Transformation of PUC19 in E. coli

In this experiment, the plasmid pUC19 carrying the gene of interest- CRY2Aa, was chemically synthesized and introduced into competent E. coli bacteria through transformation. Once the transformation procedure was completed, the E.coli was cultured and the transformed colonies were distinguished from the non-transformants by inoculating them on an agar plate infused with ampicillin.


Figure 01:Transformed white colonies on ampicillin medium

Isolation of PUC19 containing Cry2Aa from transformed E. coli and visualization on gel electrophoresis

After completion of screening, pUC19 was isolated from E. coli, and the extracted plasmid carrying Cry2Aa was then run and visualized on the 0.8% agarose gel with a marker. This confirms the presence of the plasmid in E. coli and proves its integrity.


Figure 02: Isolated PUC19 with Cry2Aa

Elution of PUC19 from the gel and restriction digestion to restrict the gene of interest

Once gel electrophoresis was performed, the isolated plasmid pUC19 was eluted from the agarose gel. Subsequent digestion by restriction enzymes BamHI and NcoI resulted in the cleavage of the Cry2Aa gene. The separated gene was then subjected to Agarose gel electrophoresis, alongside a 1000-kb DNA ladder, and visualized through a UV-gel Transilluminator.


Figure 03: Digested Cry2Aa with NcoI and BamHI

ABTS assay

ABTS assay is a common method to characterize the activity of the enzyme laccase. In this assay, ABTS which is a colorless substrate, is oxidized by laccase, leading to the formation of a yellow-green product which can be measured at 420 nm. The absorbance value of the product correlates to the activity of laccase. Laccase was added to ABTS, and at regular time intervals, the absorbance was measured, and thereby the activity of Laccase was characterized.


Figure 04: ABTS Assay

laccase activity

Figure 05: Enzymatic activity of laccase under various substrate concentrations. The laccase enzyme was assayed in the presence of substrate ABTS, and enzymatic oxidation of ABTS by laccase was observed at various time intervals continuously under standard assay conditions.