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Part:BBa_K1355003:Design

Designed by: Maria Clara Tavares Astolfi, Luna Barroco de Lacerda   Group: iGEM14_UFAM_Brazil   (2014-10-06)
Revision as of 19:25, 7 October 2014 by Lunalacerda (Talk | contribs) (→‎Design Notes)


Mercury ions accumulator device


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 988
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 586
    Illegal NgoMIV site found at 1160
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 579


Design Notes

For this genetic construct, we followed these summarized steps in the following image: MBP Cutandlinking.jpg

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; File:K346004plasmidDNA.JPG[K346004plasmidDNA] File:K1355001plasmidDNA

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

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

References