Part:BBa_K1355003:Design
Mercury ions accumulator device
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 988
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 586
Illegal NgoMIV site found at 1160 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 579
Design Notes
For this genetic construct, we followed these summarized steps in the following image:
1) Transformation of DH5-alpha with the Biobrick Metal Binding Peptide (MBP) BBa_K346004 and with the Essential Biobrick (Regulation and transport of mercury) BBa_K1355001 that contains a bidiretional promotor regulated by the MerR protein.
2) Extraction and quantification of plasmid DNA of the BBa_K346004 and BBa_K1355001;
2) Verifying the electrophoretic profile of the extracted plasmid DNA;
Figure 1: A) Plasmid DNA of K346004; B) Plasmid DNA of K1355001.
3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_K346004 with EcoRI and XbaI;
4) Checking the electrophoretic profile of digested samples;
5) Purification from the agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_K346004);
6) Ligation with T4 DNA ligase of purifieds biobricks;
7) Transformation of the ligation in DH5-alpha;
8) Extraction of plasmid DNA with our bioaccumulation construt from DH5-alpha transformed;
9) Check the electrophoretic profile to see results of samples linked (no fragments);
10) Digestion of BBa_K1355001 + BBa_K346004 with EcoRI and PstI, aiming to analyze the fragment size to be isolated;
11) Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of BBa_K1355001 + BBa_K346004 in pSB1C3;
Source
BBa_K1355001; BBa_K346004