Lactate Dehydrogenase Lab Report
Isolation, Expression and Cloning of Lactate Dehydrogenase Gene from Lactobacillus bulgaricus
Lactobacillus bulgaricus is used for industrial production of yogurt mainly because of his fermentative capability. In this experiment, the gene encoding the enzyme lactate dehydrogenase [ldhA] from the bactaria was isolated and cloned. The expressed ldhA gene was inserted into pET28b plasmid vector. The resulting recombinant pET28b-LdhA expression vector was then transformed after introduction into E. coli. The ligation gave 15 colonies of recombinant DNA which later gave 6 recombinant plasmids as revealed by gel electrophoresis.
1. Introduction
The NADH-dependent lactate dehydrogenase
(EDH) is a key enzyme in the fermentative metabolism …show more content…
This process, fermentation, is necessary in the production of yogurt. Lactobacillus bulgaricus is a fermenting bacteria that converts milk into yogurt. This lactic acid bacteria produces the lactate that is responsible for the sharp taste of yogurt.
In this experiment, the genomic sequence of the LdhA gene was cloned from Lactobacillus bulgaricus after being amplified using PCR. The amplified LdhA gene was expressed upon insertion into the vector, pET28b. The subsequent recombinant expression vector was introduced into E. coli to allow for the process called Transformation, which is the genetic modification that is caused by the direct uptake of the exogenous DNA.
2. Materials and Methods
All experiments were conducted using Standard operating procedures and culture techniques according to the instructions and recommendations provided by the New England Biolabs inc.
I. Bacterial strains and Plasmids
Lactobacillus bulgaricus was obtained from Activia® yogurt. The pET28b vector used for the insertion of the LdhA gene was obtained from NEB.
II. Growth and Isolation of Genomic …show more content…
coli for transformation. The reaction was heat-shocked at 42 ºC to aid transformation. The cells were recovered and plated on a 30 µg/mL kanamycin and the colonies were estimated after 24 hours.
VIII. Selection by Colony PCR
Transformed cells were isolated after anaerobic incubation at 37 ºC. Colonies grown after 7 days were isolated on replica plate containing kanamycin. 13 colonies of transformants with one positive control was further analyzed using colony PCR. A master mix of a 16X reaction was performed.
IX. Purification and DNA Sequence Analysis
Crude Plasmid DNA was recovered from the E. coli bacteria and was purified using the Wizard Plus SV Minipreps DNA Purification Kits. Sequencing reactions were performed by the primer walking technique using the computer analytic APE software.
3. Results
A. Amplification od ldhA gene from L. bulgaricus
Lactobacillus bulgaricus is used for industrial production of yogurt mainly because of his fermentative capability. In this experiment, the gene encoding the enzyme lactate dehydrogenase [ldhA] from the bactaria was isolated and cloned. The expressed ldhA gene was inserted into pET28b plasmid vector. The resulting recombinant pET28b-LdhA expression vector was then transformed after introduction into E. coli. The ligation gave 15 colonies of recombinant DNA which later gave 6 recombinant plasmids as revealed by gel electrophoresis.
1. Introduction
The NADH-dependent lactate dehydrogenase
(EDH) is a key enzyme in the fermentative metabolism …show more content…
This process, fermentation, is necessary in the production of yogurt. Lactobacillus bulgaricus is a fermenting bacteria that converts milk into yogurt. This lactic acid bacteria produces the lactate that is responsible for the sharp taste of yogurt.
In this experiment, the genomic sequence of the LdhA gene was cloned from Lactobacillus bulgaricus after being amplified using PCR. The amplified LdhA gene was expressed upon insertion into the vector, pET28b. The subsequent recombinant expression vector was introduced into E. coli to allow for the process called Transformation, which is the genetic modification that is caused by the direct uptake of the exogenous DNA.
2. Materials and Methods
All experiments were conducted using Standard operating procedures and culture techniques according to the instructions and recommendations provided by the New England Biolabs inc.
I. Bacterial strains and Plasmids
Lactobacillus bulgaricus was obtained from Activia® yogurt. The pET28b vector used for the insertion of the LdhA gene was obtained from NEB.
II. Growth and Isolation of Genomic …show more content…
coli for transformation. The reaction was heat-shocked at 42 ºC to aid transformation. The cells were recovered and plated on a 30 µg/mL kanamycin and the colonies were estimated after 24 hours.
VIII. Selection by Colony PCR
Transformed cells were isolated after anaerobic incubation at 37 ºC. Colonies grown after 7 days were isolated on replica plate containing kanamycin. 13 colonies of transformants with one positive control was further analyzed using colony PCR. A master mix of a 16X reaction was performed.
IX. Purification and DNA Sequence Analysis
Crude Plasmid DNA was recovered from the E. coli bacteria and was purified using the Wizard Plus SV Minipreps DNA Purification Kits. Sequencing reactions were performed by the primer walking technique using the computer analytic APE software.
3. Results
A. Amplification od ldhA gene from L. bulgaricus