JBEI Highlights June 2015

1. 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).

2. 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

3. 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 Diazotrophic
Endophyte". Genome Announc, 3(3). doi, 10.1128/genomeA.00607
Draft Genome Sequence of Raoultella
terrigena R1Gly, a Diazotrophic
Endophyte

4. 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 Robinia
pseudoacacia 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 arbuscular
mycorrhizal fungi associated with Robinia
pseudoacacia 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.