Device

Part:BBa_K2719009:Experience

Designed by: Andrea Pamela Jimenez Tapia & Rodrigo Valencia Ocampo   Group: iGEM18_TecCEM   (2018-08-08)
Revision as of 14:32, 17 October 2018 by AKARAM (Talk | contribs) (→‎Applications of BBa_K2719009)


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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).

T--TecCEM--LEPCLONED.png

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).

T--TecCEM--LEPEXTRACTION.png

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) to probe the presence of the insert.

T--TecCEM--LEPRESTRICTION.png

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

T--TecCEM--LEPRESTRICTIONC.png

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


Later 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. The lysate was loaded into a 18% polyacrylamide SDS-PAGE (Figure 4), from which a higher presence of protein was detected at 5 h after induction.


A western blot was made for this part and positive results were obtained. (Figure 5)

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