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Team:Minnesota/Project
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Overall project
Metabolic Engineering: In vivo Nanobioreactors
Modern microbial engineering methods allow the introduction of useful exogenous metabolic pathways into cells. Metabolism of certain organic compounds is sometimes limited by the production of toxic intermediates. Several bacteria have evolved protein based microcompartments capable of sequestering such reactions, thus protecting cytosolic machinery and processes from interference by these intermediates. For our project, we will identify and transform the genes encoding proteins responsible for the production and assembly of bacterial microcompartments. Additionally, we will confirm the signal sequences that target enzymes to the protein compartments by fusing this sequence to reporter genes. To demonstrate the microcompartment’s potential to serve as nanobioreactors, we will target genes encoding a short catabolic pathway into recombinant microcompartments assembled in E. coli.
Athina Software Package
Automated Theoretical Interaction Network Assembly is a web service to transform a sequence of BioBricks, or any other set of biomolecular components, to a set of reactions that can be simulated dynamically. The user simply inputs the Biobricks and their relationships and Designer builds a reaction network using biological interactions rules. This software package also includes a wiki. This is a web service to collect the kinetic parameters necessary to create a model that can be simulated.
Project Details: In vivo Nanobioreactors
For application in synthetic biology, bacterial microcompartments could be useful tools for engineering metabolic pathways for two important reasons. First, they can be used to sequester a toxic intermediate away from the cytoplasm. Second, by co-localizing enzymes inside a microcompartment the rate of diffusion of intermediate between enzymatic steps is decreased, thereby improving efficiency.
Part 2
The Experiments
Methods for obtaining Eut SMNLK construct: Specific primers containing BglII and NotI sites were designed for five genes on the Eut operon, EutL and EutK were cloned as a single PCR fragment, as were EutM and EutN. EutS was cloned separately. Each PCR product was ligated into the pucBB plasmid. The EutMN and EutS constructs were then digested with EcoRI and SpeI and gel purified to prepare entry fragments. The EutLK construct was linearized with EcoR1 and XbaI. A EutMNLK construct was obtained by ligating the EutMN fragment into the linearized pucBB-EutLK plasmid. This EutMNLK plasmid was then linearized with EcoRI and XbaI and a final ligation was performed with EutS as the entry fragment.
Expression of the compartment associated proteins was confirmed by an SDS-PAGE. To further verify that expression of these genes yields functional microcompartments, the Eut SMNLK plasmid was co-transformed with a plasmid containing GFP fused to a microcompartment specification sequence.
In order to add Eut signal sequences onto GFP so that it could be imported into the BMC it was necessary to design primers with the signal sequence and restriction sites extending beyond the hybridizing domain of the primer. This creates an obstacle when carrying out the PCR reaction because the melting temperature of the primer when hybridized to the template DNA varies greatly from that of the primer when bound to the extended second round products. In order to overcome this problem, the thermocycler program must feature a short initial round of denaturing, annealing and extension with an annealing temperature lower than the melting point of the primer portion that hybridizes to the template. After a few cycles, enough of the second round product should have accumulated for the full length of the primer to hybridize to. For the reaction to continue, a second set of cycles must be included with a higher annealing temperature.
