Part:BBa_K2719009:Experience

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Applications of BBa_K2719009

This part was cloned on pSB1C3 using EcoRI and PstI, later it was transformed on E.coli DH5a (Figure 1).

Figure 1. Leptin cloned in pSB1C3 in agarose gel 0.85%, 100V, 45 min.

Then the plasmid was extracted and run on an agarose gel electrophoresis (Figure 2).

Figure 2. Leptin extraction in agarose gel 0.85%, 100V, 45 min.

Later an restriction was performed using EcoRV and was run on an agarose gel (Figure 3, 3.1) to probe the presence of the insert.

Figure 3. Leptin restriction in agarose gel 0.85%, 100V, 45 min.

Figure 3.1. Leptin restriction simulation (SnapGene) in agarose gel 0.85%, 100V, 45 min simulated using SnapGene.

Latter the extracted plasmid was transformed on E.coli BL21 (DE3).

The E.coli BL21 (DE3) colonies were later inoculated on liquid media (seed culture) and incubated overnight. From the seed culture 1 ml was inoculated on fresh LB+CAM broth, and at regular intervals the absorbance was measured. When the absorbance reached 0.7, IPTG was added to a final concentration of 1 mM and samples were taken after 5 and 16 h. Those samples were lysed. Then, a western blot was made for this part and positive results were obtained. (Figure 4)

Figure 4. Leptin Western Blot transfer membrane results

Figure 4.1. Leptin Western Blot results after a treatment with anti-histidine antibody

There is a band in at the expected molecular weight which suggest the production of Leptin, but it is necessary to elaborate more experiments to standardize the result.

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