Achieving greater microbial aciduricity through the Escherichia coli cyclopropane fatty acid systemConary Meyer, Alex Codik, Nicole Mattson, Campbell Yore, Joseph Ayer, Danielle Mercado, Robert Kousnetzov, Santa Clara University

WHY BIOREACTORS?Bioreactor technology allows us, as synthetic biologists, to take our technology to the large scale. It denotes cell culture methods that allow for the mass produc-tion of organisms and their products by facilitating growth in various apparatuses. These systems allow for the mass production of useful products such as biofuels and therapeutics, as well as the degradation of waste products.

REFERENCES

ACKNOWLEDGEMENTS

Figure 2: To decrease the permeability of the membrane to small molecules, such as weak acids and bases, the Cycloproprane Fatty Acid (CFA) synthase uses S-Adenosyl Methionine (SAM) as a methyl donor to add a protruding carbon to the phospholipid tails. The added carbon then acts as a steric hin-derance to the incoming small molecules, decreasing its likelihood to cross the membrane.

OUR SOLUTION To help alleviate the acidification problem, we wanted to create a system that was resilient to the steep pH gradients created by the metabolic acids as well as the in-fused base. We decided to target the membrane as it is the interface for transport in the cell. We sought to bolster the membrane so that it was less permeable.

Figure 3: Schematic displaying the mechanism of the CFA acid resistance system.

OUR RESULTSBelow are the results from some of our preliminary trials as well as our experimental validation of our composite part. In the initial trials, we wanted to extrapolate a gener-al trend between the pH and growth kinetics. We found that there is a direct correlation between the pH level and the number of intact cells, derived from optical density read-ings. The growth rate also follows this relationship.

POLICY & PRACTICESOur three phase integrated human practices study 1) presents the Student Re-search Collaborative, a organizational solution designed to help iGEM teams con-tinue to develop their technology outside of the competition, 2) provides a suite of legal forms iGEM teams can use to organize corporate partnerships and a summa-ry of our team’s efforts to organize a collaboration with corporate entity, Anaerobe Systems, and 3) introduces three former iGEM participants who used their expe-rience in the competition to found their own technology startups.

Akshay SethiAmbercycle

iGEM 2012: UC Davis

Freedans FerdinandMorph Bio

iGEM 2012: University College of London

David BrownMycodev

iGEM 2012: University ofAlberta Upcycled Aromatics

University of Michigan SoftwareStrategies for resolving and avoiding IP

Conflicts

University of BielefeldBest Practices for Reducing the Risk

Associated with the Disclosing Dual Use Technologies

COLLABORATIONS

CONCLUSIONSFrom our data, we can conclude that the overexpression of CFA increases resilience to acid. The experimental data follows suit with the observed trend in the priliminary data which depicted that decreasing pH resulted in a decrease in cell count and growth rate. This correlation between the graphs leads us to believe that the cells expressing CFA for the ample amount of time are seeing the effects of cells seen exposed to less acidity. It was found that CFA requires at least 30 minutes to cyclopropanate the membrane and resume standard growth. We hypothesize that the decrease in the number of intact cells as well as the increased rate of cell lysis seen for the 0 and 15 minute induction cultures is resultant of the shift in metabolism in those cells to create CFA sythase instead of its standard pathways leading to an increased susceptibility to acid.

I would like to thank the Provost Office, Engineering and Bioengineering Departments of Santa Clara University for their generous funding of this project. Also the Biology and Bioengineering De-partments for the use of their facilities and equipment. Thank you to IDT, NEB, BioBricks Founda-tion, and the Intellectual Property Law Investment Group for sponsoring our program. We would also like to thank all of those that helped us along the way: Drs. Linda Kahl, Christelle Sabatier, Tracy Ruscetti, Jonathan Zhang, and Angel Islas.

[1] Chang, Y.-Y. and Cronan, J. E., “Membrane cyclopropane fatty acid content is a major factor in acid resistance of Esche richia Coli.” Molecular Microbiology 33 (1999): 249-59.[2] Cotter, P.D., and C. Hill., “Surviving the Acid Test: Responses of Gram-Positive to Low pH.” Microbiology and Moleclar Biology Reviews 67.3 (2003): 429-53.[3] Grogan, D., and J. Cronan., “Cyclopropane Ring Formation in Membrane Lipids of Bacteria.”Microbiology and Molecu- lar Biology 61.4 (1997): 429-41.[4] Kobayashi Hiroshi, Hiromi Saito, and Tomohito Kakegawa., “Bacterial Strategies to Inhabit Acidic Environments.” The Journal of General and Applied Microbiology J. Gen. Appl. Microbiol. 46 (2000): 235-43.[5] Li, Feng et al., “Cell Culture Processes for Monoclonal Antibody Production.” mAbs 2.5 (2010): 466-477.[6] Taylor, F., and J. E. Cronan., “Selection and Properties of Escherichia Coli Mutants Defective in the Synthesis of Cyclo propane Fatty Acids.” Journal of Bacteriology 125.2 (1976): 518-523.

THE PROBLEMOf the current issues limiting the efficiency of bioreactors, we chose to target the problem of increased acidification in continuous culture systems.

Figure 1: Depiction of the Acidification problem found in bioreactors as well as the current solution.

60 and 30 minUninduced

15 min0 min

pH 5pH 7

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pH 3

Our experimental trials followed the same protocol as the one used in the preliminary trial. Our data suggests that with ample time allotted for the membrane to be cyclopro-ponated, the cells react in a similar way as the cells shocked with less acid. There is an observable increase in the number of intact cells between the uninduced cultures and those expressing CFA for at least 30 minutes. There is also a decrease in the rate of cell lysis observed as CFA is allowed more time to alter the membrane.

FUTURE DIRECTIONSWe plan to further elucidate the phenotypic effects of CFA expression by running more trials at varying pHs to see if the same positive trend, showing a decrease in cell lysis from exposure to acid, is seen. Alongside these experiments, we would run vitality as-says by plating the cells at each time point to check for growth along with the OD read-ings. We would also run the test at high pH to determine its potentcy when faced with alkali conditions. Once we are certain of the effect in small scale, we would move to a chemostat and then a small bioreactor to see if the effect of CFA is consistent.

Figure 3: Acid shock assay of T7 expression cells. Cells were grown at 37˚C at 250 rpm, n=2 average shown. A. Number of Cells After Acid Shock of T7 Expression Cells. Time points taken every 30 minutes by spec-trophotometry, measured at 600nm. Cells were resuspended in varying pH LB’s, pH 7 (diamonds), pH 5 (squares), pH 4 (triangles), and pH 3 (squares). B. Effects of pH on the growth rate of T7 Expression cells. Cells grown in pH 7 LB until mid-log (OD600~0.6) then acid shocked into LB pH 3.

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Figure 3: Acid shock assay of CFA Mutant Cells. Acid shock occurred when cells were in mid Cells were grown at 37˚C at 250 rpm, n=3 average shown with ±1 SEM. A. Number of CFA mutant cells after acid shock. Time points taken every 30 minutes by spectrophotometry, measured at 600nm. Cells were induced with 1mM IPTG 0 (lines), 15 (circles), 30 (triangles), 60 minutes (squares), and uninduced (diamonds). B. Growth rate of CFA mutant cells that were induced before acid shock. Cells were induced with 1mM IPTG as indicat-ed minutes before acid shock.

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Phase I

This graphic depicts the stages of a student research collaborative: idea conception; dry lab research; technology development; and beginning commercialization. Blue circular arrows symbolize the nonlinear, iterative process of converting the prod-ucts of one stage to the products of next stage in the collaborative’s lifecycle. Enti-ties involved in each stage are displayed in gold boxes with arrows linking each en-tity to their contributions to the system.

Phase II

Phase III

Non-Disclosure Agreement

1. Santa Clara University iGEM Team’s goal and the relationship with Anaerobe Systems

2. How the relationship may form - what are its drivers?

Material TransferAgreement

Licensing Agreement

3. The reality of the situation – what are the consequences to infringement?

Displays the thought process of how an iGEM team selects the appropriate legal instrument to govern a relationship or situation. Important factors to consider in-clude: the goal and purpose of a potential collaboration, each party’s motivations, and the consequences of not executing a formal agreement.

We conducted thorough interviews with each entrepreneur discussing the signifi-cance of their iGEM experience, the resources student entrepreneurs need to suc-ceed, and their approach to intellectual property.