Before the BRIC-20 project could move into the SVT phase, Wyatt Lab graduate student researcher, Proma Basu, had to solve three problems introduced by the logistics of sending seeds to space to germinate and then returning them to the lab for analysis.
Complicating the issue was the fact that little work has been published on plant proteins specifically in microgravity conditions, so she had to start by testing the suitability of ground-based protocols for space.
First, Basu had to determine what extraction protocol would yield the greatest amount of protein from the smallest sample size. Most protein extraction protocols require a 5g sample. The physical limitations of the experiment meant that Basu would only get 1.5g per sample, but she still needed to extract at least 60 micrograms of proteins from each sample (or replicate). She tried many protocols before discovering a precipitation protocol that could yield enough protein from a 1.5g sample.
Next, Basu had to determine if the fixative (RNAlater) used to fix (or stop) germination of the seedlings would negatively impact proteome (total protein) analysis. The experiment required the International Space Station (ISS) crew to fix the seeds after 72-hours of germination. The product label of the RNAlater fixative – required for RNA extraction – warned it would proteins and Basu had to make certain this necessary step would not impact the proteomic analysis. After repeated protein extractions and confirmation of quantity and quality from Danforth Plant Science Center in St. Louis, Basu’s preliminary tests showed that RNAlater would not negatively impact proteomic analysis results. (That is, RNAlater does in fact degrade native proteins but since the proteomic analysis does not include native proteins the degradation would not impact her study.)
Last, Basu had some concerns about how long the seedlings would be submerged in RNAlater fixative before being deep-frozen to -80 Celsius and packaged for the month-long journey back to the Wyatt Lab on Earth. Through vital staining of RNAlater treated seedlings with Trypan Blue, Basu was able to show that 12 hours of incubation in RNAlater at room temperature (22 to 23 degrees Celsius) would cause sufficient diffusion of RNAlater into the seedlings to stop transcription.
Having determined the extraction method and that the exposure to RNAlater fixative would not impact the proteome analysis, Basu was ready to test her protocols during SVT to ensure the introduction of NASA hardware and shipping of the control samples from Kennedy Space Center to the Wyatt Lab did not introduce any further variables she would have to contend with before flying the seeds in space.