Lab Report 3: Transformation of E.

coli Cell

With Yeast Shuttle Vector pRS426

By Manuel Molina Villalba

Group Members: Gabriel Fernando, Elizabeth Blake
April 1, 2010
University of Pennsylvania
CBE 480-001 Laboratory in Biotechnology and Genetic
Engineering
Introduction

Experiments were undertaken using a yeast shuttle vector, pRS426, in order to assess the

efficiency and success of transferring the vector from S. cerevisiae, yeast, to E. coli

bacteria. This is an important procedure that makes it possible to exploit the differences

between eukaryotic and bacterial cells in many genetic engineering applications. The

shuttle vector was isolated from yeast cell cultures using a QIAprep® Spin MiniPrep Kit.

The pRS426 vector was then mixed with Chemically Competent JM109 E. coli cells to

transform these via a heat-shock procedure. The presence of the Lac-Z operon was

exploited in a blue-white screening procedure that was used to identify JM109 cells that

possibly transformed successfully. Cells that produce blue colony forming units were

cultured for further experimentation. A final isolation of the pRS426 vector from the

blue-white screened E. coli cell culture was undertaken to confirm the presence of the

vector by use of flash gel electrophoresis.

Materials and Methods

pRS426 Vector

The vector pRS426 is a phagemid vector that is 5726 base pairs long [4]. Figure 1

displays many of the vector’s features. Some of the more important features of this

vector are, in order: ori(f1) - Lac Z - T7 promoter - MCS(KpnI-SacI) - T3 promoter-LacI

- ori(pMB1) - ampR - ori (2 µm) - URA3 [4]. pRS426 contains 21 restriction sites that

can be used in many genetic engineering applications. It contains a 2µm origin of

replication site. The vector has REP3 and FRT sequences that give it a high propagation

number in yeast, which is about 20 per haploid cell [4]. Non-selective growth of host

cells containing this vector experiences loss of about 4.4 ± 1.4% of progeny per doubling
through mitotic segregation [4]. One appropriate type of selective medium lacks uracil.

Since this vector can express uracil production, the vector will be placed in a host strain

that is deficient in this amino acid. The host cell will then be grown in a defined, uracil-

lacking medium. The presence of the Lac Z operon makes this a suitable vector for blue-

white screening. For the applications of these experiments, blue colonies denote the

desired cells since these indicate the possible presence of the pRS426 vector or any other

vector containing the Lac Z with the α-fragment.

Figure 1. Map of pRS426 Vector[1]: Major

features of this vector include 21 restriction sites,
2µm origin of replication, high copy number in
yeast cells (REP3 and FRT sequences). Other
major features in order include ori(f1) - lacZ - T7
promoter - MCS (KpnI-SacI) - T3 promoter -
lacI - ori(pMB1) - ampR - ori (2 micron) -
URA3. The type of selective medium used in
the subsequent experiments lacked uracial, since
the vector can express the production of uracil.
The Lac Z operon also contains the α-fragment
that is important in the use of the blue-white
screening technique. For the applications of this
report the blue colony forming units (CFU’s)
from the screening are desired. These colonies
indicate that the CFU’s consist of cells
containing the Lac Z operon with a α-fragment.

Growth Conditions for S. cerevisiae and E. coli

Yeast, S. cerevisiae, cell cultures containing the pRS426 vector were grown in two

different defined mediums. One was a selective medium that lacked the uracil amino

acid. This medium consists of 6.7 g YNB without amino acids, 20 g d-glucose, 20 g

Bacto agar, 1.4 g Sigma drop-out mix, 532 mg histidine, leucine, tryptophan mix and

additional H2O until a 1L volume is reached. The histidine, leucine, and tryptophan mix

consists of 76 mg histidine, 76 mg tryptophan and 380 mg leucine. The other medium

was a YEPD medium that consists of 5 g of yeast extract, 10 g of peptone and 10 g of d-

glucose. These cells were incubated at 37˚C in suspension. Blue colonies that resulted
from the blue-white screening of the transformed E. coli cells were inoculated in 10mL of

Luria Bertani (LB) medium† and incubated with shaking at 37˚C overnight.

pRS426 Vector Isolation From Yeast

Yeast cell cultures were separated from their respective mediums by centrifugation at

about 4000 rpm for 5 minutes and discarding the supernatant. Yeast cell pellets were

isolated from 13mL and 5mL of yeast suspension containing selective medium and the

suspension containing YEPD medium, respectively. Each plasmid vector was isolated

from its respective cell pellet by following the “Isolation of Plasmid DNA from Yeast

Using the QIAprep® Spin Miniprep Kit” protocol[2]. The kit consisted of the

appropriate buffers to lyse the yeast cells, precipitate undesired cell material and a

QIAprep-Spin Column to separate the plasmid from the undesired cell material. The cell

pellet in acid-washed 450µm-600µm glass beads was vortexed to lyse cells by shearing.

10-30 µL of plasmid solution was isolated from the 5mL and 13mL cell suspension

sample.

E. coli Transformation by Heat Shock

50 µL of thawed chemically competent E. coli cells were mixed with 3µL of vector that

was isolated from the yeast cells suspended in selective medium. The same mixture was

made for the vector that was isolated from the yeast cells suspended in YEPD medium.

The cell mixtures were kept in ice for 10 minutes. After the 10 minutes elapsed, the cell

mixtures were heat-shocked at 42˚C for 45-50 seconds. The cell mixtures were then kept

in ice for 2 minutes. 510 µL of SOC† medium at 37˚C was added to each mixture, and

the resulting mixtures were incubated at 37˚C for 60 minutes with shaking.

Blue-White Screening
†
See Appendix A.1
10 µL of a mixture of 1µL of cells transformed with the vector isolated from the yeast in

the selective medium, and 1 mL of LB medium was plated on an LB-Amp plate. 20µL of

50 mg/mL X-Gal and 100µL of 1M IPTG inducer were also added to the LB-Amp Plate.

The same plate was made for cells transformed with the vector isolated from the yeast in

the YEPD medium. As a control 100µL and 400µL samples from each cell mixture were

plated on different LB-Amp plates. The plates were then incubated at 37˚C overnight, in

preparation for blue-white screening. The blue-white screening technique was used to

determine if the pRS426 vector is present in the transformed E. coli cells. The pRS426

vector contains a Lac promoter, operator and the α- part of the ß-galactosidase gene (Lac

Z’). ß-galactosidase is expressed in α-complementation, which means that an E. coli

deletion mutant with the Ω part Lac Z’M15 codes the second part of the ß-galactosidase

gene. Upon screening, the Lac promoter is induced with IPTG, and the X-gal substrate is

added to an agar plate with cells. Blue colonies will form as a result of X-gal being

cleaved by functional ß-galactosidase. Blue colonies will confirm the presence of an

uninterrupted Lac Z operon, indicating the possibility that desired vector is in the

transformed cells that is being screened. If the α part (Lac Z’) of the gene is not

expressed, ß-galactosidase will be non-functional with only the Ω- part. The resulting

colony will be white, indicating that the Lac Z operon is missing or interrupted.

pRS426 Vector Isolation From E. coli

An E. coli cell pellet was separated from 5mL of E. coli cell suspension by centrifuging

at 4000 rpm for 5 minutes and discarding the supernatant. After centrifuging, the plasmid

was isolated from the cell pellet by using the Qiagen plasmid prep kit according to the

manufacturers protocol [3]. The kit consisted of the appropriate buffers to lyse cells,
precipitate undesired cell material and a QIAprep-Spin Column to separate the plasmid

from the undesired cell material. 10-30µL of plasmid solution was isolated from a 5mL

cell suspension.

Flash Gel Electrophoresis

The flash gel electrophoresis technique was done on a gel from Lonza. Each of the

plasmid vectors was run at 250 V for 8 minutes. A 1 kb DNA Ladder from NE Biolabs

was used as a reference to find the mass of each band in order to deduce the

concentration of plasmid. The flash gel ladder was used to measure migration distances

in order to deduce the base pair of the vector. 4:6 vector samples consisting of 4µL of

plasmid vector sample isolated from yeast, 1µL of TAE buffer and 1µL 6x Flash Gel

Loading dye were run through the gel. A 3:6 vector sample that consisted of 3µL of

plasmid vector sample isolated from E. coli, 2µL of TAE Buffer and 1µL of 6x Flash

loading gel was also run through a gel.

Results and Discussion

The chemically competent E. coli had a poor transformation efficiency. For 28 LB-Amp

plates that were plated with transformed E. coli cells for blue-white screening, there was

a yield of two blue colonies. Successive experiments were made on inoculations from

these two colonies. Efficiency calculations on this data may be unreliable due to the

small size of the data and the lack of distribution of blue colonies amongst the plates.

From Figure 1 it can be estimated that the length of the vector lies between 1461 and

6576 base pairs. From Figure 2 it can be estimated that the length of the vector lies

between 3949 Base pairs to 9502 base pairs in length. Super-imposing these to limits to

find an area in which the vector lies in both have ranges we get an overall range or of
3949 and 6576 base pairs, which is a smaller range that is close to the expected size of

the vector.

Figure 1 Flash Gel of Yeast Shuttle Vector

pRS426, Separated from Yeast: Lane 1: 5 µL 1
Kb DNA Ladder. Lanes 2 & 4: 4 µL pRS426
DNA isolated from yeast, 1 µL TAE Buffer, and
1 µL Flash Gel loading dye. Lane 9: 5 µL 1 Kb
DNA Ladder. The length of the vector is
estimated to be between 1461 Base pairs to 6576
base pairs in length. Isolation of the vector was
difficult. The isolation may have yielded small
amounts of the desired vector, decreasing the
transformation efficiency.

Figure 2 Flash Gel of Yeast Shuttle Vector

pRS426, Separated from E. Coli: Lane 1: 5 µL
1 Kb DNA Ladder. Lanes 6 & 8: 3 µL pRS426
DNA isolated from E. coli, 2 µL TAE Buffer,
and 1 µL Flash Gel loading dye. Lanes 2-5 & 7:
3 µL pUC19 DNA, 2 µL TAE Buffer, and 1 µL
Flash Gel loading dye. This vector is estimated
to be 2897 Base Pairs in length. Lane 9: 5 µL
Flash Gel DNA Ladder. The length of the vector
is estimated to be between 3949 Base pairs to
9502 base pairs in length. Isolation of the vector
was less difficult relative to isolation from yeast.
There was a significant amount of vector in the
E. coli cells.

From Figure 1 it can be concluded that isolation of a plasmid from yeast cells is more

difficult to achieve than for E. coli cells. The isolation of the pRS426 vector proved to be

more difficult for yeast cells than for E. coli. The poor isolation of the pRS426 vector

may have led to the poor transformation that was experienced by the E. coli cells. Yeast

cells have more residual DNA than Bacteria, and therefore the plasmid may have failed

to separate in the QIAprep-Spin Column. A different separation column setup should be

considered.

From Figure 2, it can be concluded that it is more difficult to separate pRS426 from E.
coli than pUC19. The same protocol for plasmid isolation was followed for both vectors,

but the lanes for the pUC19 plasmid, lanes 2-5 and 7, show clear and defined bands while

the pRS426 plasmid sample smeared within the lane, lanes 6 and 8. Although the lanes

have material smeared within the lane, there is pRS426 band outlined between [BP

lengths]. This shows that the lysis of the E. coli cells containing pRS426 was successful

and had similar results to that the of E. coli cell lysate containing pUC19. The smeared

lanes may contain other residual DNA and cell fragments that may have failed to separate

in the QIAprep-Spin Column. This means that separation of the pRS426 plasmid vector

from the cells’ residual DNA fragment may be improved by using a different column set

up. Options that can be considered for changing the set up include using a different

column, using the same column with a different length or changing the amounts of

solution that flow through.

From the foregoing experiments we can consider the causes of having very low

transformation efficiencies. A low transformation efficiency can result from a low yield

of isolated plasmid: a lower concentration of plasmid decreases the probability of a

vector permeating the cells membrane during the transformation process. From the

super-imposed range of base pair lengths, it seems that the unknown vector may be

pRS426. From the gel results, we can consider what factors facilitate the isolation of a

plasmid: an appropriate separation column setup for the vector of interest should always

be sought after if better results are to be obtained.

Acknowledgements

Many thanks to everyone that helped guide our group through the experiments. Special

thanks to Ken Chen, or lab coordinator, for helping us set up experiments. Also, thanks
to Dr. Miriam Wattenbarger for her guidance throughout our experiments.

References

1. Lab Life. http://www.atcc.org/catalog/numSearch/numResults.cfm?atccNum=77107

2. Qiagen Sample and Assay Technologies. www.qiagen.com. 2001. User developed

Protocol: Isolation of plasmid DNA from yeast using the QIAprep® Spin Miniprep

Kit. www.qiagen.com/literature/handbooks/default.asp.

3. Qiagen Sample and Assay Technologies. www.qiagen.com. 2006. Protocol: Plasmid

DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge.

QIAprep® Miniprep Handbook. 2 ed. 22- 24.

4. Saccharomyces Genome Database (SGD) project. Vector Data Base.

http://genome-www.stanford.edu/vectordb/vector_descrip/PRS426.html.

Appendix A.1: Recipes for Luria Bertani and YEPD Medium

LB (Luria-Bertani) Liquid Medium

Tryptone 10 g/L
Yeast Extract 5 g/L
NaCl 10 g/L
 H2O 900 mL
**Adjust to pH 7.5 with 1 M NaOH. Approximately 1 mL/L broth.

LB Agar
Add 15 g/L of agar to the LB liquid medium. Autoclave.

LB-Amp Medium
Add 100 µg/ml of Ampicillin for LB-Amp medium.

Ampicillin
Make up 500 mg of Ampicillin in 20 mL of H2O (25 mg/mL) and filter sterilize, dividing
into 1 mL aliquots. Store frozen at -20 to -80°C. Add to cold liquid medium or 45°C
medium containing agar to prevent thermal degradation. Typical concentrations for
selecting cells containing plasmids are 50 to 100 µg/mL which are prepared by adding 2
to 4 mL of stock to a liter of medium.

Plates containing ampicillin can be kept at 4°C for 2 weeks; in an incubator at 37°C, the
ampicillin will be degraded in 24 hours. The frozen stock can be kept for several months.

YEPD Medium
Yeast Extract 5g
Peptone 10 g
D-glucose (Dextrose) 10 g
**Dissolve in 500 ml of H2O. Autoclave.