June 25, 2002

The key element in automation of sitting drop crystallography testing is storage of proteins. The current method of storing precious reagents, such as proteins, is a 1mL Eppendorf tube. This method of storage kills any hope of increasing the throughput of testing by the use of automation. It requires the pipetting of the protein sample on a one by one basis.

Tomtec proposes the use of a new Uniwell(tm) (pat. pend.) for storage of precious reagents such as protein aliquots. The Uniwell(tm) is shown here:
16 small wells
Uniwell(tm) strip
The 16 small wells are 2.2mm in diameter. The interconnecting channel is 0.7mm wide. The total brim volume is 1.1mL For low viscosity reagents, such as aqueous, the entire strip can be filled from one well. With higher viscosity aliquots it would be added to several wells, allowing them to flow in-between. For heavier viscosity materials that can still be pipetted, it may be necessary to shake or even centrifuge the Uniwell(tm) strip to equate the volume in all wells.

The common wells are on 4.5mm spacing. Thus they may be accessed by an 8 tip pipettor on 9mm spacing or a 16 tip pipettor on 4.5mm spacing. This provides a source for all pipetting tips to withdraw reagent. There is essentially no dead volume since each aspirating needle may access the bottom of each well. Any volume withdrawn and not consumed may be dispensed back into the Uniwell(tm).

Twenty-four Uniwells may be arranged in a carrier. The carrier has the footprint and height of a microplate meeting the SBS dimensional standard. Each Uniwell(tm) has a male and female end to force a common orientation in the frame. The assembled unit may now be handled as a microplate by all of the microplate handling devices.
Each Uniwell(tm) has an identifying barcode on the bottom. This may be a standard linear bar code label or a 2D barcode, if desired. The forced orientation of the Uniwells, contribute to the ease in reading the barcodes.

The protein samples in the assembled Uniwell(tm) format can be handled as a 384 well microplate. The common channel between wells means a 96 well pipettor may also access it. Yet each Uniwell(tm) can be removed, recapped and returned to library storage.
24 strips in Uniwell(tm) frame
Full automation of the Sitting Drop Crystallography test can be accomplished with Tomtecs Quadra-Plus. This is an automated liquid handling workstation. It has two sets of automated infeed/outfeed cassettes designed to handle anything with the SBS microplate footprint. There are two pipettor heads and an eight-station shuttle. The shuttle transfers microplates from the cassette to the pipettor stations. Each pipettor station has a full indexing stage that carries the microplate up to the pipettor tips. An ultrasonic tip washing station is used to wash both sets of tips between samples. On a recent carryover test, using saturated Tartrazine dye as a source, the absorbency after ultrasonic tip washing was the same as the blank 0.06 ABS. Using Fluoroscein the carryover was in the range of less than 1 part in ten million (10,000,000).

The choice of pipettor heads, in the Quadra-Plus, is a function of the application. There is a choice of eight pipettor heads available. For crystallography the choice comes down to three depending on how the user plans to run the experiments.

There are two basic scenarios. One is to run a limited number of proteins against a large number of reagents or mother liquors. The second is the opposite—a large number of proteins against a limited number of reagents or mother liquors.
It is assumed the reagents or mother liquors are supplied in 96 deepwell plates, whereby each well can hold from 1 of 2mL of reagent. These deepwell mother plates would infeed to the Quadra-Plus shuttle from one of the stackers. A 96 well pipettor head would automatically transfer aliquots to the main wells of the crystal plate. The reagents in the mother plate could be 96 different reagents or it could be arranged with eight rows of reagents with each row having twelve wells alike.


The proteins would be in the Uniwell(tm), arranged in a carrier frame. Each carrier frame would hold twelve Uniwells of proteins. The carrier frames would automatically infeed to the Quadra shuttle. The second pipettor head choice is a function of the testing format.
Quadra-Plus(tm)
If it were desired to test a protein against a number of reagents, the second pipettor head would be a single row pipettor, of eight tips on 9mm spacing. Assuming 1µL was used for the sitting drop, each of the eight tips would aspirate 12µL of protein from Uniwell(tm) #1. The crystal plate would be indexed under the single row pipettor, by the Quadra stage, and 1µL would be dispensed to all 96 of the protein wells. The reagent or mother liquor would be added by the other 96 well pipettor. This can be done either before or after dispensing the protein. On the next plate the same protein from Uniwell(tm) #1 could be tested with another 96 reagents, or protein from Uniwell # 2 could be tested with the same reagents as protein from Uniwell(tm) #1. Complete flexibility is provided.

In the other testing format, the second pipettor would be Tomtec?s 96SV head. This is a 96 well pipettor capable of dispensing a 1µL drop with a CV of better than 5%. It is the same positive displacement design as the single row pipettor described above, except there are 96 instead of eight. On this format there would be twelve proteins in the Uniwell(tm) frame assembly. The deepwell reagent mother plates would be arranged to have eight rows of reagent, with all twelve wells in the row having the same reagent. Rather than from a deepwell plate this could also be from special reservoirs. The 96SV head would then aspirate 1µL per tip from the protein Uniwell(tm) and dispense it to the sitting drop wells in the crystal plate. This format then tests a matrix of twelve rows of protein against eight rows of reagent. If a plate with multiple sitting drop wells were used, then the next Uniwell(tm) frame assembly would be the aspirating source for those wells.

In summary, complete flexibility of testing format is provided to the user by the choice of the pipetting head chosen combined with the matrix of rows and columns of reagents and proteins. In the last scenario of testing, assume it was desired to test 96 proteins against 32 individual reagents or mother liquors. The 96 proteins would be arranged 24 Uniwells in four carrier frames. There would be four 96 deepwell mother plates each with eight rows of reagents (32 total). The time to prepare these 3,072 reactions automatically is 34 minutes, which includes tip washing between reagents.