1. Eric Han | @01305985 | (512)829-2531 | Dr.Gonzalez | Biology Independent Study | Spring
Semester
Saccharomyces boulardii
LAB REPORT ON THE GENETIC MODIFICATION OF YEAST AND POSSIBLE
APPLICATIONS

2. PAGE 1
Abstract:
Saccharomyces boulardii is a species of yeast that is commonly found in
tropical fruits. The ability to survive high temperatures in the tropics allows this
yeast to survive within a human host. The yeast is considered a probiotic organism,
which means that these microorganisms have the potential to provide health
benefits. We are attempting to genetically modify this diploid yeast to secrete
certain types of proteins. The yeast are able to live and survive in the human
gastrointestinal tract for several days. Once consumed this yeast should secrete the
encoded protein through the presence of a plasmid
An attempt to construct a recombined vector using the In-Fusion® cloning
technique has been made. The In-Fusion® technique is used to join any piece of
DNA that has 15 homologous base pairs at their linear end. Initially the vector that
is targeted for modification has to be linearized. The gene of interest then has to
be amplified with specific primers containing the 15 base pairs homologous to the
vector ends. The amplified gene is then recombined with the linearized vector on
the 15 homologous base pairs and this results in a fused vector (Hamilton, 2007).

3. PAGE 2
Experiment and Procedure:
The In-Fusion® Protocol Overview:
1. The vector is linearized with a restriction enzyme at the insertion site
(Stu I).
2. PCR primers were designed by Dr. Gonzalez with 15 homologous base pair
to the linearized vector ends.
Upper Primer:
5’ ATG GAT CCT AGG AGG AGG CCA GCT GCT CGA GAA 3’
Lower Primer:
5’ TGT CAG TAC TGA AGG GCC AGC TGT ACG TTAT CGA TCT
AGA G 3’
3. The Hepatitis B surface antigen (HbsAg) gene was amplified with PCR using
the KOD polymerase with these primers. (See PCR procedures)
4. The amplified DNA is present, as verified by electrophoresis (Figure 1).
5. The DNA in the gel is extracted and purified. (See next section)
6. The purified insert is combined with the vector at a 2:1 molar ratio.

4. Figure 1: The first well is the
ladder. The next 4 lanes are
the amplified inserts 764bp.
7. The In-Fusion® cloning reaction is set up as follows (10 µL total in tube):
2 µL of 5X In-Fusion® buffer
1 µL of In-Fusion® Enzyme
1.6 µL of vector
0.2 µL of insert
5.2 µL of dH2O
8. The reaction is incubated for 15 minutes at 37 °C followed by 15 minutes at
50 °C.
9. TE buffer is put in the In-Fusion® solution until it reached a volume of 50 µL
and then mixed gently.
10. The cells were transformed with 2.5 µL of the diluted mixture.
The procedure for DNA gel extraction:

5. PAGE 1
1. The DNA gel slice was placed in a pre-weighed tube. The weight of the tube
with the gel was taken and subtracted from the weight of the pre-weighed
tube of the gel.
2. We multiplied the weight of the gel by 3 and multiply this number by 1000.
This is the quantity, in µL of QG buffer used to dissolve the gel.
3. The tube is warmed in a heating block at 50 °C for 10 minutes. Every couple
of minutes the solution within the tube was mixed.
4. The solution within the tube was pipetted into the column. The capacity of
the column is around 800 µL. Since there was not enough solution that can
fit in the column, additional columns were used.
5. The solution was centrifuged for 1 minute at 6,500 rpms.
6. The flow through was discarded.
7. 500 µL of QG buffer was then added in each column to wash the membrane
of agarose. Each column was spun down for 1 minute at 13,000 rpms.
8. PE buffer containing ethanol is used to wash the column. After placing the
buffer into the solution, a timer was set at 2 minutes and the solution was
left to incubate with the buffer.
9. The solution was then spun down for 1 minute at 13,000 rpms.
10. Once the flow through is out of the column. The column was spun down
again without any wash for 1 minute at 13,000 rpms.
11. The column was then kept at room temperature for 5 minutes to dry.

6. PAGE 2
12. 50 µL de-ionized water was then pipetted to the column and the column
was placed in a 70 °C heating block and left open for a few minutes. The
column was then spun down for 1 minute at 13,000 rpms.
13. Once the centrifugation was finished the final product was placed in the
spectrometer to determine the concentration of the DNA. The DNA was
diluted 1/10 for a final volume of 80 µL which was placed in a sub-micro
spectrometer cell. The concentration was measured with the spectrometer
and was converted using this formula: Concentration (µg/ml) = (A260
reading – A320 reading) × dilution factor (10) × 50µg/ml
Conclusion:
The In-Fusion® technique did not produce the correct vector we needed.
There were no colonies observed. From Dr. Gonzalez’s speculation, the reason why
the experiment was unsuccessful was due to the fact that the vector recombined
with a 5 base pair gap with a 10 base pair overhang at both ends of the insert at the
insertion site (Stu I). The G and C rich region of the Tef promoter probably base
paired to the G and C rich region of the adjacent Tef promoter in the kanMX and
formed a stem loop structure containing an A and T rich region between the two,
popping the insert out, leaving linear DNA which is degraded by the cell. The stem
loop, base pair gaps, and base pair overhangs at the ends of the insert resulted in
torsional strain which caused the inserted HbsAg insert to break away from the

7. PAGE 3
modified vector. Because of this, the intact vector is not available for the E. coli to
use, thus no transformants were observed.
References:
Hamilton, M.D. et al. (2007) Nucleic Acids Res. 35(1):143–151.

8. PAGE 4
Insert Electrophoresis

9. PAGE 5