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Work Progress - 10.7.17

The document discusses the isolation and characterization of the urease gene cluster from Klebsiella pneumoniae MTCC109, which is known to produce urease. It details the growth conditions, genomic DNA isolation, and the amplification of the urease gene cluster for cloning into vectors. Additionally, it lists the reagents procured and those still pending for the experiments.

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kirankumarkakumani
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12 views11 pages

Work Progress - 10.7.17

The document discusses the isolation and characterization of the urease gene cluster from Klebsiella pneumoniae MTCC109, which is known to produce urease. It details the growth conditions, genomic DNA isolation, and the amplification of the urease gene cluster for cloning into vectors. Additionally, it lists the reagents procured and those still pending for the experiments.

Uploaded by

kirankumarkakumani
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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RECOMBINANT UREASE PRODUCTION

• Culture selected for the isolation of urease gene cluster is Klebsiella pneumoniae MTCC109

• Klebsiella aerogenes is renamed as Klebsiella pneumoniae.

• Urease gene cluster is highly conserved between various strains of Klebsiella pneumoniae

Blast results of urease gene cluster of Klebsiella pneumoniae


Klebsiella pneumoniae MTCC109 was grown in the below listed medium composition
• Beef extract – 1.0g
• Yeast extract – 2.0 g
• Peptone – 5.0 g
• Nacl – 5.0 g
• D H20 – 1L

• Klebsiella pneumoniae MTCC 109 produces Urease enzyme


• We have tested whether MTCC109 produces urease by growing the culture on
christensen’s urea agar (CUA)
• If the microbe produces urease, it cleaves the urea present in the medium and
raises the pH due to which the phenol red turns from orange to pink

Fig:1 Growth of MTCC109 on different


growth media
A- MTCC 109 grown on MTCC medium
B- MTCC 109 grown on CUA medium
Isolation of Genomic DNA from Klebsiella pneumoniae MTCC 109

The genomic DNA of Klebsiella pneumoniae was isolated according to Tiller et al.,(2000)

Concentration and Purity of Genomic DNA sample Isolated from Klebsiella pneumoniae

Sample Concentration (ng/ul) 260/280


1 411.182 1.7
2 458.3 1.6
pACYC-Duet -1 or pET Duet-1 ????
Amplification of pUC 18 and pET duet-1

M 1 2 3 4 5

5000 bp 5420bp
2500 bp 2686bp

Lane M : 1Kb marker


Lane 1 : pUC18 from sigma
Lane 2 & 3 : pUC 18 isolated from DH5α
Lane 4 : pET-duet-1 from Novagen
Lane 5 : pET-duet-1 isolated from DH5α
Amplification of Urease gene cluster to be cloned in to
pACYC-Duet -1 or pUC 18

M 1 2 3 4 5 6
Lane M : 1Kb marker
Lane 1,2&3 : Urease gene cluster to be
cloned in to pACYCDuet-1
Lane 4,5&6: Urease gene cluster to be
cloned in to pUC18
5000 bp
Reagents and Chemicals Procured
Reagents received Reagents

Yet to receive

Klebsiella pneumoniae pACYC –Duet

Oligonucleotides

Restriction enzymes FD
NdeI, EcoRI and XhoI

Plasmid purification kits and


Gel purification kits

Phenol:chloroform:methanol

pUC18

BL21 (DE3)
THANKYOU

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