Part:BBa_K1355004:Design

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;

4) Checking the electrophoretic profile of digested samples;

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. 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) Eletrophoretic profile of BBa_K346004 (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 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: Eletrophoretic profile of the BBa_K1355003 do not digested; digested only with EcoRI; digested only with PstI; and digested with EcoRI + PstI, respectively.