Part:BBa_K1355001:Design

For this genetic construction, we followed these summarized steps in the following image:

Read more about the design of this genetic construction on the extended version below:

1) Transformation of DH5-alpha with the pBSK plasmid wich contains the BBa_K1355001 - bidirectional promotor regulated by the MerR protein.

2) Extraction and quantification of the BBa_K1355001 plasmid DNA;

3) Verifying the electrophoretic profile of the extracted plasmid DNA;

Figure 1: Electrophoretic profile of the BBa_K1355001 plasmid DNA.

4) Restriction enzyme digestion of the BBa_K1355001 in pBSK with EcoRI and PstI and of BBa_J04450 in pSB1C3 with EcoRI and PstI aiming to isolate the biobrick fragment and isolate the plasmid backbone, respectively.

5) Checking the electrophoretic profile of digested samples;

Figure 2: A) Electrophoretic profile of BBa_K1355001 digested with EcoRI + PstI; B) Electrophoretic profile of the BBa_J04450 digested with EcoRI + PstI.

6) Purification from agarose gel of the fragment (BBa_K1355001) and the pSB1C3 plasmid backbone (BBa_J04450);

7) Checking the electrophoretic profile of purified samples;

Figure 3: A) Electrophoretic profile of BBa_K1355001 (fragment) purified; B) Electrophoretic profile of BBa_J04450 (pSB1C3 plasmid backbone) purified.

8) Ligation of the pSB1C3 plasmid backbone digested with the fragment (BBa_K1355001) using T4 DNA ligase;

9) Transformation of the ligation in DH5-alpha;

Figure 4: DH5-alpha transformed with Essential Biobrick (BBa_K1355001) in pSB1C3 grown in chloramphenicol;

10) Extraction of plasmid DNA with our Essential Biobrick from DH5-alpha transformed;

11) Check the electrophoretic profile to see results of samples linked;

Figure 5: Electrophoretic profile of the BBa_K1355001 extracted plasmid DNA.

Figure 4: DH5-alpha transformed with the Essential Biobrick - BBa_K1355001