A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection

Outcomes• Structural studies reveal that an unprecedented

12 Å translocation of pigment accompanies the activation of the OCP for photoprotection.

• XF identifies amino acid residues that drive the dramatic reconfiguration of protein-pigment interactions for the photoprotective response.

• Such functionally relevant plasticity in protein-chromophore interactions has not previously been described for any pigment-protein system.

1) Crystal structures of the Orange Carotenoid Protein (OCP) and Red Carotenoid Protein (RCP) binding canthaxanthin (CAN). (A) Superimposed ribbon structures of OCPCAN (grey) and RCPCAN (red). The CAN is shown in orange sticks in OCP, purple sticks in RCP. (B) CAN structures in OCP and RCP show increased planarity of the polyene chain in RCP and distinctly different β-ring configurations.

Leverenz, et al. (2015) “A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection ” Science, 348(6242), 1463‐66, doi: 10.1126/science.aaa7234 

Background• Pigment-protein and pigment-pigment

interactions are critical for light harvesting and photoprotective functions essential to photosynthesis.

• The primary photoprotective mechanism in cyanobacteria is activated by the photoactive Orange Carotenoid Protein (OCP).

Significance• These results reveal an unprecedented type of conformational switch for

photoprotection and prompt re-examination of other photoprotective and light harvesting systems.

2) Solvent accessibility changes in the OCP complex as measured by x-ray hydroxyl radical footprinting. A) Peptide modification as a function of x-ray irradiation dose for W41, residue clusters W41-F42-Y44-M47 and P276-W277-F278, and M284. (B) Structural view of the CAN binding residues of the OCPCAN structure .

Approach• Determine the atomic resolution structure of an

active form of OCP combined with solution-state structural data from x-ray radiolytic labeling and proteomic analysis (X-ray Footprinting, XF).

Automating Synthetic Biology using a Droplet Microfluidic Platform

Outcomes• Microfluidic chip successfully integrated DNA assembly and transformation (by electroporation) of cells• Multiple assembly methods such as golden gate, Gibsn and yeast were implemented. • Chip-based assembly matches well with conventional DNA assembly method

Shih et al. (2015). "A Versatile Microfluidic Device for Automating Synthetic Biology". ACS Synth Biol. doi, 10.1021/acssynbio.5b00062

Background• Current synthetic biology

experimental protocol too slow, uses large amount of reagents, and manual.

• Alternative high throughput platforms are needed that use less reagents and can be automated

Approach• Develop a droplet microfluidic

platform to carry out DNA assembly and transformation. Automate the entire process for hands-free operation.

Significance• Chip-based method is faster and drastically reduces the reagent consumption• Has the potential to significantly reduce the design-build-test cycle for synthetic biology

Assembled 16 cassettes using Golden Gate, Gibson, and yeast –

95% sequence matching

1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).

Background• Raoultella terrigena is a nitrogen fixing bacterium that can be found as endophyte in

plants.• Endophytes are important for plants and may be beneficial for nutrient acquisition

and stress tolerance.

Approach and Outcomes• Raoultella terrigena was isolated from sterile tobacco roots. It ability to fix nitrogen

has been confirmed• The draft genome sequence revealed a 5.7-Mb genome with 57.84 mol% G+C

content• R1Gly also contains all necessary genes for the tryptophan-dependent production of

the plant hormone IAA and of 2,3-butanediol and acetoin volatiles previously shown to promote growth in Arabidopsis

Significance• Endophytes are important for plant growth and development • Understanding the genomics and physiology of nitrogen-fixing endophytes such

as Raoultella terrigena may help in the development of bioenergy crops that can grow on marginal land with limited inputs.

Schicklberger et al. (2015). "Draft Genome Sequence of Raoultella terrigena R1Gly, a DiazotrophicEndophyte". Genome Announc, 3(3). doi, 10.1128/genomeA.00607

Draft Genome Sequence of Raoultellaterrigena R1Gly, a DiazotrophicEndophyte

1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).

Background• Arbuscular mycorrhizal fungi (AMF) colonize most plants and

contribute to uptake of nutrients and soli contaminants.• The diversity of AMF under different environmental conditions

has not been well investigated

Approach and Outcomes• Metagenomic analyses were used to profile the AMF

community in soil with different level of heavy metal contamination (Pb, Zn, Cd).

• Chemical and physical parameters in the soil were determined.

• All soils had abundant AMF and efficient root colonization.• AMF diversity was much lower in have metal contaminated

soils, which were dominated by a single genotype of Glomus• AMF communities were also affected by other soil

parameters, especially pH

Significance• AMF can potentially be employed to improve

bioremediation by black locust or other tolerant plants.• Heavy metal contaminated soils can be used for growing

non-food crops, for production of biofuels and/or wood

Yang et al. (2015). "Community structure of arbuscular mycorrhizal fungi associated with Robiniapseudoacacia in uncontaminated and heavy metal contaminated soils". Soil Biol Biochem 86, 146‐158

Robinia pseudacacia (Black Locust) is a useful plant that is tolerant to soil pollutants. The roots are associated with arbuscular mycorrhizal fungi.

Community structure of arbuscularmycorrhizal fungi associated with Robiniapseudoacacia in uncontaminated and heavy metal contaminated soils

Many different mycorrhizal fungi were associated with the plants. In general, the heavy metal contaminated soils had the lowest species diversity. Not only heavy metals, but also other soil parameters affect the fungal communities. pH was the most important parameter besides heavy metals.