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

(→‎Design Notes)
(→‎Design Notes)
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[[File:DNApRTPMBP.jpg]]
 
[[File:DNApRTPMBP.jpg]]
  
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Figure 1: A) Plasmid DNA of K1355001; B) Plasmid DNA of K346004.
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Figure 1: A) Electrophoretic profile of BBa_K1350033 plasmid DNA in pSB1C3; B) Eletrophoretic of BBa_K346004 plasmid DNA in pBSK.
  
  
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[[File:DigstRTPMBP.jpg]]
 
[[File:DigstRTPMBP.jpg]]
  
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Figura 2: A) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI; B) Restriction enzyme digestion of the BBa_K346004 with EcoRI and XbaI.
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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.
  
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5) Purification from the agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_K346004);  
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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;  
 
4) Checking the electrophoretic profile of purified samples;  
  
 
[[File:PuriRTPMBP.jpg]]
 
[[File:PuriRTPMBP.jpg]]
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Figure 3: A) Eletrophoretic profile of BBa_K346004 (linearized vector) purified; B) Eletrophoretic profile of BBa_K1355001 (fragment) purified.
  
 
6) Ligation with T4 DNA ligase of purifieds biobricks;
 
6) Ligation with T4 DNA ligase of purifieds biobricks;
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LALALALA
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9) Check the electrophoretic profile to see results of samples linked (no fragments);
 
9) Check the electrophoretic profile to see results of samples linked (no fragments);

Revision as of 22:36, 7 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 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;

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

DNApRTPMBP.jpg

Figure 1: A) Electrophoretic profile of BBa_K1350033 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;

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 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