to accommodate the contact thermocouple, and then placed on ice for 2 minutes. A syringe
10 aligner measured UV intensity, which is variable with the age was placed at the inlet of each device and used air pressure to
and quality of the UV bulb. After the necessary heat, evacuate the device of bacteria, and pool was collected at the
developing and cleaning treatments, the wafer is then placed 60 outlet and incubated in 50 µ L LB-Amp media for 1 hour. The
Amp
into a vacuum chamber for silanizing. Silanizing the wafer appropriate dilutions were made and the culture was plated on
allows cured PDMS to be more readily removable from the agar-Amp plates and allowed to grow overnight. Pictures were
Amp
15 surface of the wafer and is essential for soft lithography. taken the following day and colonies were counted by the
counte
ImageJ software.
Results and Discussion
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
65 solutions into microdevices. A picture using phase contrast
microscopy was taken demonstrating successful vacuum
loading (Figure 4). A total of 28 devices were then
).
successfully used in transformation runs and had enough
ormation
colonies on their corresponding plates to be counted.
70 Transformation efficiency is determined quantitatively as the
total colony count on each plate, with higher counts equating
Fig. 2 Close-up channel dimensions of a 50 µm device
s
to higher transformation efficiencies.
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,
20 resulting in a 50 g base: 5 g curing agent mixture. This
solution was degassed by vacuum and then poured over the
ution
clean wafer, and allowed to cure overnight on a hot
plate. Once cured, the PDMS layer was carefully peeled off
the wafer. Individual devices were cut out from the PDMS
25 sheet and 1 mm holes were punched at the inlet and outlet.
hed
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
slides
30 were then bonded together to produce a final useable
microdevice.
Experimental Procedure Fig. 4 Phase microscopy image of E. coli loaded in a 50 µm device
After our devices were created, we began running chemical 65 The first set of experiments we tried to perform included: 3
transformation trials (Figure 3). 20 µ L of chemically
. x 50 µ m, 3 x 100 µ m, 3 x 250 µ m, and 3 x 500 µ m channel
2 | Bioengineering University of California, Berkeley, College of Engineering 2010
e

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 µ m, 2 x
100 µ m, 2 x 250 µ m, and 2 x 500 µ m devices were able to
5 generate any measurable data (Figure 5). Some devices were
not able to load completely in a reasonable amount of time
and had to be discarded. In addition, our initial batch of 50
µ m channel width devices were not bonded very well to the
glass slides, and popped off when we attempted to use air
10 pressure to empty the device of E. coli.
40 Fig. 6 Colony count data gathered from the second set of
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 µ m, 4 x 100 µ m, 4 x 250 µ m, and 4 x 500 µ m channel
45 width devices. The 50 µ m and 500 µ m channel widths
performed the best at an average of 250 and 300 colonies
respectively, while the 100 µm and 250 µ m channel widths
had 100 and 180 colonies each (Figure 7). Unfortunately this
data still does not agree with our previous runs, and we must
50 end this project with inconclusive results.
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 µ m
devices had an average of 900 colonies while the 250 µ m and
15 500 µ m devices had an average of 800 and 600 colonies,
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
20 findings.
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
25 experimental error, we decided that we should only focus on
two channel widths and make sure that we believe our results.
We ran trials with 4 x 100 µ m and 4 x 250 µ m devices in the Fig. 7 Colony count data gathered from the third set of transformations
same fashion as the first set of runs and gathered the colony
data (Figure 6). The data shows that the average colony Different dilution factors were used for each run prior to
30 number from the 100 µ m and 200 µ m devices are 1000 and plating, so the colony counts between runs are very different
1200 respectively, which is in direct contradiction of the trend in our data. However, only the relative difference in colony
observed in the first set of runs. This new data suggests that 55 counts between individual devices within runs matters, and
there is relatively little difference between the transformation from the three sets of runs that we performed, there was no
efficiency of the 100 µ m and 200 µ m channel width devices. clear trend indicating the effect of channel width on
35 Judging from the extreme variability of the individual trials in transformation efficiency. One reason for this could be due to
the second run (1500 colonies in trial 1 and 400 colonies in experimental error. The transformation has been shown to be
trial 4 of the 250 µ m set), it appeared that our experimental 60 very robust even at a 10x dilution factor across all device
methods were still unable to generate consistent results. widths, indicating that slight errors in experimental procedure
such as inexact transfer volumes can result in high variability
University of California, Berkeley, College of Engineering 2010 Bioengineering | 3

4. in colony counts. For example trial 1 of the 50 µ m 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
5 channel width may have had on these colony counts would
have been masked by the extreme variability introduced by
experimental error.
Conclusion
Colony count data collected from three separate runs of
10 multiple transformation trials did not reveal a clear trend
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
15 transformation efficiency. Variability in colony counts
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
20 improve both device design and the experimental procedure
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.
25 References
a
College of Engineering, Bioengineering Department, University of
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4 | Bioengineering University of California, Berkeley, College of Engineering 2010