The Effect of PDMS-Based Micro Device Channel Width on Plasmid DNA Transformation Efficiency in E....

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



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



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



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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