Difference between revisions of "Part:BBa K1355004:Design"
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2) Verifying the electrophoretic profile of the extracted plasmid DNA; | 2) Verifying the electrophoretic profile of the extracted plasmid DNA; | ||
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Figure 1: | Figure 1: | ||
Revision as of 22:31, 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;
Figure 1:
3) Restriction enzyme digestion of the BBa_K1355001 with SpeI and EcoRI and of BBa_K1355000 with EcoRI and XbaI aiming to isolate the biobrick fragment and linearize the vector, respectively;
4) Checking the electrophoretic profile of digested samples;
Figure 2: (1) Electrophoretic profile of BBa_K1355000 do not digested and (2) digested with XbaI and EcoRI; (3) Eletrophoretic profile of the BBa_K1355001 do not digested and (4) digested with EcoRI and XbaI.
5) Purification from agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_K1355000);
4) Checking the electrophoretic profile of purified samples;
PuriRTPMBP.jpg
Figure 3: (1) Eletrophoretic profile of BBa_K1355000 (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 bioremediator (DH5-alpha transformed with BBa_K1355004)
8) Extraction of plasmid DNA with our bioremediator constrution from DH5-alpha transformed;
9) Check the electrophoretic profile to see results of samples linked;
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.