Team:MichiganState/Design

Design

Project goals

  1. Culture the endophytic bacteria in the roots of grass
  2. Characterize the endophytic bacteria to find good candidates for transformation
  3. Develop a transformation system for at least one of the endophytic bacteria
  4. Clone or synthesize an ACC deaminase from bacterial or fungal sources
  5. Transform the bacteria to allow for fluorescent labeling and ACC deaminase expression
  6. Demonstrate that the transformed bacteria could reinfect a grass and persist in roots
  7. Engineer a circuit to regulate expression as a response to abscisic acid
  8. Demonstrate plant growth promotion in a model grass

Goal 1: Culture the endophytic bacteria in the roots of grass

Our team aimed to find bacteria which could promote plant growth through the enhancement of plant growth promotion traits. We collected grasses, both wild and cultivated, and cultured the bacteria inside the roots after surface-sterilization. This gave us bacteria which were intimately associated with the plant and ideally more permanent, as opposed to the more ephemeral association with bacteria on the root surface or soil.

Goal 2: Characterize the endophytic bacteria to find good candidates for transformation

We aimed to find bacteria that could be easily engineered to symbionts within the plant, an affect the plant’s phenotype as desired. We therefore characterized the antibiotic resistance, growth rate, and ACC deaminase activity of the strains we isolated. For our project, we aimed for susceptibility to chloramphenicol and streptomycin (corresponding to the two selection genes we used on vectors). The strain FCP2-01, originating from switchgrass, was ideal based on these characters.

Goal 3: Develop a transformation system for at least one of the endophytic bacteria

We aimed to contribute a endophytic bacteria of grasses as a part to the Registry. The critical component of this goal is a simple and effective transformation protocol. We explored techniques including conjugation and electroporation. After consultation with Peiqi Zhang, a graduate student in Dr. Ken Obasa’s lab at University of Florida, we developed an effective electroporation protocol. Our attempts at electroporation were based on protocol used successfully on other organisms. We evaluated parameters such as cell washing number and solutions, DNA concentration, pre- and post-electroporation incubation, and voltage of electroporation.

Goal 4: Clone or synthesize an ACC deaminase from bacterial or fungal sources

Dr. Gregory Bonito’s Lab at Michigan State University maintains a culture collection of fungi and bacteria isolated from plants and soil. Several of these isolates have confirmed or suspected acdS genes (coding for ACC deaminase), including Fusarium oxysporum, Trichoderma sp., Ralstonia picketii, and Burkholderia sp. From our own culturing, we isolated Pseudomonas and Burkholderia cultures which expressed ACC deaminase activity. We were successfully able to clone acdS from Pseudomonas sp. 1C2P-04, isolated from a wild grass species.

Goal 5: Transform the bacteria to allow for fluorescent labeling and ACC deaminase expression

We first attempted to transform the bacteria using a simple fluorescent expression vector, and we used BBa_K608011 for this purpose. After many attempts were were unable to see expression with this vector, and we attempted to express acds in bacteria and detect its expression by plating on DF-ACC agar. These trials were unsuccessful, and we transitioned back to proof-of-concept using fluorescent markers. After finally observing fluorescent colonies, we proceeded to construct the fusion protein acdS-eGFP in the part BBa_K2633000. This part was first built using cloned sequences and was not restriction legal. A later synthesized version was constructed. While our transformed bacteria have been observed to be fluorescent, we have not observed ACC deaminase activity in the transformed bacteria.

Goal 6: Demonstrate that the transformed bacteria could reinfect a grass and persist in roots

Multiple attempts were made to infect Brachypodium distachyon, a model grass species, with our transformed bacteria. Early on we experienced poor germination rates, likely because the seeds were of poor quality. After our first plants had grown to maturity and produced seeds, we then had high quality seed for growing plants. We then inoculated plants with wild-type, eGFP expressing, and acdS-eGFP expressing FCP2-01, using seed soaking. Seed soaking was used instead of addition to soil to address concerns about transfer between plants and the spraying of transformed bacteria. We observed these plants each week for 1-5 weeks post inoculation, and took micrographs using confocal microscopy.

Goal 7: Engineer a circuit to regulate expression as a response to abscisic acid

Plants produce abscisic acid in response to drought conditions, and this molecule leads to a dimerization of two proteins. We aimed to use this interaction to regulate expression of ACC deaminase in bacteria. We did not get to developing this circuit due to time constraints. We hypothesize that this regulation can reduce impacts on normal plant physiology in the absence of drought conditions.

Goal 8: Demonstrate plant growth promotion in a model grass

This was our ultimate project goal, which we may have achieved if plants germinated in the early trials and our transformation techniques were developed earlier. Our plan was to use the inoculation technique in Goal 6, apply stresses to the plants, and measure characters of the plants after a growth period of 8 weeks. This would allow us to measure yield, biomass, and ethylene pathway genes and metabolites. Evidence for plant growth promotion will increment from sterile environment in growth chamber, to non-sterile in growth chamber, to field trials.