Task Progress:
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Seed line development The T-DNA insertion mutant line has had its knockout status for the PDC1 gene verified through expression monitoring, along with seed bulking. Expression monitoring was completed through a comparison of wildtype and mutant lines following a 4°C induction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. This analysis displayed a distinct reduction in expression of the PDC1 gene, consistent with a knockout status. The green fluorescent protein (GFP) reporter lines are in varying stages of development. Two lines are currently undergoing expression verification tests to ensure visible production of the GFP protein. These lines have been confirmed using qRT-PCR analysis, and microscopy analysis to visualize the GFP protein and monitor its abundance. One line has been transformed and is at the transition from the T1 to T2 generations. This line was generated in-house through restriction enzyme mediated vector generation, which was produced using the promoter sequence cloned from the Columbia-0 background driving the expression of GFP. Screening of expression is currently being conducted on multiple lines to ensure presence and correct expression of the GFP protein. The inductions are carried out through crimping of leaves followed by microscopy. pPDC1:GFP is currently in vector development.
Testing Short-duration hypobaric treatments have been conducted. These treatments are conducted using clear hypobaric chambers retrofitted with a MSR Electronics data logger, to track barometric pressure, and a vacuum pump to draw out the gases from the chamber. Treatments typically last for 12-24 hours and are subjected to varying reduced pressures from 10-80kPA. These are general atmosphere reductions, not controlled gases. The purpose of these treatments has been to examine expression of the reporter lines in a hypobaric environment. Further analysis of the pdc1 knockout line using the hypobaric chambers, followed by expression verification through qRT-PCR, is also currently underway.
Hardware Progress The hypobaric experiment, which has been designated APEX-11 (Advanced Plant Experiments), is scheduled to use the CIR (Combustion Integrated Rack) facility and the VEGGIE growth chamber. The plants will grow for 10 days in the NASA Vegetable Production System (Veggie growth chamber) using a still-under-development plant growth rack. The Veggie rack will be designed for 40 plates, which will allow for a shortened experimental procedure process. There is also currently a separate rack in development for hypobaric treatments inside the CIR. In April 2023, Dr. Paul traveled to NASA Glenn Research Center outside of Cleveland, OH to work on a plan to integrate science into the CIR. Dr. Paul traveled with several plates to serve as mockup plates that will grow the plants for APEX-11. These plates were used to test the prototype rack. Plates were mounted on the rack and loaded into the CIR for design tests.
Future Plan Current plans call for Science Verification Test (SVT) to being in Q4 2023, with Experiment Verification Test (EVT) and Flight operations in 2024. SVT stage of operation is ready to move forward on the biology side; however, hardware operations are still in planning stages.
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