Difference between revisions of "Team:Virginia/Poster"

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<ul><b>Part Expression Optimization</b> - We used the Anderson RBS/Promoter libraries and the Salis Lab RBS Calculator to effectively control part expression.
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<b>Part Expression Optimization</b> - We used the Anderson RBS/Promoter libraries and the Salis Lab RBS Calculator to effectively control part expression.
 
<li><b>Relevance</b> - This process directly assisted our wet lab team by using quantification to determine the RBS\Promoter sequences for each part.</li>
 
<li><b>Relevance</b> - This process directly assisted our wet lab team by using quantification to determine the RBS\Promoter sequences for each part.</li>
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<li><b>Model Validity</b> – We utilized a mass action model to validate the RBS/Promoter combinations for parts required in creation of DNA Scaffolds. </li>  
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<li><b>Model Validity</b> – We utilized a mass action model to validate the RBS/Promoter combinations for parts required in creation of DNA Scaffolds. </li> <br><br>
 
<b>BMC Pore Dynamics</b> - We used PyMOL to visualize BMC pore interactions for acetate in comparison to natural BMC metabolites. These pore energies were then modeled using a mass action, three-compartment system to quantify BMC concentrations at quasi-steady state.
 
<b>BMC Pore Dynamics</b> - We used PyMOL to visualize BMC pore interactions for acetate in comparison to natural BMC metabolites. These pore energies were then modeled using a mass action, three-compartment system to quantify BMC concentrations at quasi-steady state.
 
<li><b>Relevance</b> - This process generated a bounded estimation for the acetate concentration available for our compartmentalized pathway.</li>
 
<li><b>Relevance</b> - This process generated a bounded estimation for the acetate concentration available for our compartmentalized pathway.</li>
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<li><b>Model Validity</b> - The compartment model is derived from Arrhenius and Fick’s Law equations and uses pore energies determined in silico. Further validation of this approach will require in vitro testing.</li>
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<li><b>Model Validity</b> - The compartment model is derived from Arrhenius and Fick’s Law equations and uses pore energies determined in silico. Further validation of this approach will require in vitro testing.</li><br><br>
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<b>Reaction Kinetics<b> - We used Michaelis-Menten equations to determine theoretical resveratrol concentration over time in our BMC system and a shell-free system.
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<b>Reaction Kinetics</b> - We used Michaelis-Menten equations to determine theoretical resveratrol concentration over time in our BMC system and a shell-free system.
 
<li><b>Relevance</b> - This process quantified the expected increase of resveratrol production in the BMC system compared to a shell-free system. MANIFOLD improves the resveratrol titer by a factor between 206.5 and 434.2 compared to a shell-free system after 12 hours.</li>
 
<li><b>Relevance</b> - This process quantified the expected increase of resveratrol production in the BMC system compared to a shell-free system. MANIFOLD improves the resveratrol titer by a factor between 206.5 and 434.2 compared to a shell-free system after 12 hours.</li>
 
<li><b>Model Validity</b> - Reaction kinetic models utilize parameters established in vitro and shell-free titer appears consistent with previous studies.(https://aem.asm.org/content/77/10/3451) The model relies on inputs from the pore dynamics methodology suggesting the necessity of in vitro studies for further model validation.</li></ul></div>
 
<li><b>Model Validity</b> - Reaction kinetic models utilize parameters established in vitro and shell-free titer appears consistent with previous studies.(https://aem.asm.org/content/77/10/3451) The model relies on inputs from the pore dynamics methodology suggesting the necessity of in vitro studies for further model validation.</li></ul></div>

Revision as of 04:20, 10 November 2020

Manifold: Protein Shells with Encapsulated DNA Scaffolds for Increasing Efficiency of Biosynthetic Pathways
Presented by Team Virginia 2020

Team: J. Ball, V. Gutierrez, C. Haws, A. Kola, S. Link, C. Marino, E. Micklovic, D. Patel, J. Polzin, A. Pradhan, P. Revelli

Advisors: K. G. Kozminski, J. A. Papin

Abstract:
The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.
Inspiration
The field of synthetic biology has seen great success in utilizing bacteria and yeast to produce chemicals of medical and industrial value, but these methods still have the potential to be improved. In many cases, prokaryotes are structurally simpler and generally easier to engineer than eukaryotes, but do not possess a simple way to compartmentalize reaction pathways like eukaryotic organelles do. We strove to combine the ease of use of prokaryotes with the compartmentalization capability of eukaryotes by developing a method to synthetically isolate a simple biosynthetic pathway - resveratrol synthesis - within a prokaryotic system.
4 Big Problems in Biosynthesis
  • Flux imbalances occur when the amount of substrate available does not match the efficiency of the enzyme. In multi-enzyme pathways, this results in an overabundance or lack of intermediates, even with careful promoter and ribosome binding site choice.
  • Loss of intermediates can occur as pathway intermediates cross a membrane or move to a region of the cell where pathway enzymes are not present and thus, overall yields are reduced.
  • Pathway competition also reduces yield. If an enzyme, substrate, intermediate, or coenzyme of the pathway of interest is utilized by a process native to the cell it will be less likely to be available for the desired pathway.
  • Toxic intermediates can cause harm or cell death before any product can be made making biosynthesis highly inefficient or impossible for many pathways.
Proposed Solution
    Manifold is a platform technology designed to improve the efficiency of biosynthesis in prokaryotes by combining DNA scaffolds with BMC shells.
  • Short double-stranded DNA scaffolds with zinc-finger binding motifs are produced in vivo by reverse transcriptases.
  • Scaffolds localized to the lumen of a PDU BMC shell via an interaction with a zinc-finger fusion shell protein.
  • As the shell assembles, zinc-finger fusions of the pathway enzymes bind to the scaffolds, allowing for localization of the enzymes to the BMC interior.
  • The combination of scaffolds and BMCs creates a comprehensive solution to the compartmentalization and organization needs.
  • Proteinaceous BMC shells solve the problems of lost intermediates, pathway competition, and toxic intermediates by sequestering the pathway.
  • Scaffolds provide a convenient way to target enzymes to the interior of the BMC in controllable ratios to prevent flux imbalances.

    • Manifold can optimize metabolic flux by creating pathway orthogonality, which will actualize potential efficiency of existing biosynthetic pathways and allow for the development of previously impossible ones.
Idea
    Resveratrol biosynthesis was chosen for a proof of concept due to its simplicity and capacity to be improved through scaffolding. (Cite)
  • Biosynthesis of resveratrol from p-Coumaric acid requires two enzymes, 4CL and STS, and the coenzyme malonyl-CoA.
    • Figure of resveratrol pathway
  • Steric hindrance of malonyl-CoA makes it unlikely to diffuse into the BMC through a pore.
    • Hindrance pore model
  • Malonyl-CoA must be available within the BMC to allow for resveratrol production.
  • ACC and ACS produce malonyl-CoA from acetate.
    • Figure of acetate pathway
  • Introduction of an additional scaffold with ACC and ACS fusion enzymes allows malonyl-CoA recycling within the BMC.
Modeling
    Part Expression Optimization - We used the Anderson RBS/Promoter libraries and the Salis Lab RBS Calculator to effectively control part expression.
  • Relevance - This process directly assisted our wet lab team by using quantification to determine the RBS\Promoter sequences for each part.
  • Model Validity – We utilized a mass action model to validate the RBS/Promoter combinations for parts required in creation of DNA Scaffolds.


    • BMC Pore Dynamics - We used PyMOL to visualize BMC pore interactions for acetate in comparison to natural BMC metabolites. These pore energies were then modeled using a mass action, three-compartment system to quantify BMC concentrations at quasi-steady state.
  • Relevance - This process generated a bounded estimation for the acetate concentration available for our compartmentalized pathway.
  • Model Validity - The compartment model is derived from Arrhenius and Fick’s Law equations and uses pore energies determined in silico. Further validation of this approach will require in vitro testing.


    • Reaction Kinetics - We used Michaelis-Menten equations to determine theoretical resveratrol concentration over time in our BMC system and a shell-free system.
  • Relevance - This process quantified the expected increase of resveratrol production in the BMC system compared to a shell-free system. MANIFOLD improves the resveratrol titer by a factor between 206.5 and 434.2 compared to a shell-free system after 12 hours.
  • Model Validity - Reaction kinetic models utilize parameters established in vitro and shell-free titer appears consistent with previous studies.(https://aem.asm.org/content/77/10/3451) The model relies on inputs from the pore dynamics methodology suggesting the necessity of in vitro studies for further model validation.
Section 2.1
Use this section to explain whatever you would like! Suggestions: Safety, Human Practices, Measurement, etc.
Section 2.2
Use this section to explain whatever you would like! Suggestions: Safety, Human Practices, Measurement, etc.
Section 3
Use this section to explain whatever you would like! Suggestions: Safety, Human Practices, Measurement, etc.
Results
What did your team achieve? What do you plan to work on moving forward?
References and Acknowledgements
If not already cited in other sections of your poster, what literature sources did you reference on this poster? Who helped or advised you?