Part:BBa_K2719005:Experience

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Applications of BBa_K2719005

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

Figure 1. Colonies transformed with the Tenascin 5 Domain V Expression device

Then the plasmid was extracted and run on an agarose gel electrophoresis (Figure 2). Latter an restriction was performed using NotI and was run on an agarose gel (Figure 3) to probe the presence of the insert. After the extracted plasmid it was transformed on E.coli BL21 (DE3).

Figure 2. 0.85% agarose gel with GelRed: MW) 1Kb plus from NEB; 7) Tenascin 5 Domain V Expression device (BBa_K2719005) V'; 8) Tenascin 5 Domain V Expression device (BBa_K2719005) V

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.

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