As a subsection of the MoClo library expansion project we will be investigating the application of Histidine Kinase systems, specifically in the E. coli we are accustomed to using in lab. Histidine Kinases are a specific class of transmembrane protein that are composed of a sensing, linking, and effector domains. The sensing domain undergoes conformational changes in response to an extracellular stimuli (i.e. a specific wavelength of light, a threshold osmolarity, etc.), thereby enabling the effector domain that resides on the inside of the membrane to undergo autophosphorylation. Once phosphorylated the effector domain displays kinase activity, enabling it to activate a response regulator molecule within the cytoplasm. Once activated, the response regulator alters transcription either by directly acting as a transcription factor or serving as a part of a larger cascade with the same ultimate outcome. This signal transduction pathway serves as a link between environmental conditions and cellular activity by adjusting the transcription of specific proteins.

While H-K two component systems have a natural purpose, past experiments have revealed that they can be manipulated so as to accomplish a specified goal. For example, Levskaya et al. generated a chimeric histidine kinase protein by identifying overlapping regions of proteins present in Synechocystis and Escherichia coli. By varying the length of the linking domain between the two proteins, Levskaya eventually grew bacteria that produced a pigment depending on the presence of red light, thus creating an effective bacterial camera using the “dark detector” (Levskaya et. al 2005). More recently, combining the dark detector with a Quorum sensing system led to the construction of an effective edge detector(Tabor et. al 2009). These displays of logic through cellular response give Histidine Kinases promise as another system through which to exert control over bacterial transcription.

For our work we will be attempting to recreate these proven systems. In order to do so, we will create MoClo parts for the protein domains and cognate promoters necessary for the functionality of the pathways. After replicating proven systems we plan to move toward generating our own H-K systems by combining different sensor and effector domains. Finally, using the Flow Cytometer will allow us to generate transfer functions for our histidine kinases and quantify their cellular responses. Ultimately, we hope to reinforce the usefulness of the modularity of MoClo and quantify the effectiveness of histidine kinase manipulation for the alteration of gene expression.

Reference

[1] Levskaya et. al "Synthetic Biology:engineering Escherichia coli to see light" Nature 2005;438:441–442

[2]Tabor et. al "A Synthetic Genetic Edge Detection Program" Cell. 2009 June 26; 137(7):1272-1281