Ricerc@Sapienza

The Oryx 4 crystallization robot is an instrument that allows the automated production of crystallization plates. In a crystallization plate, 24, 48 or 96 different crystallization conditions are tested simultaneously. For each condition a micro / nano volume of a solution containing the target protein is mixed with a suitable amount of crystallization solution present in the single well of the plate. Subsequently each condition is sealed and, by vapor diffusion, the concentration of the individual components of the drops thus obtained varies very slowly over time (from a few days to several weeks) until in one or more conditions the formation of protein crystals is observed. Normally, to obtain initial crystallization conditions it is necessary to test from 200 to 1000 different conditions for protein sample (about 2-10 plates of 96).

The use of a crystallization robot reduces the time needed to setup the crystallization trial of 100 times and the protein amount of 10 times (manual setup: 12conditions/h ; automatic setup: up to 1000 condition/h). In other words, using a crystallization robot allows to sample the crystallization space of a protein (960 tests) in about an hour, whereas it would require 2 weeks of work to do it by hand. For this reason, to remain competitive with international laboratories, the use a crystallization robot is mandatory.

Careful market research has indicated the Oryx4 robot produced by Douglas Instruments among the best robots on the market for flexibility and to have the best value/cost ratio.

All Oryx systems use multi-hole dispensing tips, which have several independent channels to deliver small volumes. Each channel provides a different solution. The solutions do not mix in the tip: they mix in the drop after they have been dispensed; this means that no dead volume remains.

The Oryx4 robot equipped with the LCP module has the following features:

Soluble Protein Dispensing:

• Oryx4 can accurately and reproducibly set up sitting drop crystallization experiments. The same system can set up nano-drops for screening (eg 100 + 100 nl) or micro-drops for optimization and data collection (e.g. 2 + 2 μl). For crystal harvesting and soaking experiments a higher volume (up to 8+8μl) is possible.

• Oryx4 can set up hanging drop experiments with 24-well Linbro or SBS plates and glass (or plastic) cover slides. Up to 5 drops can be dispensed per slide, and the volume of protein, reservoir solution and additive (such as seed stock) in each drop can be varied at will.

• Oryx4 wastes virtually no protein sample or seed-stock - only 0.5 μl are lost for a 96-well plate. Contact dispensing means that almost no protein remains in the tip at the end of the experiment. Also, since only one (multi-channel) tip is used, all of the protein for an experiment can be placed in a single PCR tube, which also reduces waste.

• Oryx4 can perform microseed matrix screening (MMS) experiments using a simple one-pass routine. Contact dispensing enables a suspension of crystal seeds to be dispensed, without risk of blockages.

• Oryx4 possesses powerful but user-friendly software for designing screening, MMS microseeding, additive, 2-d grids (protein is varied against precipitant or additive), Combinatorial (see below), Additive Scatter (see below), microbatch-under-oil, hanging drop and sitting drop experiments. No programming or scripting by the user is required.

• The software allows individual wells to be selected for dispensing. For example, wells that gave heavy precipitate can be ‘cherry-picked’ with a mouse and repeated with a lower protein concentration.

• Oryx4 can use virtually all crystallization plates, including non-SBS compatible plates. The system is extremely flexible with regard to plates. New plates can be added as required by the user.

• Oryx4 is not dependent on consumables (other than crystallization plates).

• Oryx4 can perform Combinatorial experiments in which ingredients can be reshuffled and/or appropriate dilutions of seed stocks can be found. Additive Scatter experiments are similar except that the seeds/additives are scattered evenly around the plate.

• Oryx4 can dispense a vapor diffusion experiment and a microbatch-under-oil experiment in parallel.

• Oryx4 can fill reservoirs for simple optimization experiments.

Membrane protein dispensing using Lipidic Cubic Phase (LCP):

• Oryx4 LCP functionality sets up crystallization experiments with LCP, dispensing to sandwich plates, sitting drop plates or Linbro plates etc. with cover slides. The volume of the LCP bolus can vary from around 5 nl to 8 μl.

• Two dispensing methods are available – flattened boluses are generally used for volumes below 200 nl, with conical boluses for larger volumes.

• LCP screening, additive screening and combinatorial optimization experiments are performed.