Cell lines

Bacteria used, their genotypes and functions for our Project.

• Escherichia coli DH5α

Genotype: F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

Nalidixic acid resistant.

We used this strain of E. coli for DNA cloning resuspended from parts kit. In addition to this role, it was chosen for our tests with mercury.

• Escherichia coli DH10B

Genotype: F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-

Antibiotic Resistance: streptomycin.

We used this strain of E. coli for DNA cloning resuspended from parts kit Escherichia coli RR1

Genotype: F- mcrB mrr hsdS20(rB- mB-) recA+ leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ-

Antibiotic Resistance: streptomycin.

We used this strain of E. coli for DNA cloning resuspended from parts kit Escherichia coli RR1

• Escherichia coli ER2925

Genotype: ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10)TetS endA1 rpsL136 dam13::Tn9 xylA-5 mtl-1 thi-1 mcrB1 hsdR2

Antibiotic Resistance: kanamycin, streptomycin.

After DNA cloning resuspended from parts kit by strains of E. coli RR1, DH5α and DH10B, we used this strain of E. coli to demethylate the XbaI restriction site, enabling our genetic constructs to be efficient, taking into account that the same dam is negative.

• Escherichia coliJM110

Genotype: rpsL thr leu thi lacY galK galT ara tonA tsx dam dcm glnV44 Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)

Antibiotic Resistance: streptomycin.

After DNA cloning resuspended from parts kit by strains of E. coli RR1, DH5α and DH10B, we used this strain of E. coli to demethylate the XbaI restriction site, enabling our genetic constructs to be efficient, taking into account that the same dam is negative.

DNA Synthesis

2. Plasmids:

Biobricks and plasmids used and their roles in Mercury Bacter project.

• BBa_K346004:

RBS (B0034) + MBP (lead metal binding peptide egineered from PbrR) + Terminator (B0015). This part functions as a basic lead binding peptide part. This part was used in our project for bioaccumulative construction. For this purpose, it was done ligation to bidirectional promoter regulated by Merr protein (BBa_K1355001), building the Mercury ions accumulator device (BBa_K1355003).

• BBa_E0840:

GFP generator. BBa_E0840 takes as input a transcriptional signal (PoPS) and produce as output the fluorescent protein GFP. This part was used in our project for biosensor construction. For this purpose, it was done ligation to bidirectional promoter regulated by MerR protein (BBa_K1355001), building the Mercury ions accumulator device (BBa_K1355002).

• p26G

Plasmid with pUC replication origin, ampicillin resistance gene, and high level and late Green Fluorescent Protein expression. This plasmid was used in our project as a positive control for quantification of GFP in spectrofluorometer.

• BBa_K1355000 - pBSK.

Plasmid with pUC replication origin, ampicillin resistance gene and translatiol unit of Mercury reductase (MerA). This plasmid was used in our project for cloning and synthesis of biobrick BBa_K1355000. From this plasmid, we constructed bioremediador Mercury Bacter, by doing the ligation of reductase Mercury translational unit to BBa_K1355001 (bidirectional promoter regulated by protein MerR).

• BBa_K1355001 – pBSK.

Plasmid with pUC replication origin, ampicillin resistance gene and bidirectional promoter regulated by protein MerR together with Hg transporters and MerP and merT. This plasmid was used in our project for cloning and synthesis of biobrick BBa_K1355001. From this plasmid, we constructed Mercury Bacter bioremediator- bioaccumulating - biosensor, doing the ligation of this part to biobricks BBa_E0840, BBa_K346004 and BBa_K1355000.