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8/3/2019 The Effect of PDMS-Based Micro Device Channel Width on Plasmid DNA Transformation Efficiency in E. Coli
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PAPER
University of California, Berkeley, College o
Effect of PDMS-based Mi
Transformation Efficienc
Albert Peng,a
Simrunn Girn,a
Regine
Submitted 9th December 2010
The effect of PDMS-based microdevice chan5
in E. coli was studied in this project. Four dif
250 µm, and 500 µm channel widths were use
soft lithography fabrication techniques to cre
transformation trials using optimal macroscal
devices, and data was collected from agar pla10
software package. Although we have successf
microscale environment, our data suggests th
by experimental error is large enough such th
transformation efficiency is masked.
Introduction15
Plasmid DNA transformation is a key m
concept of introducing new functionality to
strains by importing desired DNA molecules
transformation in E. coli is generally a
chemical and electrical means, and various st20
performed to maximize transformation effi
methods. While there are advantages and
both techniques, chemical transformation is c
accessible than electroporation and is the m
study.25
Although heat shock chemical transform
used and accepted,6 it is relatively unclear h
During chemical transformation, it is theorizein a and E. coli solution envelop the
thus producing a net positive charge on the s30
the negatively charged plasmid DNA.1,3,4 A
then opens pores on the cell surface and fa
through the cell membrane due to the close
plasmid DNA to the cell. An ice incubation s
reduce the thermal motion of the DNA an35
binding to the cell membrane.1 Finally
incubation in rich LB media allows the cells
the previous disturbances to cellular process
survivability of the culture. In addition, this
could allow further uptake of plasmid into the40
second heat shock step.1
Traditional transformation optimization stu
always been done at macroscale.5 Appli
genomic and cDNA library construction t
transformation with low DNA copy, so it is45
parameters that maximize transformation ef
transformation has been shown to be possibl
the influence of channel width on transfor
has never been studied. Since the exact mech
DNA uptake in E. coli during chemical t50
unknown, it is important to study all the pos
University of Calfornia, Be
f Engineering 2010
crodevice Channel Width on Plas
in E. coli
Labog,a
and Yiqing Zhaoa
el width on GFP plasmid transformation efficiency
ferent device designs consisting of 50 µ m, 100 µ m,
d in conjunction with standard photolithography and
te PDMS microdevices. Multiple chemical
e heat shock parameters1 were performed with these
te cultures and subsequently analyzed by the ImageJ
ully demonstrated chemical transformation in a
t variability in transformation efficiency introduced
at any potential influence channel width may have on
lecular biology
xisting bacteria
into cells. DNA
complished by
udies have been
ciency for both
isadvantages to
heaper and more
in focus of this
ation is widely
ow it functions.
d that
ionscell membrane
rface, attracting
heat shock step
cilitates passage
proximity of the
tep is thought to
d allow further
a warm
to recover from
s and promotes
incubation step
cell as sort of a
dies have almost
ations such as
ypically require
ecessary to find
ficiency.1 While
at microscale,2
ation efficiency
nism of plasmid
ansformation is
sible parameters
that may affect transformation efficie
size in which transformation occurs.60
chemical transformation procedure i
competent E. coli cells and GFP. Whi
are specific to the strain of E. coli a
these experiments, the results gather
future work with other strains of bacte65
Materials and Methods60
Device Fabrication
The design of our 50 µm, 100 µm,75
channel width devices was drawn usin
an external manufacturer to prod
photolithography (Figure 1). When d
needed to incorporate three functions:
GFP loading, a heat shock chamber w80
dimensions, and an outlet to collect
chosen for the transformation chamb
while maintaining the designated cha
the devices were designed to have ide
each in order to make channel width85
between devices. This resulted in fewe
channel width devices compared wi
width devices.
Fig. 1 A 50 µm channel width
rkeley | BioE 121L
Bioengineering | 1
mid DNA
cy, such as the channel
or our study a standards used with chemically
ile the results we obtain
d GFP plasmid used in
ed could be useful for
ria and plasmids.
250 µm, and 500 µm
g AutoCAD and sent to
uce mylar masks for
esigning our device we
an inlet for E. coli and
ith the required channel
the pool. S-curves were
er to maximize volume
nel widths. In addition,
tical volumes of 3.4 µL
the only varying factor
r S-curves for the larger
th the smaller channel
device design
8/3/2019 The Effect of PDMS-Based Micro Device Channel Width on Plasmid DNA Transformation Efficiency in E. Coli
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2 | Bioengineering
Fig. 3 E
A standard contact photolithography pr
negative SU-8 2035 photoresist was then d
previously created mylar mask and a 4” silicon
photolithography was used to keep production c5
maintaining high resolution of features.
parameters were chosen to create a single fin
height of 50 µm for all devices (Figure 2), and t
UV exposure times were chosen to accommod
aligner measured UV intensity, which is variabl10
and quality of the UV bulb. After the n
developing and cleaning treatments, the wafer is
into a vacuum chamber for silanizing. Silaniz
allows cured PDMS to be more readily remov
surface of the wafer and is essential for soft lith15
Fig. 2 Close-up channel dimensions of a 50 µm
The silanized wafer with all the device feat
used as a mold for PDMS soft lithography. A
base to curing agent was weighed out and thor
resulting in a 50 g base: 5 g curing agent20
solution was degassed by vacuum and then p
clean wafer, and allowed to cure overnight
plate. Once cured, the PDMS layer was carefu
the wafer. Individual devices were cut out fr
sheet and 1 mm holes were punched at the in25
Devices and microscope glass slides were t
cleaned and chemically cleaned by acetone, IPA
and subjected to UVO treatment to modif
chemistry to facilitate bonding. The PDMS devi
were then bonded together to produce a30
microdevice.
Experimental Procedure
After our devices were created, we began run
transformation trials (Figure 3). 20 µL
University of California, Berkeley, College
perimental procedure used for transformation
cedure with
ne using the
afer. Contact
osts low while
Spin coating
al photoresist
he appropriate
te the contact
e with the age
cessary heat,
then placed
ing the wafer
able from the
graphy.
device
ures was then
10:1 ratio of
ughly mixed,
mixture. This
ured over the
n a hot
lly peeled off
m the PDMS
let and outlet.
en both tape
and DI water,
the surface
ces and slides
final useable
ning chemical
f chemically
competent E. coli was thawed on ice50
which 2 µL GFP plasmid obtained t
added and mixed by gentle tapping. A
for 30 minutes, 5 µL of this solution w
each device. Vacuum loading was done
device was loaded. The devices were55
plate set at for 30 seconds
thermocouple, and then placed on ice fo
was placed at the inlet of each device an
evacuate the device of bacteria, and po
outlet and incubated in 50 µL LB-Amp60
appropriate dilutions were made and the
agar-Amp plates and allowed to grow ov
taken the following day and colonies
ImageJ software.
Results and Discussion
Prior to performing any transformati
attempted to vacuum load our devices
that vacuum loading is a viable te
solutions into microdevices. A picture65
microscopy was taken demonstrating
loading (Figure 4). A total of 28
successfully used in transformation rcolonies on their corresponding pl
Transformation efficiency is determined70
total colony count on each plate, with h
to higher transformation efficiencies.
Fig. 4 Phase microscopy image of E. coli loa
The first set of experiments we tried t65
x 50 µm, 3 x 100 µm, 3 x 250 µm, and
of Engineering 2010
for 30 minutes, after
rough miniprep was
ter incubating on ice
s vacuum loaded into
on ice until the entire
then placed on a hot
as monitored by a
2 minutes. A syringe
d used air pressure to
l was collected at the
edia for 1 hour. The
culture was plated on
ernight. Pictures were
were counted by the
on experiments, we
with E. coli to show
hnique to introduce
using phase contrast
successful vacuum
devices were then
ns and had enoughtes to be counted.
quantitatively as the
igher counts equating
ded in a 50 µm device
o perform included: 3
3 x 500 µm channel
8/3/2019 The Effect of PDMS-Based Micro Device Channel Width on Plasmid DNA Transformation Efficiency in E. Coli
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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 were5
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 theglass 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 widthscoincide 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 on25
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 colonynumber from the 100 µm and 200 µm devices are 1000 and30
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 in35
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 widths45
performed the best at an average of 250 and 300 coloniesrespectively, 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 toplating, 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, and55
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 device60
widths, indicating that slight errors in experimental procedure
such as inexact transfer volumes can result in high variability
8/3/2019 The Effect of PDMS-Based Micro Device Channel Width on Plasmid DNA Transformation Efficiency in E. Coli
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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 would5
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 trend10
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 counts15
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 procedure20
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.
References25
a College of Engineering, Bioengineering Department, University of
California, Berkeley,CA, 94704, USA.
1 Mahipal Singh, Arpita Yadav, Xiaoling Ma and Eugene Amoah.
Plasmid DNA Transformation in Escherichia Coli: Effect of Heat
Shock Temperature, Duration, and Cold Incubation of CaCl2 Treated30
Cells. International Journal of Biotechnology and Biochemistry,
Volume 6 Number 4 (2010) pp. 561–568.
2 Sha Li, L. Meadow Anderson, Jui-Ming Yanga, Liwei Lin, HawYang. DNA transformation via local heat shock. APPLIED
PHYSICS LETTERS 91, 2007.35
3 W. Edward Swords. Chemical Transformation of E. coli. Methods in
Molecular Biology, 2003, Volume 235, 49-53, DOI: 10.1385/1-
59259-409-3:49.
4 Dagert M, Ehrlich SD. Prolonged incubation in calcium chloride
improves the competence of escherichia coli cells. Gene. 197940
May;6(1):23-8.
5 Huff JP, Grant BJ, Penning CA, Sullivan KF. Optimization of routine
transformation of escherichia coli with plasmid DNA.
BioTechniques. 1990 Nov;9(5):570,2, 574, 576-7.
6 Bergmans HE, van Die IM, Hoekstra WP. Transformation in45
escherichia coli: Stages in the process. J Bacteriol. 1981
May;146(2):564-70.
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