Part:BBa_K1355004:Design
Design notes
For this genetic construction, we followed these summarized steps in the following image:
- We couldn't separate the biobrick from the plasmid backbone because both of the vector pBSK and the fragment (biobrick BBa_K1355004) have around 3.000 base pairs!
Read more about the design of this genetic construction on the extended version below:
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;
3) Verifying the electrophoretic profile of the extracted plasmid DNA;
Figure 1: Electrophoretic profile of the BBa_K1355001 and of BBa_K1355000 plasmid DNA.
4) 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;
5) 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) Electrophoretic profile of the BBa_K1355001 do not digested and (4) digested with EcoRI and XbaI.
6) Purification from agarose gel of the fragment (Biobrick BBa_K1355001) and the linearized vector (BBa_K1355000);
7) Checking the electrophoretic profile of purified samples;
Figure 3: (1) Electrophoretic profile of BBa_K1355000 (linearized vector) purified; (2) Electrophoretic profile of BBa_K1355001 (fragment) purified.
8) Ligation of the linearized vector with fragment using T4 DNA ligase;
9) Transformation of the ligation in DH5-alpha;
Figure 4: Mercury Bacter Hg bioremediator (DH5-alpha transformed with BBa_K1355004)
10) Extraction of plasmid DNA with our bioremediator constrution from DH5-alpha transformed;
11) Check the electrophoretic profile to see results of samples linked;
Figure 5: Electrophoretic profile of BBa_K1355004 plasmid DNA in pBSK.
12) Restriction enzyme digestion of BBa_K1355001 + BBa_K1355000 (BBa_K1355004) with EcoRI + PstI aiming to analyze the fragment size to be isolated;
13) Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of BBa_K1355001 + BBa_K1355000 in pBSK;
Figure 6: A) - C) Electrophoretic profile of the BBa_K1355003 do not digested and B) - D) digested with EcoRI + PstI.
We have a tricky molecular question! We produced the enzymatic system to isolate our biobrick, but it didn’t isolate!!! Why?!?! We thought a lot about it and we found out!!
The answer is: the BBa_K1355004 (BBa_K1355001 + BBa_K1355000) fragment has 3.057 base pairs and the vector pBSK has 2.861, that’s 196 base pairs of difference! The electrophoretic profile shows only one big band in 3.000 base pairs (comparing it to the marker). That both are there, but we couldn’t separate it!
To prove this we digested the vector PBSK with the biobrick BBa_K1355004 aiming to linearize and to isolate the fragment using EcoRI and EcoRI + PstI, respectively. The linear band should present double of band’s size digested to isolate vector from fragment. That means, the sample digested with EcoRI + PstI will show a big band in 3.000 base pairs comparing it to the marker and the sample only digested with EcoRI will show double size of that, in 6.000 base pairs! Let's check this out?!
14) Restriction enzyme digestion of BBa_K1355001 + BBa_K1355000 (BBa_K1355004) only with EcoRI and with EcoRI + PstI;
15) Checking the electrophoretic profile of the digested sample to answer the molecular question:
Figure 7: A) BBa_K1355004 in pBSK do not digested; B) digested only with EcoRI and C) digested with EcoRI + PstI
B) One band in 6.000 base pairs comparing it to the marker, the BioBrick only linearize by EcoRI. C) Two bands in 3.000 base pairs comparing it to the marker! We proved our hypothesis! But how are we going to separate our biobrick BBa_K1355004 from the pBSK vector for cloning it in PSB1C3, considering that both of the vector and the fragment have around 3.000 base pairs? We solved it and developed the following strategy:
16) Restriction enzyme digestion of BBa_K1355004 in pBSK and of the BBa_J04450 in pSB1C3 with EcoRI + PstI.
17) Checking the electrophoretic profile of the digested samples:
Figure 8: (A) BB_K1355004 in pBSK do not digested; and (B) digested with EcoRI + PstI; (C) BBa_J04450 in pSB1C3 do not digested; and (D) digested with EcoRI + PstI.
- Do not forget! One big band on the 3.000 base pairs comparing it to the marker because both of the vector and the fragment has around 3.000 base pairs, as you can see in the sample (B) - figure 8 and sample (C) - figure 7.
18) Purification from agarose gel of BBa_K1355004 digested (pBSK + biobrick) and the vector/plasmid backbone of the BBa_J04450 - pSB1C3.
Figure 9: A) BBa_K1355004 digested with EcoRI + PstI vector + fragment purified; B) BBa_J04450 digested with EcoRI + PstI vector purified.
19) Ligation of the purifieds samples (BBa_K1355004 in pBSK digested with EcoRI and PstI + pSB1C3 plasmid backbone digested with EcoRI and PstI) using T4 DNA ligase;
20) Transformation of the ligation in DH5-alpha;
21) Selection of 19 colonies of transformed DH5-alpha with ligation system grown in chloramphenicol;
22) Colony PCR of the 19 colonies using the VR - VF2 primers for pSB1C3 plasmid;
21) Checking the electrophorectic profile to obtain results showing that the amplified samples is the junction of BBa_K1355004 with pSB1C3 plasmid backbone;
Figure 10: (1) - (19) Colony PCR of 19 colonies transformed with ligation system.
As we can analyse, only the samples (8) - (11) - (16) - (17) amplified using the VR - VF2 primers. We selected only the 8 and 11 samples to continue the procedure, because they have around 3.000 base pairs comparing it to the marker.
22) Plasmid DNA extraction of the BBa_K1355004 in pSB1C3 amplified using VR - VF2 (8 and 11 colonies);
23) Checking the electrophoretic profile;
Figure 11: Electrophoretic profile of BBa_K1355004 plasmid DNA in pSB1C3.
24) Restriction enzyme digestion of BBa_K1355004 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);
25) 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_K1355000 in pSB1C3 and that the linearized vector (sample digested only with EcoRI or PstI) is our biobrick in pSB1C3;
Figure 12: (A) Electrophoretic profile of the BBa_K1355004 do not digested; (B) digested only with EcoRI; (C) digested only with PstI; and (D) digested with EcoRI + PstI, respectively.
There is our new biobrick part bioremediator device!
The fragment - our biobrick, the junction of BBa_K1355001 + BBa_K1355000 - in the digestion with EcoRI + PstI (sample D) has 3.057 base pairs and the vector pSB1C3 has 2.070 base pairs. The linearized vector contains about of 5.000 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!