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Lab Report 3
Lab Report 3
coli Cell
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
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
- ori(pMB1) - ampR - ori (2 µm) - URA3 [4]. pRS426 contains 21 restriction sites that
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
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
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.
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.
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
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
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.
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.
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
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.
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
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
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
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
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
Protocol: Isolation of plasmid DNA from yeast using the QIAprep® Spin Miniprep
Kit. www.qiagen.com/literature/handbooks/default.asp.
DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge.
http://genome-www.stanford.edu/vectordb/vector_descrip/PRS426.html.
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.