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

(→‎Design Notes)
 
(15 intermediate revisions by one other user not shown)
Line 3: Line 3:
  
 
<partinfo>BBa_K1355003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1355003 SequenceAndFeatures</partinfo>
−
 
  
 
===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.   
Line 17: Line 18:
 
[[File:DNApRTPMBP.jpg]]
 
[[File:DNApRTPMBP.jpg]]
  
−
Figure 1: A) Electrophoretic profile of BBa_K1355001 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;  
Line 26: Line 27:
 
[[File:DigstRTPMBP.jpg]]
 
[[File: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.
+
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);  
Line 34: Line 35:
 
[[File:PuriRTPMBP.jpg]]
 
[[File:PuriRTPMBP.jpg]]
  
−
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.
  
 
6) Ligation of the linearized vector with fragment using T4 DNA ligase;
 
6) Ligation of the linearized vector with fragment using T4 DNA ligase;
Line 41: Line 42:
  
  
−
[[File:MercuryBacterBIOACC]]
+
[[File:MercuryBacterBIOACC.jpg]]
  
  
 
Figure 4: Mercury Bacter Hg bioaccumulator (DH5-alpha transformed with BBa_K1355003)
 
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;
 
8) Extraction of plasmid DNA with our bioaccumulation construt from DH5-alpha transformed;
  
Line 55: Line 55:
 
Figure 5: Electrophoretic profile of BBa_K1355003 plasmid DNA in pSB1C3.
 
Figure 5: Electrophoretic profile of BBa_K1355003 plasmid DNA in pSB1C3.
  
−
10) Restriction enzyme digestion of BBa_K1355001 + BBa_K346004 with EcoRI and PstI, aiming to analyze the fragment size to be isolated;  
+
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 is the junction of BBa_K1355001 + BBa_K346004 in pSB1C3;
+
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.
  
−
Figure 6: Electrophoretic profile of BBa_K1355003 digested with EcoRI and PstI.
+
'''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