Anne Ruffing

Principal Member of the Technical Staff

Anne-ruffing

Anne Ruffing joined Sandia in early 2010. As a Truman Fellow, Anne led her own research project on the Genetic Engineering of Cyanobacteria for Biodiesel Feedstock Production. In February of 2013, Anne converted to a technical staff position, in which she continues to research cyanobacteria and algae for biofuel production. Recent research in Anne’s laboratory applies metabolic engineering and synthetic biology approaches to develop novel biotechnologies for national security applications. Prior and ongoing projects in her laboratory include: (1) improving energy security by enabling photoautotrophic biofuel production, (2) developing novel biomonitoring technologies for the detection of chemical targets, and (3) applying synthetic biology for the development of biodefense technologies.

Education

Bachelor’s Degree: Chemical Engineering, University of Dayton (2000-2004)

Doctoral Degree: Chemical Engineering, Georgia Institute of Technology (2004-2010)

Postdoctoral Fellowships: President Harry S. Truman Fellowship in National Security Science and Engineering, Sandia National Laboratories (2010-2013)

During her graduate studies at the Georgia Institute of Technology, Ruffing worked with a polysaccharide-producing soil bacterium, Agrobacterium sp. ATCC 31749. Her thesis included metabolic engineering of the Agrobacterium for production of medically-important oligosaccharides, investigation of Agrobacterium physiology, and genomic and transcriptomic studies of polysaccharide production. As a Truman Fellow at Sandia National Laboratories, Ruffings’ work focused on the genetic modification of several cyanobacteria for the production of free fatty acids (FFAs) as feedstock for biodiesel production. This work characterized the physiological impact of FFA production, identified transcriptional changes associated with enhanced FFA production, and demonstrated the importance of cyanobacterial host selection.

Research Interests

Genetic Engineering of Cyanobacteria and Algae for Biofuel Production

Unlike eukaryotic algae, cyanobacteria are not natural lipid producers, yet cyanobacteria can be easily manipulated through genetic modification, have smaller genomes, and are regulated by prokaryotic genetics, making cyanobacteria ideal hosts for rational strain development. Ruffing’s research group has engineered the metabolisms of two model cyanobacteria, Synechococcus elongatus PCC 7942 and Synechococcus sp. PCC 7002, for the production of free fatty acids, a precursor for biodiesel production. The engineered strains were characterized using Sandia’s hyperspectral fluorescence imaging technology in addition to other traditional techniques, such as RNA-seq, to assess the effect of free fatty acid production on the cyanobacterium’s physiology and metabolism. The group seeks to continue to develop cyanobacteria for biofuel production through the application of high-throughput genome engineering technologies and integration with metabolic modeling and simulation.

Eukaryotic algae, such as Nannochloropsis gaditana, have shown promise for algal biofuel production, as they are natural lipid accumulators and demonstrate robust growth under outdoor production conditions. While additional strain development is necessary to achieve the desired characteristics for biofuel production, synthetic biology tools for genetic modification of eukaryotic algae are scarce. Ruffing’s group is currently developing tools for the genetic manipulation of N. gaditana, including acoustic-based delivery of DNA (i.e., sonoporation), characterized expression parts, and CRISPR-Cas9 editing technology. These tools will be used to engineer N. gaditana for robust growth under a range of environmentally relevant conditions.  

Algae Pond
Biotechnology and Bioengineering journal cover from 2012

Biosensors for the Detection of Chemicals

Biological organisms naturally sense chemicals in their environment through highly specific protein interactions. By leveraging and engineering these natural sensing systems, we can develop biological sensors that produce an easy-to-detect output signal in response to specific chemicals. Ruffing’s laboratory focuses on the development of biological sensors for the detection of metals, gases, and other small molecule targets. They use synthetic biology approaches to construct whole cell biosensors using natural proteins that have activity with a target chemical. This is combined with traditional and high-throughput protein evolution approaches to improve sensor function or evolve entirely novel detection capabilities. Additionally, they study natural responses in bacteria, algae, and plants to identify natural signatures that can be leveraged for chemical detection.

Synechococcus sp. PCC 7002 Illustration

Engineering Natural Microbial Defense

Mechanisms to Inhibit Pathogenic Bacteria Bacteria have evolved natural mechanisms to inhibit or kill off competitor bacterial species that extend beyond traditional antibiotics. Ruffing’s group seeks to engineer bacterial systems with these natural inhibition mechanisms for selective targeting of pathogenic bacteria. Their work has primarily focused on engineering contact-dependent growth inhibition (CDI), a protein-based method of growth inhibition that has been demonstrated in several pathogenic bacteria. They aim to express heterologous CDI operons in non-pathogenic E. coli to selectively target and inhibit pathogenic bacteria, including Pseudomonas aeruginosa, Burkholderia pseudomallei, and Yersinia pestis.

Illustration of expressing CDI in E. coli

Awards, Honors, and Memberships

  • President Harry S. Truman Fellow in National Security Science and Engineering (2009)
  • Philanthropic Educational Organization (P.E.O.) Scholar Award (2008-2009)
  • Outstanding Teaching Assistant Award, Georgia Institute of Technology (2007)
  • National Science Foundation Graduate Research Fellowship (2005-2008)

Selected Publications

  • Haddal CC, Bull DL, Pacheco Hernandez PM, Kistin Keller EJ, Heimer BW, Ruffing AM, and Aamir MS. Global security implications of chemical and biological innovation. European Commission Joint Research Centre’s Future Technology Assessment Conference Proceedings. June 2018. Brussels, Belgium.
  • Ruffing AM, Jensen TJ, and Strickland LM. Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis. Microbial Cell Factories. 2016. 15:190. doi: 10.1186/s12934-016-0584-6
  • Ruffing AM and Kallas T. Editorial: Cyanobacteria: The Green E. coli. Frontiers in Bioengineering and Biotechnology. 2016. Research Topic ebook. 4:7. doi: 10.3389/fbioe.2016.00007
  • Ruffing AM and Trahan CA. Biofuel toxicity and mechanisms of biofuel tolerance in three model cyanobacteria. Algal Research. 2014. 5:121-132.
  • Ruffing AM. Improved free fatty acid production in cyanobacteria with Synechococcus sp. PCC 7002 as host. Frontiers in Bioengineering & Biotechnology. 2014. 2:17.
  • Ruffing AM. RNA-Seq analysis and targeted mutagenesis for improved free fatty acid production in an engineered cyanobacterium. Biotechnology for Biofuels. 2013. 6:113.
  • Ruffing AM. ­­Borrowing genes from Chlamydomonas reinhardtii for free fatty acid production in engineered cyanobacteria. Journal of Applied Phycology. 2013. 25(5): 1495-1507.
  • Ruffing AM and Jones HDT. Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942. Biotechnology and Bioengineering. 2012. 109(9): 2190-2199. (Cover, Spotlight)
  • Ruffing AM and Chen RR. Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis. Microbial Cell Factories. 2012. 11:17. Reichardt TA, Garcia OF, Collins AM, Jones HDT,
  • Ruffing AM, and Timlin JA. Spectroradiometric monitoring of Nannochloropsis salina growth. Algal Research. 2012. 1(1): 22-31.
  • Ruffing AM. Engineered Cyanobacteria: Teaching an old bug new tricks. Bioengineered Bugs. 2011. 2(3).
  • Ruffing AM, Castro-Melchor M, Hu W-S, and Chen RR. Genome sequencing of the curdlan-producing Agrobacterium sp. ATCC 31749. Journal of Bacteriology. 2011. 193(16): 4294-4295.
  • Ruffing AM and Chen RR. Citrate stimulates oligosaccharide synthesis in metabolically engineered Agrobacterium sp. Applied Biochemistry and Biotechnology. 2011. 164(6): 851-866.
  • Ruffing AM and Chen RR. Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope. Microbial Cell Factories. 2010. 9(1).
  • Ruffing AM and Chen RR. Metabolic engineering of microbes for oligosaccharide and polysaccharide synthesis. Microbial Cell Factories. 2006. 5: 25-33.
  • Ruffing AM, Mao Z, and Chen RR. Metabolic engineering of Agrobacterium sp. for UDP-galactose regeneration and oligosaccharide synthesis. Metabolic Engineering. 2006. 8(5): 465-473.

Selected Book Chapters

  • Ruffing AM. Metabolic Engineering and Systems Biology for Free Fatty Acid Production in Cyanobacteria. Book chapter in Cyanobacteria: Omics and Manipulation. Editor: Dmitry A. Los. Caister Academic Press. Poole, UK. 2017. p161-186. doi: 10.21775/9781910190555.08.
  • Ruffing AM. Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production. Book chapter in Liquid, Gaseous and Solid Biofuels – Conversion Techniques. Intech, Rijeka, Croatia. 2013.
  • Ruffing AM and Chen RR. Metabolic Engineering of Microorganisms for Oligosaccharide and Polysaccharide Production. Book chapter in Microbial Production of Biopolymers and Polymer Precursors: Applications and Perspectives. Horizon Bioscience, Wymondham, UK. 2009. p197-228.
  • Ruffing AM and Chen RR. Metabolic Engineering and Other Methods of Strain Improvement. Book chapter in Advances in Fermentation Technology. Asiatech Publishers, Inc. New Delhi. 2008. p119-144.

Selected Patents

  • Reichardt TA, Collins AM, Garcia OF, Ruffing AM, Timlin JA, and Jones HDT. Spectroradiometric Monitoring of Algal Growth. 2013. US patent application: 13869854.
  • Carnes E, Ruffing AM, and Ashley C. Delivery Platforms for the Domestication of Algae and Plants. 2015. US patent application: 14/871,748.