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

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<partinfo>BBa_K1355003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1355003 SequenceAndFeatures</partinfo>
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===Design Notes===
 
===Design Notes===
−
For this genetic construct, we followed these summarized steps in the following image:  
+
For this genetic construction, we followed these summarized steps in the following image:  
 
[[File:MBP Cutandlinking.jpg]]
 
[[File:MBP Cutandlinking.jpg]]
 +
 +
Read more about the design of this genetic construction on the extended version below:
  
 
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.   
 
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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[[File:DNApRTPMBP.jpg]]
 
[[File:DNApRTPMBP.jpg]]
  
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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.
+
Figure 1: A) Electrophoretic profile of BBa_K1355001 plasmid DNA in pBSK; B) Electrophoretic of BBa_K346004 plasmid DNA in pSB1C3.
  
  
−
3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_K346004 with EcoRI and XbaI;  
+
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;  
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[[File:DigstRTPMBP.jpg]]
 
[[File: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.
+
Figure 2: A) Electrophoretic profile of BBa_K1355001 digested with SpeI and EcoRI; B) Electrophoretic 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);  
 
5) Purification from agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_K346004);  
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[[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.
+
Figure 3: A) Electrophoretic profile of BBa_K346004 (linearized vector) purified; B) Electrophoretic profile of BBa_K1355001 (fragment) purified.
  
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6) Ligation with T4 DNA ligase of purifieds biobricks;
+
6) Ligation of the linearized vector with fragment using T4 DNA ligase;
  
 
7) Transformation of the ligation in DH5-alpha;  
 
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;
 
  
 +
[[File:MercuryBacterBIOACC.jpg]]
  
  
 +
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);
 
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;
+
[[File:DNApBIOACC.jpg‎]]
  
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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;
+
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;
 +
 
 +
[[File:RTPMBPdigestions.jpeg]]
 +
 
 +
Figure 6: Electrophoretic profile of the BBa_K1355003 do not digested; digested only with EcoRI; digested only with PstI; and digested with EcoRI + PstI, respectively.
 +
 
 +
'''There is our new biobrick part bioaccumulator device!'''
 +
 
 +
The fragment - our biobrick, the junction of BBa_K1355001 + BBa_K346004 - in the digestion with EcoRI + PstI (sample 3) has  1.736 base pairs and the vector pSB1C3 has 2.070 base pairs. The linearized vector contains about of 3.806 base pairs.
 +
 
 +
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_K346004
 
BBa_K1355001; BBa_K346004
−
 
−
===References===
 

Latest revision as of 23:09, 17 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

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

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 pBSK; B) Electrophoretic of BBa_K346004 plasmid DNA in pSB1C3.


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

Figure 2: A) Electrophoretic profile of BBa_K1355001 digested with SpeI and EcoRI; B) Electrophoretic 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) Electrophoretic profile of BBa_K346004 (linearized vector) purified; B) Electrophoretic 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;


MercuryBacterBIOACC.jpg


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: Electrophoretic profile of the BBa_K1355003 do not digested; digested only with EcoRI; digested only with PstI; and digested with EcoRI + PstI, respectively.

There is our new biobrick part bioaccumulator device!

The fragment - our biobrick, the junction of BBa_K1355001 + BBa_K346004 - in the digestion with EcoRI + PstI (sample 3) has 1.736 base pairs and the vector pSB1C3 has 2.070 base pairs. The linearized vector contains about of 3.806 base pairs.

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_K346004