Utilizing MAGE to optimize E. coli for production of...
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Utilizing MAGE to optimize E. coli for production of bioplastics Background Methods Results Future Work and Outreach Acknowledgements Poly(lactic acid) (PLA) Poly(lactic acid) (PLA) is a versatile plastic because it is: • Biodegradable- microorganisms break it down into water and carbon dioxide. • Biocompatible- immune response is minimal • Bioresorbable- allows it to be resorbed into the body • Thermoplastic- enables melting and molding in a cast or deposition from a 3D printer Problems with Conventional Syntheses Cost Chemically synthesized PLA chemically are quite expensive: one gram of pure PLA costs around $90. Environmental Impact The processing and purifying steps require many chemicals that are environmentally unfriendly. Biosynthesis of PLA The KAIST research group under Sang Yup Lee successfully produced PLA with high efficiency by introducing two heterologous genes into E. coli: propionate CoA transferase and a polyhydroxyalkanoate synthase. Multiplex automated genome engineering (MAGE) MAGE is “recombineering on steroids.” The single-stranded DNA- binding protein, Beta, guides ssDNA fragments to the lagging strand of the replication fork for genetic incorporation. Repeated cycles of transformation with mutagenic oligos generate incredible diversity among cell populations that can be used to rapidly optimize biological pathways or generate new genetic codes. Synthesizing and Assembling the Heterologous Enzymes Detecting PLA production As a method of detection, we decided to use the fluorescent dye Nile red, an intracellular lipid strain. Nile red does not affect the growth of bacteria, is specific to PHA’s, and its fluorescence is quenched in water. Bioassay using Fluorescence Activated Cell Sorting (FACS) In order to quickly screen the large diversity created using MAGE, we employed fluorescence-activated cell sorting (FACS). FACS was able to sort cells above a certain gate of fluorescence, allowing us to select for those cells with the highest efficiency of PLA production Spiekermann et al. 1999 Graph 2: 15 cell in P2 Graph 3: 98 cell in P2 FACS Sorting after MAGE We would like to thank the Isaac’s lab. Also, we would like to thank our numerous sponcers Yale West Campus, Yale MB&B, Yale MCDB Yale Physics, Yale SEAS, Yale Sackler Institute, Keck Biotechnology, IDT, New England Biolabs, Biomatters, Mendeley. References Jung, Y. K., Kim, T. Y., Park, S. J. and Lee, S. Y. "Metabolic Engineering of Escherichia Coli for the Production of Polylactic Acid and Its Copolymers. ." Biotechnol. Bioeng (2010): 161-71. Print. Spiekermann, P., Rehm, B. H., Kalscheuer, R., Baumeister, D., Steinbuchel, A. "A Sensitive, Viable-Colony Staining Method Using Nile Red for Direct Screening of Bacteria That Accumulate Polyhydroxyalkanoic Acids and Other Lipid Storage Compounds." Arch Microbiol 171.2 (1999): 73-80. Print. Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM. "Programming Cells by Multiplex Genome Engineering and Accelerated Evolution." Nature (2009): 894-98. Print. Yang, T. H., Kim, T. W., Kang, H. O., Lee, S. H., Lee, E. J., Lim, S. C., Oh, S. O., Song, A. J., Park, S. J., Lee, S. Y. "Biosynthesis of Polylactic Acid and Its Copolymers Using Evolved Propionate Coa Transferase and Pha Synthase." Biotechnol Bioeng 105.1 (2010): 150-60. Print. Yang, T. H., et al. "Tailor-Made Type Ii Pseudomonas Pha Synthases and Their Use for the Biosynthesis of Polylactic Acid and Its Copolymer in Recombinant Escherichia Coli." Appl Microbiol Biotechnol 90.2 (2011): 603-14. Print. Biomedical Disposables 3D Printing Gene Knockouts (Cumulative) Growth Rate (1/hr) None 1.6531 adhE 1.6531 ackA 1.6531 pfl 1.6531 frd 1.6531 pta/eutD 1.6531 ppc 1.6471 Flux Balance Analysis In order to make sure that knocking out these genes would not impact the fitness of the E. coli, we used flux balance analysis. The chart to the right shows that none of the KO caused a fitness hit besides ppc. KO oligos (11) ackA frdB frdD pflA pflB adhE frdA frdC ATOB PTA EUTD RBS oligos (20) LDHA ACS ATOB EUTD PTA MAEA MAEB PYKF ENO PGM PGK GAPA TPIA FBAA FBAB PFKA PFKB GLK PGL RPOS MAGE Targets Validating PLA production When our plasmid was transformed into E. coli, it showed an increased Nile red fluorescence compared to the wild type strain We designed oligos to target 31 different genetic loci, including 11 to knock out genes, and 20 to tune the ribosome binding sites. Results from FACS sorted cells After a single iteration of MAGE and FACS sorting, we saw a twofold increase in Nile red fluorescence. Initially, the wild type had very few cells with significant levels of fluorescence. We hypothesize that the second smaller peak represents those cells that have accumulated enough PLA to be detected. The distribution of the second peak has a larger range after MAGE, indicating a diversity in the ability to produce PLA. Glycolysis Citric Acid Cycle Synthesized as 500 base Gblocks gene fragments with overlapping ends for Gibson Assembly Yale iGEM Wildtype Baseline w/ Plasmid 6 MAGE Cycles Graph 1: 1 cell in P1 PLA Secretion System To reduce the cost and environmental impact of chemical extraction methods, we will attempt to export PLA granules using a hlyA tag mediated export of the granule associated protein phasin Linton 2010 Customized Copolymer Synthesis By using MAGE on the active site of the PHA Synthase and supplementing the cells with 3HB, we can create a population of cells producing diverse PHA/P(3HB) copolymers with diverse physical and chemical properties Outreach to Local Business Met with the Cyalume Vice President of Technology, Earl Cranor, to discuss the issues affecting companies trying to develop biodegradable products and how we can attempt to use our plastic to make a prototype Sprout During the summer, we taught a 4 week, hands on class in synthetic biology to local middle and high school students that culminated in a presentation of our own research 2 # ∗ # # # 2 11 ∗ 2 10 20 = 3.29 ∗ 10 63
Transcript of Utilizing MAGE to optimize E. coli for production of...
Utilizing MAGE to optimize E. coli for
production of bioplastics
Background Methods Results Future Work and
Outreach
Acknowledgements
Poly(lactic acid) (PLA)
Poly(lactic acid) (PLA) is a versatile plastic because it is:
• Biodegradable- microorganisms break it down into water and carbon dioxide.
• Biocompatible- immune response is minimal
• Bioresorbable- allows it to be resorbed into the body • Thermoplastic- enables melting and molding in a cast or
deposition from a 3D printer
Problems with Conventional Syntheses Cost Chemically synthesized PLA chemically are quite expensive: one gram of pure PLA costs around $90. Environmental Impact The processing and purifying steps require many chemicals that are environmentally unfriendly.
Biosynthesis of PLA
The KAIST research group under Sang Yup Lee successfully produced PLA with high efficiency by introducing two heterologous genes into E. coli: propionate CoA transferase and a polyhydroxyalkanoate synthase.
Multiplex automated genome engineering (MAGE) MAGE is “recombineering on steroids.” The single-stranded DNA-binding protein, Beta, guides ssDNA fragments to the lagging strand of the replication fork for genetic incorporation. Repeated cycles of transformation with mutagenic oligos generate incredible diversity among cell populations that can be used to rapidly optimize biological pathways or generate new genetic codes.
Synthesizing and Assembling the Heterologous Enzymes
Detecting PLA production As a method of detection, we decided to use the fluorescent dye Nile red, an intracellular lipid strain. Nile red does not affect the growth of bacteria, is specific to PHA’s, and its fluorescence is quenched in water.
Bioassay using Fluorescence Activated Cell Sorting (FACS) In order to quickly screen the large diversity created using MAGE, we employed fluorescence-activated cell sorting (FACS). FACS was able to sort cells above a certain gate of fluorescence, allowing us to select for those cells with the highest efficiency of PLA production
Spiekermann et al. 1999
Graph 2: 15 cell in P2
Graph 3: 98 cell in P2
FACS Sorting after MAGE
We would like to thank the Isaac’s lab. Also, we would like to thank our numerous sponcers Yale West Campus, Yale MB&B, Yale MCDB
Yale Physics, Yale SEAS, Yale Sackler Institute, Keck Biotechnology, IDT, New England Biolabs, Biomatters, Mendeley.
References Jung, Y. K., Kim, T. Y., Park, S. J. and Lee, S. Y. "Metabolic Engineering of Escherichia Coli for the Production of Polylactic Acid and Its Copolymers. ." Biotechnol. Bioeng (2010): 161-71. Print.
Spiekermann, P., Rehm, B. H., Kalscheuer, R., Baumeister, D., Steinbuchel, A. "A Sensitive, Viable-Colony Staining Method Using Nile Red for Direct Screening of Bacteria That Accumulate Polyhydroxyalkanoic Acids and Other Lipid Storage Compounds." Arch Microbiol 171.2 (1999): 73-80. Print.
Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM. "Programming Cells by Multiplex Genome Engineering and Accelerated Evolution." Nature (2009): 894-98. Print.
Yang, T. H., Kim, T. W., Kang, H. O., Lee, S. H., Lee, E. J., Lim, S. C., Oh, S. O., Song, A. J., Park, S. J., Lee, S. Y. "Biosynthesis of Polylactic Acid and Its Copolymers Using Evolved Propionate Coa Transferase and Pha Synthase." Biotechnol Bioeng 105.1 (2010): 150-60. Print.
Yang, T. H., et al. "Tailor-Made Type Ii Pseudomonas Pha Synthases and Their Use for the Biosynthesis of Polylactic Acid and Its Copolymer in Recombinant Escherichia Coli." Appl Microbiol Biotechnol 90.2 (2011): 603-14. Print.
Biomedical Disposables 3D Printing
Gene Knockouts
(Cumulative)
Growth Rate (1/hr)
None 1.6531
adhE 1.6531
ackA 1.6531
pfl 1.6531
frd 1.6531
pta/eutD 1.6531
ppc 1.6471
Flux Balance Analysis In order to make sure that knocking out these genes would not impact the fitness of the E. coli, we used flux balance analysis. The chart to the right shows that none of the KO caused a fitness hit besides ppc.
KO oligos (11)
ackA frdB frdD pflA pflB adhE frdA frdC
ATOB PTA
EUTD
RBS oligos (20) LDHA ACS ATOB EUTD PTA MAEA MAEB PYKF ENO PGM
PGK GAPA TPIA FBAA FBAB PFKA PFKB GLK PGL RPOS
MAGE Targets
Validating PLA production When our plasmid was transformed into E. coli, it showed an increased Nile red fluorescence compared to the wild type strain
We designed oligos to target 31 different genetic loci, including 11 to knock out genes, and 20 to tune the ribosome binding sites.
Results from FACS sorted cells After a single iteration of MAGE and FACS sorting, we saw a twofold increase in Nile red fluorescence.
Initially, the wild type had very few cells with significant levels of fluorescence. We hypothesize that the second smaller peak represents those cells that have accumulated enough PLA to be detected. The distribution of the second peak has a larger range after MAGE, indicating a diversity in the ability to produce PLA.
Glycolysis
Citric Acid Cycle
Synthesized as 500 base Gblocks gene fragments with overlapping ends for Gibson Assembly
Yale iGEM
Wildtype
Baseline w/ Plasmid
6 MAGE Cycles
Graph 1: 1 cell in P1
PLA Secretion System To reduce the cost and environmental impact of chemical extraction methods, we will attempt to export PLA granules using a hlyA tag mediated export of the granule associated protein phasin
Linton 2010
Customized Copolymer Synthesis By using MAGE on the active site of the PHA Synthase and supplementing the cells with 3HB, we can create a population of cells producing diverse PHA/P(3HB) copolymers with diverse physical and chemical properties
Outreach to Local Business Met with the Cyalume Vice President of Technology, Earl Cranor, to discuss the issues affecting companies trying to develop biodegradable products and how we can attempt to use our plastic to make a prototype
Sprout During the summer, we taught a 4 week, hands on class in synthetic biology to local middle and high school students that culminated in a presentation of our own research
2# 𝑜𝑓 𝐾𝑂 𝑜𝑙𝑖𝑔𝑜𝑠 ∗ # 𝑜𝑓 𝑝𝑜𝑠𝑠𝑖𝑏𝑙𝑒 𝑛𝑢𝑐𝑙𝑒𝑜𝑡𝑖𝑑𝑒𝑠# 𝑜𝑓 𝑑𝑒𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑒 𝑛𝑢𝑐𝑙𝑒𝑜𝑡𝑖𝑑𝑒 𝑠𝑖𝑡𝑒𝑠 # 𝑜𝑓 𝑅𝐵𝑆 𝑜𝑙𝑖𝑔𝑜𝑠
211 ∗ 210 20 = 3.29 ∗ 1063
