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

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3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_K346004 with EcoRI and XbaI;  
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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;  
 
4) Checking the electrophoretic profile of digested samples;  

Revision as of 22:26, 8 October 2014

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 construction, 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;

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

DNApRTPMBP.jpg

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 aiming to isolate the biobrick fragment and linearize the vector, respectively;

4) Checking the electrophoretic profile of digested samples;

DigstRTPMBP.jpg

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;

PuriRTPMBP.jpg

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;


File:MercuryBacterBIOACC


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);

DNApBIOACC.jpg

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;

RTPMBPdigestions.jpeg

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

References