Part:BBa_K2048002
Long GolS (P118A)
This composite part contains Tet promoter, teto,GolS(P118A),GolB and LacZ genes. GolS is a MerR family, gold responsive transcriptional regulator of gol and ges genes. This circuit contains mutant GolS P118A which is highly specific to gold. GolB is a promoter that gets activated by GolS factor. This circuit produces golS under the regulation of TetO promoter. Synthesized GolS protein binds to GolB promoter and synthesizes LacZ which generates a visible color.
iGEM Concordia University Collaboration
As part of our collaboration with iGEM UofT, we at iGEM Concordia University performed a liquid X-gal assay with DH5alpha E. coli cells transformed with this construct as well as GolS without the P118A point mutation (BBa_K2048001). Since DH5alpha cells do not express a Tet repressor, they produce GolS constitutively, and as such these cells grown with X-gal and gold ions produce the blue pigment as a result of X-gal being cleaved by beta-galactosidase.
We grew untransformed E. coli cells with no antibiotic, E. coli cells transformed with GolS grown in chloramphenicol, and E. coli cells transformed with GolS_P118A grown with chloramphenicol to an OD of 3. We diluted our cells into a final volume of 600 µL of M9 media, having an OD of 1.5. We used Au (III) ions at 1 µM and 5 µM, Cu(II) ions at 1 µM and 10 µM, Ag (I) ions at 1 µM and 5 µM, and Al (III) ions at 1 µM and 5 µM. Pictures were taken at different time points after the addition of ions, shown in the pictures below. Reactions were incubated at 37℃, 300 RPM. M9 indicates just M9 buffer, M9X indicates M9 buffer with 4.8 µL of 20 mg/mL X-gal. All wells with indicated ions contained M9X.
Throughout the entire process, none of the untransformed DH5ɑlpha cells constructs expressed any blue color, indicating that blue color formation was due to the presence of GolS and GolS_P118A. Blue color seems to have appeared in the GolS M9X construct, as well as all ion constructs, possibly indicating that GolS reacted with some ions in the M9 media (which contains calcium and magnesium). However, a darker blue color was observed with the 5 µM Au GolS construct, indicating higher LacZ activity caused by increased GolS activity in the presence of Au, as compared to the other ions. This is a confirmation that GolS is performing its intended function of binding to gold ions and permitting transcriptional activation.
Interestingly, we have found that the GolS_P118A construct can clearly bind to gold, copper, and silver ions. Silver ion binding by GolS and has not been documented in the iGEM registry, so we have characterized GolS to another degree. Both GolS and GolS_P118A do not show any binding affinity for Al ions.
We also wanted to obtain quantitative values from this data, so we attempted to extract the blue insoluble pigment in DMSO. We first spun down the cells (4000 RPM, 5 min), got rid of the supernatant, and lysed the cells with a commercial E. coli lysis buffer. We then added the commercial neutralization buffer, spun the cells again, removed the supernatant, and resuspended the pellet in DMSO, which was completed dissolved. We then performed an absorbance spectrum to determine the wavelength of absorption of the blue pigment.
We found that the solubilized blue pigment had absorbance maximum at 640 nm. As can be seen in the spectrum above, the untransformed DH5alpha incubated with Au ions (which did not have blue pigment) did not absorb at 640nm. This indicates that the relative amount of blue product from X-gal cleavage can be quantified, and by extension, so can the ability for GolS to activate translation.
We measured the absorbances of the solubilized blue pigments in each well at 640nm. The absorbances are written under each well. As is evident, the deeper the blue, the higher the absorbance. These quantified absorbance values add onto the qualitative data we have generated for our collaboration.
Sequence and Features
- 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 426
- 1000COMPATIBLE WITH RFC[1000]
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