Difference between revisions of "Part:BBa K1355004:Design"

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===Source===
  
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__NOTOC__
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BBa_K1355001, BBa_K1355000
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<partinfo>BBa_K1355004 short</partinfo>
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<partinfo>BBa_K1355004 SequenceAndFeatures</partinfo>
 
  
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== Design notes ==
  
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===Design Notes===
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For this genetic construction, we followed these summarized steps in the following image:
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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 MerA Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for merA Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of merA 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 + merA 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 +merA Biobrick ;
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ESQUEMA
  
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1) Transformation of DH5-alpha with the Biobrick - Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015) - BBa_K1355000 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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===Source===
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2) Extraction and quantification of plasmid DNA of the BBa_K1355000 and BBa_K1355001;
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2) Verifying the electrophoretic profile of the extracted plasmid DNA;
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[[File:DNApRTPMERA]]
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Figure 1:
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3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_K1355000 with EcoRI and XbaI;
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4) Checking the electrophoretic profile of digested samples;
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DigstRTPMBP.jpg
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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 agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_K346004);
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4) Checking the electrophoretic profile of purified samples;
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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.
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6) Ligation of the linearized vector with fragment using T4 DNA ligase;
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7) Transformation of the ligation in DH5-alpha;
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File:MercuryBacterBIOACC
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Figure 4: Mercury Bacter Hg bioaccumulator (DH5-alpha transformed with BBa_K1355003)
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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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DNApBIOACC.jpg
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Figure 5: Electrophoretic profile of BBa_K1355003 plasmid DNA in pSB1C3.
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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);
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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;
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RTPMBPdigestions.jpeg
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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.
  
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BBa_K1355001, BBa_K1355000
 
  
 
===References===
 
===References===
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oioi
 

Revision as of 22:23, 8 October 2014

Source

BBa_K1355001, BBa_K1355000


Design notes

For this genetic construction, we followed these summarized steps in the following image:

ESQUEMA

1) Transformation of DH5-alpha with the Biobrick - Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015) - BBa_K1355000 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_K1355000 and BBa_K1355001;

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

File:DNApRTPMERA

Figure 1:

3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_K1355000 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 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.


References