Lumazine Synthase Microcompartment for Compartmentalization

Our project this year is to use existing biological pathways and mechanisms for the cleaning contaminated water such as the tailings ponds. We are using the protein catechol-2,3-dioxygenase as the hub that other chemicals that are harmful to the environment breaks down into. With a working system of breaking down these harmful chemicals into biologically useful molecules there needs to be a way to apply the pathway for the cleaning of the contaminated tailings ponds. To apply the system to the tailings we can do so by isolating the pathway (enzymes) from the cell and applying it like a dry powder or by applying the bacteria directly to the water.

To apply the pathways (in a dry powder form) to the tailings ponds we need a method to keep the relevant enzymes in proximity to one another and to isolate them from the cell. To do this we are creating microcompartments that will function to isolate enzymes from the cell's natural pathways. Compartmentalization is a method that is used within nature to concentrate substances, reduce pathway cross-talk, isolate toxic components and bring components into close proximity for increased pathway efficiency as is seen in eukaryotic cells. The chassie most used among iGEM teams is Eschericia coli which is a prokaryote that, by definition, does not have any compartments. By introducing synthetic compartment we will be utilizing a natural characteristic that has evolved to increase efficiency.

The compartment we are using is made up from a single protein (lumazine synthase or LS) that forms an icosahedral by assembling 60, 120 or 180 of the monomers and if found in Aquifex aeolicus (Seebeck et al., 2006). This protein has been characterized and shown that it forms this structure with a cavity that is able to encapsulate other molecules. In the previous characterization it was shown that by selectively mutating five of the interior amino acids of the compartment to glutamate and by attaching a positively charged arginine tag to the C-terminus of the protein for targeting you can selectively target the tagged protein into the compartment (Lethbridge 2009 Modeling) (Seebeck et al., 2006). We will be using these features to selectively target our catechol-2,3-dioxygenase into the compartment.

Figure 1. Test construct for the characterization of the lumazine synthase (LS) microcomparments.

To characterize the means of targeting the tagged protein we will be using another expression construct as shown in Figure 1 that contains the IPTG inducible LS that also has two fluorescent proteins – cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) – that are controlled by an arabinose induced inverter. This would allow us to selectively express the LS by adding IPTG and repressing the fluorescent protein expression by adding arabinose. We have chosen to work with CFP and YFP due their ability to undergo fluorescence resonance energy transfer (FRET) that will allow us to observe their colocalization within the LS microcompartment (for a general overview of FRET visit Wikipedia or Lethbridge 2009 FRET). By observing FRET within the microcompartment it will demonstrate our ability to selectively localize multiple proteins within it.

With our ability to control the production of LS and the florescent proteins separately we will be able to determine the optimal conditions that will allow the colocalization of the proteins. By varying both the concentrations of arabinose and IPTG as well as the times of induction we will be able to determine the optimal conditions. Examples of conditions we will attempt in the future are shown in Figure 2. This will allow us to determine if the microcompartments form before entry of the proteins, after or simultaneously for a better understanding of the system.

(A)	(B)
(C)	(D) coming soon!
Figure 2. Theoretical lumazine synthase (LS) and fluorescent protein (FP) expression under different IPTG and arabinose (ara) concentrations. (A) Increasing concentrations of both ara and IPTG. As the concentration of IPTG increases the production of LS increases; as the concentration of ara increases the production of FPs decreases. (B) When initially induced with IPTG and ara, the production of LS would increase over time. The production of FPs would not occur until the ara is degraded by the cell (reducing it’s concentration), then the production of FPs would increase. (C) When there is no ara present the production of FP would immediately occur. LS production would not occur until IPTG was present. The conditions shown in B and C would allow us to determine the optimal expression conditions to get the maximum localization of protein within the compartment. We will determine this by using FRET between CFP and YFP. (D) The LS microcompartment with YFP and CFP localized within.

By characterizing LS microcompartment formation we will be able to optimize the system getting maximum protein withing the compartment. We will then use the comparment to isolate the catechol-2,3-dioxygenase (Figure 3), purify the complex and use for application on tailings ponds water. Once we have demonstrated that we are successfully able to isolate the catechol-2,3-dioxygenase we will then add more enzymes for the bioremediation of the tailings ponds.

Figure 3. Lumazine synthase microcomparment formation with catechol-2,3-dioxygenase within the compartment.

This work will be a proof of principle that will show the compartmentalization of the proteins within the LS microcomparment for use in other systems. By isolating pathways within the compartment it will increase the pathway's efficiency by localizing the products of each reaction to an area where the next enzyme in the pathway is located. We will apply this principle to our system so that we can isolate the microcompartments containing the pathway from the cell and apply it to the tailings. A tool such as we are working to produce and characterize for selectively targeting proteins into a compartment has many applications for any lab that is using molecular biology techniques such as isolateing a toxic protein from the cell and increasing a pathways efficiency.

References:
Seebeck et al. (2006). A simple tagging system for protein encapula8on. J. Am. Chem. Soc. 128, 4516‐4517