Team:WLC-Milwaukee/Experiments
Wet Lab
WLC-Milwaukee’s wet lab work was done primarily in the summer, with a few lingering experiments being completed during the school year, into October. We focused primarily on designing a feasible and functional engineered E. Coli for use in sensing lead content in a sample of water. After deciding to pursue a test kit based around a sense of smell, the wet lab team began searching for viable parts to use.
We decided to use genes from the heavy metal resistant bacteria Cupriavidus metallidurans. Specifically, we chose this organism because of its inducible promoter and transport protein, which are sensitive to lead. We utilized this promoter, pbrR, as the basis for our project. When lead binds to the promoter, transcription is initiated for the genes attached to the promoter. Additionally, we used the gene pbrT, which ensured the uptake of lead by the cell. This provides a simple way for odor to be produced when the promoter is exposed to lead, given the gene downstream from the promoter codes for a type of odor.
This leads to the selection of a gene that will produce some sort of odor. We selected the gene benzoate/salicylate carboxyl methyltransferase, or BSMT1 (BBa_J45004). This gene creates the enzyme methyltransferase, which converts salicylate into methylsalycylate, the chemical that has a wintergreen odor. By adding this gene adjacent to the lead inducible promoter, the assembled machine will produce wintergreen odor when exposed to lead.
Our wet lab team met with Dr. John Werner early in the summer to begin lab work. We began by ligating our desired genes into the psb1c3 plasmid and moved this plasmid into an E. Coli strain by electroporation. Specifically, we inserted BSMT1, pbrT, pbrR, the pbrABCD operon promoter, and the T7 promoter into the plasmid. We verified this with DNA gel electrophoresis as well as sending our clones for sequencing.
Specifically, the function of our system is as follows.
- The pbrT gene produces the pbrT protein, which brings lead into the E. Coli cell.
- Both the pbrR and pbrT genes are under the control of the T7 promoter, a promoter from the T7 bacteriophage.
- This T7 promoter constantly expresses the pbrT and pbrR genes, which in turn constantly produce the pbrT and pbrR proteins.
- Two pbrR proteins join to form a dimer.
- Lead binds to this pbrR dimer, and the pbrR protein then binds to the pbrABCD promoter.
- This promoter expresses the BSMT1 gene, which produces the enzyme that converts salicylate (salicylic acid) into methyl salicylate.
- This methyl salicylate has a wintergreen odor; therefore wintergreen odor should only be detected when lead is present.
This summer, the wet lab team also did work with previous parts from WLC-Milwaukee (BBa_K1900000 and BBa_K 1900001). In this work, the tolC genes from Klebsiella pneumoniae and Serratia marcescens were cloned into a plasmid containing the pTrc promoter. This plasmid was then transformed into an E. coli strain that lacked a chromosomal copy of tolC. We then performed disk sensitivity assays to determine how well the S. marcescens and K. pneumoniae TolC protein could perform in an E. Coli cell. Please see the results section for more information on these experiments.
