Difference between revisions of "Part:BBa K1355002:Design"
Mctastolfi (Talk | contribs) (→Design Notes) |
Mctastolfi (Talk | contribs) (→Design Notes) |
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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. | ||
| − | 2) Extraction and quantification of | + | 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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4) Checking the electrophoretic profile of purified samples; | 4) Checking the electrophoretic profile of purified samples; | ||
| − | + | FOTO | |
Figure 3: A) Eletrophoretic profile of BBa_E0840 (linearized vector) purified; B) Eletrophoretic profile of BBa_K1355001 (fragment) purified. | Figure 3: A) Eletrophoretic profile of BBa_E0840 (linearized vector) purified; B) Eletrophoretic profile of BBa_K1355001 (fragment) purified. | ||
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| − | + | FOTO | |
| − | Figure 4: Mercury Bacter Hg | + | Figure 4: Mercury Bacter Hg biodetector (DH5-alpha transformed with BBa_K1355002) |
| − | 8) Extraction of plasmid DNA with our | + | 8) Extraction of plasmid DNA with our biodetector constrution 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); | ||
| − | + | FOTO | |
Figure 5: Electrophoretic profile of BBa_K1355002 plasmid DNA in pSB1C3. | 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, 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, 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 (digestion 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_E0840 in pSB1C3 and that the linearized vector (sample digested only with EcoRI or PstI) is our biobrick 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_E0840 in pSB1C3 and that the linearized vector (sample digested only with EcoRI or PstI) is our biobrick in pSB1C3; | ||
| − | + | FOTO | |
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. | 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. | ||
Revision as of 17:24, 8 October 2014
Mercury ions detector device
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 988
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 586
Illegal NgoMIV site found at 1160 - 1000INCOMPATIBLE 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:
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;
Figure 1: A) Electrophoretic profile of BBa_E0840 plasmid DNA in pSB1C3; B) Eletrophoretic 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;
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);
4) Checking the electrophoretic profile of purified samples;
FOTO
Figure 3: A) Eletrophoretic profile of BBa_E0840 (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;
FOTO
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 (no fragments);
FOTO
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, 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 (digestion 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_E0840 in pSB1C3 and that the linearized vector (sample digested only with EcoRI or PstI) is our biobrick in pSB1C3;
FOTO
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.
Source
BBa_K1355001, BBa_E0840