Part:BBa_K1355000:Design
Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1200
Illegal NgoMIV site found at 1262 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
For this genetic construction, we followed these summarized steps in the following image:
Read more about the design of this genetic construction on the extended version below:
1) Transformation of DH5-alpha with BBa_K1355000 - Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015) - in the pBSK plasmid.
2) Extraction and quantification of the BBa_K1355000 plasmid DNA;
3) Verifying the electrophoretic profile of the extracted plasmid DNA;
Figure 1: Electrophoretic profile of the BBa_K1355000 plasmid DNA.
4) Restriction enzyme digestion of the BBa_K1355000 in pBSK with EcoRI and PstI and of BBa_J04450 in pSB1C3 with EcoRI and PstI aiming to isolate the biobrick fragment and isolate the plasmid backbone, respectively.
5) Checking the electrophoretic profile of digested samples;
Figure 2: A) Electrophoretic profile of BBa_K1355000 do not digested and (B) digested with EcoRI + PstI; C) Electrophoretic profile of the BBa_J04450 do not digested and D) digested with EcoRI + PstI.
6) Purification from agarose gel of the fragment (BBa_K1355000) and the pSB1C3 plasmid backbone (BBa_J04450);
7) Checking the electrophoretic profile of purified samples;
Figure 3: A) Electrophoretic profile of BBa_K1355000 (fragment) purified; B) Electrophoretic profile of BBa_J04450 (pSB1C3 plasmid backbone) purified.
8) Ligation of the pSB1C3 plasmid backbone with the fragment BBa_K1355001 using T4 DNA ligase;
9) Transformation of the ligation in DH5-alpha;
10) Selection of 3 colonies of transformed DH5-alpha with ligation system grown in chloramphenicol;
11) Colony PCR of the 3 colonies using the VR - VF2 primers for pSB1C3 plasmid;
12) Checking the electrophorectic profile to obtain results showing that the amplified samples is the junction of BBa_K1355000 with pSB1C3 plasmid backbone;
Figure 4: (A) - (C) Colony PCR of 3 colonies transformed with ligation system.
13) Plasmid DNA extraction of the BBa_K1355000 in pSB1C3 amplified using VR - VF2 (B and C colonies);
14) Checking the electrophoretic profile;
Figure 5: Electrophoretic profile of the BBa_K1355000 in pSB1C3 extracted plasmid DNA.
How it be observed, the two samples (B - C colonies) showed a different electrophorectic profile comparing each other. Analyzing the electrophorectic profile of the samples digested with EcoRI + PstI we could select the right one: the C colony! So, from here on we just presented the results from the right one. Check it out!
15) Restriction enzyme digestion of BBa_K1355000 in pSB1C3 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);
16) Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment (sample digested with EcoRI + PstI) is the BBa_K1355000 in pSB1C3 and that the linearized vector (sample digested only with EcoRI or PstI) is the junction of our biobrick in pSB1C3;
Figure 6: (A) Electrophoretic profile of the BBa_K1355000 do not digested; (B) digested only with EcoRI; (C) digested only with PstI; and (D) digested with EcoRI + PstI, respectively.
There is our new part - MerA translational unit - Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015)!
Figure 7: DH5-alpha transformed with Essential Biobrick (BBa_K1355001) in pSB1C3 grown in chloramphenicol;
The fragment - our biobrick BBa_K1355000 - in the digestion with EcoRI + PstI (sample D) has 1.778 base pairs and the vector pSB1C3 has 2.070 base pairs. The linearized vector contains about of 4.000 base pairs.
To finalize our molecular characterization - design, we also make the Sanger method of DNA sequencing.
Check it out our experience with this MerA translational unit linked with the BBa_K1355001 bidirectional promotor regulated by the MerR protein, composing the BBa_K1355004 (Hg bio-remediator) device!
We analyzed all Mercury-resistance (Mer) operon genes, in these following strains available at NCBI: (1) Escherichia coli 042, (2) Escherichia coli O26 , (3) Escherichia coli ACN001, (4) Escherichia coli 838C-R1, (5) Escherichia coli APEC-O103, (6) Escherichia coli UMNK88, (7) Escherichia coli AR060302, (8) Escherichia coli PMV-1. It is showed that the mer genes from strains (1) - (2) - (3) - (4) - (5) is conserved among them. However strains (6) - (7) and (8) did not show any similarity. Therefore we used sequences from (1) to (5) due the consensus in E.coli.
Source
MerA gene sequence is found in the O26-CRL plasmid from Escherichia coli O26. We also added strong RBS from protein 10 found in phage 17 and the double terminator (BBa_B0015) to ensure efficient termination of transcription and messenger RNA recognition.