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) Electrophoretic profile of BBa_K1355001 plasmid DNA in pSB1C3; B) Eletrophoretic of BBa_K346004 plasmid DNA in pBSK.
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;
Figura 2: A) Electrophoretic profile of BBa_K1355001 digested with SpeI and EcoRI; B) Eletrophoretic profile of the BBa_K346004 digested with EcoRI and XbaI.
5) Purification from agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_K346004);
4) Checking the electrophoretic profile of purified samples;
Figure 3: A) Eletrophoretic profile of BBa_K346004 (linearized vector) purified; B) Eletrophoretic profile of BBa_K1355001 (fragment) purified.
6) Ligation of the linearized vector with fragment using T4 DNA ligase;
7) Transformation of the ligation in DH5-alpha;
Figure 4: Mercury Bacter Hg bioaccumulator (DH5-alpha transformed with BBa_K1355003)
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);
Figure 5: Electrophoretic profile of BBa_K1355003 plasmid DNA in pSB1C3.
10) Restriction enzyme digestion of BBa_K1355001 + BBa_K346004 (BBa_K1355003) with EcoRI + PstI, only with EcoRI and only with PstI aiming to analyze the fragment size to be isolated (digestion with EcoRI + PstI) or the size of the linearized vector (only with EcoRI or PstI);
11) Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment (sample digested with EcoRI + PstI) is the junction of BBa_K1355001 + BBa_K346004 in pSB1C3 and that the linearized vector (sample digested only with EcoRI or PstI) is our biobrick in pSB1C3;
Figure 6: Eletrophoretic profile of the BBa_K1355003 do not digested; digested only with EcoRI; digested only with PstI; and digested with EcoRI + PstI, respectively.
Source
BBa_K1355001; BBa_K346004