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.

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

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.

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.

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

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

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)

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 bpFragments of corresponding sizes

were produced from new vector YPADC.

M: DNA marker 1kb1: xbal and hindIII digested recombinant plasmid YPADC2: recombinant plasmid YPADC3: xbal and hindIII digested plasmid yEp3524: xbal and hindIII digested PCR PADC5: D2000 DNA marker.

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

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

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.

M: D2000 DNA marker.1: W303-1A colony PCR2: W303-PADC colony PCR3: W303-PADC colony PCR

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)

RESULT:

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

Analysis of Mutant StabilitySelected 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%

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.

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

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.

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)

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.