| Q | A |
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| What happened to the proposal? | As of 21 May, it is accepted at NIH and assigned to a special study section. The initial peer review should be completed by the end of November 2009 and a funding decision made shortly after the appropriate National Advisory Group meets in January 2010. |
| We are assuming that we could not do yeast or Dictyostelium expression - is this correct? | It's not clear that the sophisticated procedures available would significantly enhance throughput for yeast. This is something to be explored when the system is established. For Dicty, the issue would presumably be that the current setup is for free cells in suspension, not surface-associated, and that may not be what's needed for your Dicty systems. Finally, there would have to be an operational view of how many different cell systems could be routinely incorporated without cross contamination issues. |
| Can it work with insect cells? | We understand from GNF that Sf9 culture is well established. |
| Is this more for screening through a lot of different things or for scale up? | It's more for screening, but on a pretty large scale and complete to protein purification . We understand that 100 - 500 μ g / culture plate are standard and at GNF they will do a batch of four for scale up, but consider that larger scale needs are best done in a larger bioreactor. However, Scale up in the PEPP versus off line bioreactors/flasks is a question of cost effectiveness, that users could self determine. |
| how would this facility work- we would be able to provide bacculoviral constructs, and viral titering, Sf9 or Sf21 infections, and two step HPLC would be performed? | The initial setup using the GNF procedures would include Sf9. Other systems would be incorporated as the facility progressed. The initital operation would include a single step slurry separation based on His tag. Final products are produced in 96 well format plates suitable for any further automated purification |
| Do you have any idea what the charges for this might be? | On the basis of p 27 of http://www.nysbc.org/HTB/Knuth09.pdf , we estimate about $12 per sample local supply cost (excluding plasmid) . The resource cost with operations and Facility and Administration cost would increase this.
Over the long term, it will likely operate on a fee-for-service basis. |
| this is the first I have heard of your current facility. | OK, we are falling down on publicity -- see http://www.nysbc.org/start/index.htm |
| I do NMR and I dont think this could help me isotopically label. Any comments? | 1) finding the right construct in a eukaryotic screen is likely to be generally useful and be a lead for cell free or prokaryotic expression , and can identify useful complexes, post-translation mods etc.
2) for a few, high profile, cases, the economics of euk expression may be manageable once you know you have high expression. Refs -- LIBRARY:OhkiDohi08.pdf etc . But realistically we'd have to say this is a research objective once the system is in place. Note that we will have the 1.7 mm TCI 800 probe so small quantities of labeled material are useful.
3) You can think big, e.g. inducible expression of unlabeled materials interacting with injected/ transported labeled materials a la recent Nature papers. |
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