Difference between revisions of "Part:BBa K1355003:Design"

Revision as of 21:44, 11 October 2014

Mercury ions accumulator device

Design Notes

For this genetic construction, 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 pBSK; B) Eletrophoretic of BBa_K346004 plasmid DNA in pSB1C3.

3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_K346004 with EcoRI and XbaI aiming to isolate the biobrick fragment and linearize the vector, respectively;

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.

There is our new biobrick part bioaccumulator device!

The fragment - our biobrick, the junction of BBa_K1355001 + BBa_K346004 - in the digestion with EcoRI + PstI (sample 3) has 1.736 base pairs and the vector pSB1C3 has 2.070 base pairs. The linearized vector contains about of 3.806 base pairs.

To finalize our molecular characterization - design, we also make the Sanger method of DNA sequencing.

Check it out our experience with this biobrick device!

Source

BBa_K1355001; BBa_K346004

References