Difference between revisions of "Team:MIT/Experts"

In addition to our research in the human practices aspect of our project, we interviewed experts in the areas we were working in to best design our project for practical use. We incorporated the advice of Professor Kristala Jones Prather of the MIT Department of Chemical Engineering, Ben Woolston of Professor Gregory Stephanopoulos’s lab in the MIT Department of Chemical Engineering, and Jose A. Gomez of the Process Systems Engineering Laboratory in the MIT Department of Chemical Engineering.

Professor Kristala Jones Prather

Professor Prather is a professor in the Chemical Engineering Department at MIT. Her research focuses on using biocatalysis to optimize the production of small molecules in recombinant microorganisms.

Before the conversation, we aimed to focus on building continuous cultures because:

• They have higher productivities because batch cultures waste time with cleaning, sterilizing, filling, and extracting from the reactors
• There is no turnover time

During the conversation with Professor Prather, we learned that continuous cultures:

• Are rarely used in industry
• Are susceptible to contamination
• Are susceptible to genetic instability
• Suffer productivity losses due to genetic changes
• With the slow growth rate of C. hutchinsonii, we would need a very large industrial system before we could have pumps that could accurately make a continuous culture

We used this information to edit our original circuit design (See below, “The Impact on our Original Plan”). The interview with Prather aided in our decision to focus on building a circuit for batch co-cultures in place of continuous co-cultures.

Ben Woolston

Ben Woolston is a Ph.D candidate in the Stephanopoulos lab at MIT. He talked to us about the current state of the art of consolidated bioprocessing (CBP) to create fuel and where co-cultures stand.

During the conversation with Ben Woolston, we learned:

• Different metrics of success for bioprocessing:
  • Productivity - how quickly can your cell produce the goods
  • Yield - how much fuel at the end per how much cellulose at the beginning
  • Titer - gram/liter
  • Stability aspect
• State of the art of CBP, biofuels and co-cultures:
  • Not yet at industrial level
  • Companies tried but quit making butanol from lignocellulosics
  • No one has taken CBP to scale
  • Not cheap to produce enzymes to process lignocellulosics
  • Organisms are intolerant to their metabolic byproducts
  
  Jose A. Gomez
  
  Jose A. Gomez is one of the main developers of DFBALab, a collection of MATLAB functions that we used to run our dynamic flux balance analysis simulations (Gomez, J.A., Höffner, K. and Barton, P. I. (2014)). He advised us on how to best use DFBALab with our system. He helped us obtain a valid whole-genome scale metabolic model for C. hutchinsonii and advised us on how to implement various aspects of our system.
  

Initially, we planned to make a continuous co-culture. This plan seemed appealing for an industrial-sized co-culture, so that cellulosic feedstock, metabolic byproducts, and oxygen could be pumped in and out of the tank so that the co-culture could be the most productive. Our original circuit was designed to be optimal for a continuous co-culture. Image 1 below shows our original circuit. It consisted of a two-way communication system, in which each organism provides necessary nutrients and/or signals to the other. C. hutchinsonii produces Lux AHL constitutively. E. coliexpresses a suicide gene (RelE) when E. coli concentration is high and relies on the AHL signal from C. hutchinsonii to express an antidote gene (RelB). Due to the strong RBS, E. coli requires only a low concentration of the LuxR AHL to rescue itself. A strong LuxR signal (suggesting an abundance of C. hutchinsonii) will cause the LasR AHL to be sent from E. coli, activating the C. hutchinsonii suicide gene (also RelE).

Possible population states and outcomes:

• E. coli high/ C. hutchinsonii low - Plas (and RelE) effectively constitutively active in E. coli. E. coli won’t get enough antitoxin to save self, C. hutchinsonii won’t kill self. E. coli decreases, C. hutchinsonii increases.
• C. hutchinsonii high/E. coli low - C. hutchinsonii sends Lux AHL to E, promoting E. coli to save self. C. hutchinsonii stays high, E. coli increases.
• E. coli high/C. hutchinsonii high - Both signals active. C. hutchinsonii sends Lux AHL to E. coli, promoting E. coli to save self. E. coli sends Las AHL to C. hutchinsonii. E. coli increases slightly, C. hutchinsonii decreases slightly.