Reporter: Our next step is to start creating a standard curve in a calcium chloride buffer solution, supplemented with magnesium ions for beta-galactosidase. After this, we will create another curve using cells induced to produce beta-galactosidase as an in vivo assay in our new buffer system. After this, we will move on to testing these curves in tailings pond samples.
qRT-PCR: We are in the process of biobricking two promoter elements in addition to the submitted part and performing more electrochemical assays to test the reporter system in the presence of naphthenic acids. Future characterization experiments would explore enrichment of opposite strand transcripts of the fad-2 operon. Lastly, there were other candidate genes to investigate by bioinformatics screen that we were unsuccessful in developing PCR primers for. We are interested in sequencing the environmental isolate strains LD1 and LD2 to refine our screen and perhaps be able to design better primers for this line of investigation.
"Sensory Element Fishing": With our preliminary pull-down result, the next step is to repeat these several times in order to confirm any interactors that we are seeing. We also wish to biotinylate more naphthenic acid-like compounds in order to create a biotinylated library of naphthenic acid molecules to further our search for sensory elements. We also want to expand our "fishing" model to search for other types of sensory elements (i.e. ChIP-SEQ).
Chassis: In terms of tools, further characterization of the HspA70/RbcS2 promoter and algal luciferase is needed. The addition of a few more selectable markers suitable for use in algae would be useful. Ultimately, we also want to finish the construction and screening of chloroplast and nuclear promoter libararies in Dunaliella tertiolecta. Now that our conjugation system is up and working, we also want to start attempting to conjugate our putative system (BBa_K640006) from E. coli and into Pseudomonas.
Prototype: The next step would be to miniaturize our circuit and create a printed circuit board. Printed circuit boards have the advantage of integrated wires, meaning the wires are small copper lines on the board instead of physical wires sticking up from the board. We will also improve the software interface that controls our potentiostat.