Idea

Mercury, nickel, and lead were targeted for sequestration by our strain of bacteria by utilizing the efficient binding properties of the pea metallothionein and specificity of the respective metal transport proteins, merT/merP1, nixA2,and CBP4. First, the yeast metallothionein3 as well as each of the three heavy metal transport proteins were biobricked. Then, our wetlab sub team combined the parts to develop functional composite constructs. The idea of utilizing of metallothioneins in parallel with metal transporters for sequestration has been studied in depth for mercury and nickel. However, this idea has never been investigated for lead primarily due to a lack of characterization of the lead transporter.

To test the sequestration efficiency of each metal, E.coli BL21-A1 was transformed with the pea metallothionein as well the respective metal transporter for the targeted metal. The sequestering strains can be placed into fiber reactors to develop functional sequestering filters (see drylab).

Components

We obtained the sequences for many of our parts from the Wilson lab , and subsequently synthesized the genes through GenScript.
-sources
-component plasmids
-composite plasmids

No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some advantage from it? But who has any right to find fault with a man who chooses to enjoy a pleasure that has no annoying consequences, or one who avoids a pain that produces no resultant pleasure? On the other hand, we denounce with righteous indignation and dislike men who are so beguiled and demoralized by the charms of pleasure of the moment, so blinded by desire, that they cannot foresee.

Results

To assess the efficiency of our system, we used three main methods of determining whether the heavy metals were indeed being sequestered by our bacteria. First, we used a spectrophotometer to analyze the growth rates of E. coli cultures expressing either the vector only, one of the metal transporters, or both a transporter as well as the metallothionein. This test was conducted with the prediction that the bacteria without our constructs would be mildly sensitive to growth in metal-containing media, the bacteria with the transport protein only would be more sensitive to growth in metal-containing media, and the bacteria transformed with both the metal transporter and metallothionein protein would be the least sensitive (and thus grow at the greatest rate) in metal-containing media. . Next, we sampled the supernant of cell cultures that expressed the constructs, and analyzed it for total heavy metal content using the Phen(ylalanine? Phenol? Phenolphthalein?) Green assay[source to company]. Finally, we also submitted that supernatant for analysis at the Cornell Nutrient Analysis lab, in order to obtain specific, quantitative data about the concentration of each heavy metal found in solution.
-Nutrient Lab results
-Phen Green experiments metals
-Growth assays

References

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1. Ref 1
2. Ref 2
3. Ref 3