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

 
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===Design Notes===
 
===Design Notes===
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For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the MBP Biobrick (BBa_K346004); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for MBP Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Purification of the Fragment of interest  from the gel; Ligation the gel purification of MBP gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + MBP Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +MBP Biobrick ;
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For this genetic construct, we followed these steps:  
  
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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. 
  
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2) Extraction and quantification of plasmid DNA of the BBa_K346004 and BBa_K1355001;
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 +
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;
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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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6) Ligation with T4 DNA ligase of purifieds biobricks;
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7) Transformation of the ligation in DH5-alpha;
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8) Extraction of plasmid DNA with our bioaccumulation construt from DH5-alpha transformed;
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 +
9) Check the electrophoretic profile to see results of samples linked (no fragments);
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 +
10) Digestion of BBa_K1355001 + BBa_K346004 with EcoRI and PstI, aiming to analyze the fragment size to be isolated;
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 +
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===
 
===Source===

Revision as of 04:02, 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 steps:

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;

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