Gene cloning is used to make copies of individual genes separated from other genes. Cloning provides a pure sample of a gene that can then be used in research. This document describes cloning the PADC gene from Bacillus subtilis into the yeast strain W303-1A. The PADC gene was amplified via PCR and inserted into the shuttle vector YEp352. Successful transformation into yeast was confirmed by PCR and increased PADC enzyme activity in the mutant strain. Analysis found increased levels of 4-vinyl guaiacol and esters, improving the aroma of beers brewed with the mutant strain.

1. GENE cloning
Gene cloning is the act of making copies of a
single gene. Cloning can provide a pure
sample of an individual gene, separated from
all the other genes that it normally shares the
cell with. Once a gene is identified, clones
can be used in many areas of biomedical and
industrial research. Genetic engineering is the
process of cloning genes into new
organisms, or altering a genetic sequence to
change the protein product.

3. Materials And Methods
REAGENTS: 1) Taq Enzyme
2) 10X buffer Taq w/o MgCl2
 Restriction Enzymes: 1) Xbal and Hind III
2) T4 DNA ligase
 Scarlett Barley Malt was provided by Zhong
liang Malt Division
 Wheat Malt obtained from Weyermann
 Hops supplied from Barth-Haos Group(
containing 4.9% α-acid Analytica-EBC)
 High performance liquid chromatography grade
4VG was purchased from sigma

4. Strain Plasmid & Medium
 Bacillus.subtilis, E.coli DH5α and plasmid YEp352( shuttle vector
for E.coli and S.cerevaise
 Top fermenting Yeast Strain W303-1A obtaining from Doemens
 Yeast Cells were cultured on yeast peptone dextrose(YPD) medium.
(10g/l yeast extract; 20g/l & quccose)
 E.coli DH5α-cells cultured in luria Bertani (LB) medium(5g/l yeast
extract, 10 g/l tryptone & 10 g/l sodium chloride)
 For selection of E.coli transformants: 100 ug/l ampicillin was
included in LB medium.
 For the selection of Yeast transformants: SC-ura medium (6.7 g/l
yeast nitrogen base w/o AA; 20 g/l gluocose & 8.3g/l of each of growth
factors Leucine, Histidine & Tryptophan)
 Agar : 20g/l
 Bacteria and yeast cultured at 37 and 30 degree C resp.

5. CLOnING AND SEQUENCING
PROCEDURE
 The Polymerase Chain Reaction(PCR) method used for
amplification of PADC gene
 Primer: (Synthesized by invitrogen)
 Genomic DNA for B.subtilis used as template to amplify
PADC
gene.

6.  PCR programmed as:
• Template denaturation at 94 degree C for 3min.
• Denaturation at 94 degree C for 30 sec.
• Primer annealing at 52.6 degree C for 30 sec.
• Primer extension at 72 degree C for 45 sec( 30 cycles).
• Elongation at 72 degree C for 10 min .
 Volume of PCR reaction: mixture :
was 50 μl
• Consisting of 5 μl 10X Taq buffer
• 4 μl of 1.25 mM nucleotides (dNTPs)
• 1 μl of each primer
• 1 μl of template
• 1μl of easy Taq enzime
• 37 μl of double distilled water
Used wizard kit ; for the purification of PCR

7. Result of Amplification of PADC
gene:
 PCR reaction to amplify PADC gene with genomic DNA from
B. Subtiliss as template and primers:
Presence of PADC gene was confirmed, of
504bp

8. Purified PADC gene and Shuttle Plasmid YEp352 were
digested with Xbal and Hind III separetely
Then were ligated with T4 DNA ligase to generate plasmid Yep PADC
Which was transformed into Ecoli DH5α
To obtain the plasmid YPADC
(Sequenced by invitrogen)

9. Analysis of Restriction endonucleases of
the constructed plasmid YPADC
 To idenify the authenticiy of
construction to further select positive
transformants.
 Theoretical size of fragment with
Xbal= 5,181 bp
HindIII= 504 bp
Fragments of corresponding sizes were
produced from new vector YPADC.

10. M: DNA marker 1kb
1: xbal and hindIII digested recombinant plasmid YPADC
2: recombinant plasmid YPADC
3: xbal and hindIII digested plasmid yEp352
4: xbal and hindIII digested PCR PADC
5: D2000 DNA marker.

11. Transformation: into top-
fermenting Yeast Strain W303-
1A
 Standard method for yeast transformation was used:
The vector YPADC was transformed into Yeast Strain W303-1A
Transformants were selected on SC-ura plates
The new mutant Strain-named as W303 + PADC
* W303 + PADC = mutant strain

12. RESULTS:
White colonies emerged in 5 days on SC-ura plate. NO
colonies on the wild.
Indicated that the new plasmid YPADC was
succsesfully transformed into the starin
W3030=1A

13. PCR Analysis of Mutant
Colonies
The PADC genes were confirmed by PCR colonies
from transformed strains on SC-ura plates.
RESULTS:
Size of W303+padc colonies PRC product=
504bp. In accordance with that of PADC gene
fragment.
It showed PADC gene had integrated into the
appropriate chromosome of mutant strain.
Fragments not produced from wild type strain of
W303-1A.

14. M: D2000 DNA
marker.
1: W303-1A colony
PCR
2: W303-PADC
colony PCR
3: W303-PADC
colony PCR

15. Phenolic Acid Decarboxylase
activity assay of mutant
Strain
 PADC activity was described as:
 The ferulic acid degradation and 4VG production
( was monitered by UV spectrophotometer)
 Ferulic acid shows a peak at its maximum absorbance
at wavelength (285 nm)
 4VG shows a peak of its maximum absorbance at 258
nm.
Thus; the disappearance of 285nm peak and appearance
of 258nm peak indicate the degradation of ferulic acid
and formation of 4VG.
(expressed as: micromoles of substrate degraded per
min per milligram of protein)

16. RESULT:
The PADC activity of mutant strain was
increased by 2.1 fold higher, that of wild type.

17. Analysis of Mutant
Stability
Selected transformants were innoculated into 10ml
YPD broth at 30 degree C
After incubating for 10 generations
Colonies innoculated on
SC plate SC-ura Plate
Genetic stability was calculated :
Mutant Stability = (Colonies on SC-ura /Colonies on SC) X
100%

18. RESULTS:
 After incubating for 10 generation:
no of colonies on SC and SC-ura plate
were 62 and 33 resp.
thus, transformant stability- 52.23%
 Which is benefited for the continious
brewing beers with mutant strains.

19. 4VG and Volatile compound
Analysis
 4VG levels were determined by Shimadzu HPLC
 HPLC system consisted of:
 LC – 10 Avp pump
 a 7725i sample injector
 Shimadzu UV-vis detector
 N2000 data analyzer
The Samples were then analyzed using a 15cm X
4.6 mm i.d. packed column at a flow rate of
1ml/min eluted with water or phosphoric acid
UV detector operated at 230 nm
The volatile compound were analyzed with a Perkin-
Elmer autosystem XL-gas Chromatography

20. RESULT:
 After integration confirmed from PCR;
* Some volatile components also
detected.
* Also the concentration of 4VG and
Ester in top fermented beers brewed
with mutant strains had notable
increase.
* Good to enhance the aroma of top
fermented beers.

23. Sensory Evaluation
 Trained tasting Panel was used for the evaluation of fresh
top fermented beers according to analysis of attributes
including:
 Cloves-like flavours
 Head-ache sense
 ester aroma
 Taste
 Bitterness and Aftertaste
 Headache sense was evaluated in 30 min after tasting
Rating Scale Range:
 from 1 (Very Bad or weak) to 5 (good and
very strong)

24. RESULTS:
 Fresh top-fermented beers brewed
with mutant strain reveal:
- more clover like flavor and ester
aroma.
Which reflected higher 4VG and ester
levels.
- Headache sense of beer with mutant
was little stronger then wild.
Conclusion: higher alcohol in top
fermented beer with mutants.