Difference between revisions of "Part:BBa K1355002:Design"

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For this genetic construction, we followed these summarized steps in the following image:  
 
For this genetic construction, we followed these summarized steps in the following image:  
  
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[[File:RESUMO]]
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[[File:RESUMO.jpg]]
 +
 
 +
Read more about the design of this genetic construction on the extended version below:
 +
 
 
1) Transformation of DH5-alpha with the Green Fluorescent Protein (GFP) translational unit BBa_E0840 and with the Essential Biobrick (Regulation and transport of mercury) BBa_K1355001 that contains a bidiretional promotor regulated by the MerR protein.   
 
1) Transformation of DH5-alpha with the Green Fluorescent Protein (GFP) translational unit BBa_E0840 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_E0840 and BBa_K1355001;
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2) Extraction and quantification of the BBa_E0840 and BBa_K1355001 plasmid DNA;
  
 
2) Verifying the electrophoretic profile of the extracted plasmid DNA;  
 
2) Verifying the electrophoretic profile of the extracted plasmid DNA;  
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[[File:DNApRTPGFP.jpg]]
 
[[File:DNApRTPGFP.jpg]]
  
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Figure 1: A) Electrophoretic profile of BBa_E0840 plasmid DNA in pSB1C3; B) Eletrophoretic of BBa_K1355001 plasmid DNA in pBSK.
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Figure 1: A) Electrophoretic profile of BBa_E0840 plasmid DNA in pSB1C3; B) Electrophoretic of BBa_K1355001 plasmid DNA in pBSK.
  
  
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4) Checking the electrophoretic profile of digested samples;  
 
4) Checking the electrophoretic profile of digested samples;  
  
 +
[[File:DigstRTPGFP.jpg]]
  
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Figura 2: A) Electrophoretic profile of BBa_K1355001 digested with SpeI and EcoRI; B) Electrophoretic profile of the BBa_E0840 digested 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_E0840 digested with EcoRI and XbaI.
+
  
 
5) Purification from agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_E0840);  
 
5) Purification from agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_E0840);  
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4) Checking the electrophoretic profile of purified samples;  
 
4) Checking the electrophoretic profile of purified samples;  
  
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[[File:PuriRTPMBP.jpg]]
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[[File:PurificadoRTPGFP.jpeg]]
  
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Figure 3: A) Eletrophoretic profile of BBa_E0840 (linearized vector) purified; B) Eletrophoretic profile of BBa_K1355001 (fragment) purified.
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Figure 3: A) Electrophoretic profile of BBa_K1355001 (fragment) purified; B) Electrophoretic profile of BBa_E0840 (linearized vector) purified.  
  
 
6) Ligation of the linearized vector with fragment using T4 DNA ligase;
 
6) Ligation of the linearized vector with fragment using T4 DNA ligase;
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[[File:MercuryBacterBIOACC]]
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[[File:biosensor.jpg]]
  
  
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Figure 4: Mercury Bacter Hg bioaccumulator (DH5-alpha transformed with BBa_K1355002)
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Figure 4: Mercury Bacter Hg biodetector (DH5-alpha transformed with BBa_K1355002)
 
    
 
    
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8) Extraction of plasmid DNA with our bioaccumulation construt from DH5-alpha transformed;
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8) Extraction of plasmid DNA with our biodetector constrution 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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9) Check the electrophoretic profile to see results of samples linked;
  
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[[File:DNApBIOACC.jpg‎]]
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[[File:rtpgfp.jpg]]
  
 
Figure 5: Electrophoretic profile of BBa_K1355002 plasmid DNA in pSB1C3.
 
Figure 5: Electrophoretic profile of BBa_K1355002 plasmid DNA in pSB1C3.
  
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10) Restriction enzyme digestion of BBa_K1355001 + BBa_E0840 (BBa_K1355002) 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);  
+
10) Restriction enzyme digestion of BBa_K1355001 + BBa_E0840 (BBa_K1355002) with EcoRI + PstI, and only with EcoRI aiming to analyze the fragment size to be isolated (digestion with EcoRI + PstI) and the size of the linearized vector (digestion only with EcoRI);  
  
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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_E0840 in pSB1C3 and that the linearized vector (sample digested only with EcoRI or PstI) is our biobrick in pSB1C3;
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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_E0840 in pSB1C3 and that the linearized vector (sample digested only with EcoRI) is our biobrick in pSB1C3;
  
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[[File:RTPMBPdigestions.jpeg]]
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[[File:DigstRTPGFPP.jpg]]
  
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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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Figure 6: (1) Electrophoretic profile of the BBa_K1355003 do not digested; (2) digested only with EcoRI and (3) digested only digested with EcoRI + PstI, respectively.
 +
 
 +
'''There is our new biobrick part biodetector device!'''
 +
 
 +
The fragment - our biobrick, the junction of BBa_K1355001 + BBa_E0840 - in the digestion with EcoRI + PstI (sample 3) has 2.136 base pairs and the vector pSB1C3 has 2.070 base pairs, that’s a difference of 66 base pairs! There is the reason why the two bands in the electrophoretic profile are so close to each other. Be careful when using this part device!
 +
 
 +
To finalize our molecular characterization - design, we also make the Sanger method of DNA sequencing.
 +
 
 +
Check it out our experience with this biobrick device!
  
 
===Source===
 
===Source===
  
 
BBa_K1355001, BBa_E0840
 
BBa_K1355001, BBa_E0840
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−
===References===
 

Latest revision as of 23:10, 17 October 2014

Mercury ions detector 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 BsaI.rc site found at 1862
    Illegal SapI site found at 579


Design Notes

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

RESUMO.jpg

Read more about the design of this genetic construction on the extended version below:

1) Transformation of DH5-alpha with the Green Fluorescent Protein (GFP) translational unit BBa_E0840 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 the BBa_E0840 and BBa_K1355001 plasmid DNA;

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

DNApRTPGFP.jpg

Figure 1: A) Electrophoretic profile of BBa_E0840 plasmid DNA in pSB1C3; B) Electrophoretic of BBa_K1355001 plasmid DNA in pBSK.


3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_E0840 with EcoRI and XbaI aiming to isolate the biobrick fragment and linearize the vector, respectively;

4) Checking the electrophoretic profile of digested samples;

DigstRTPGFP.jpg

Figura 2: A) Electrophoretic profile of BBa_K1355001 digested with SpeI and EcoRI; B) Electrophoretic profile of the BBa_E0840 digested with EcoRI and XbaI.

5) Purification from agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_E0840);

4) Checking the electrophoretic profile of purified samples;

PurificadoRTPGFP.jpeg

Figure 3: A) Electrophoretic profile of BBa_K1355001 (fragment) purified; B) Electrophoretic profile of BBa_E0840 (linearized vector) purified.

6) Ligation of the linearized vector with fragment using T4 DNA ligase;

7) Transformation of the ligation in DH5-alpha;


Biosensor.jpg


Figure 4: Mercury Bacter Hg biodetector (DH5-alpha transformed with BBa_K1355002)

8) Extraction of plasmid DNA with our biodetector constrution from DH5-alpha transformed;

9) Check the electrophoretic profile to see results of samples linked;

Rtpgfp.jpg

Figure 5: Electrophoretic profile of BBa_K1355002 plasmid DNA in pSB1C3.

10) Restriction enzyme digestion of BBa_K1355001 + BBa_E0840 (BBa_K1355002) with EcoRI + PstI, and only with EcoRI aiming to analyze the fragment size to be isolated (digestion with EcoRI + PstI) and the size of the linearized vector (digestion only with EcoRI);

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_E0840 in pSB1C3 and that the linearized vector (sample digested only with EcoRI) is our biobrick in pSB1C3;

DigstRTPGFPP.jpg

Figure 6: (1) Electrophoretic profile of the BBa_K1355003 do not digested; (2) digested only with EcoRI and (3) digested only digested with EcoRI + PstI, respectively.

There is our new biobrick part biodetector device!

The fragment - our biobrick, the junction of BBa_K1355001 + BBa_E0840 - in the digestion with EcoRI + PstI (sample 3) has 2.136 base pairs and the vector pSB1C3 has 2.070 base pairs, that’s a difference of 66 base pairs! There is the reason why the two bands in the electrophoretic profile are so close to each other. Be careful when using this part device!

To finalize our molecular characterization - design, we also make the Sanger method of DNA sequencing.

Check it out our experience with this biobrick device!

Source

BBa_K1355001, BBa_E0840