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Advantages:
All systems (OryxNano, Oryx4 and Oryx8):
Sitting drop down to 100+100nl for screening
Up to 8+8µl drops for crystal harvesting and soaking
Contact dispensing allows "MMS" and regular microseeding
Only 10.1µl of protein for 96 wells (100+100 nl)
Simple optimization:
- 2D grids
- Cross-Matrix (Combinatorial) experiments
In addition - Oryx4 and Oryx8:
Hanging drops up to 8+8µl
Additive experiments
Microbatch-under-oil down to 100+100nl
Oryx4 can be upgraded to Oryx8
In addition - Oryx8 only:
Autodesign optimization capability:
- 7-dimensional optimization
- Central Composite and Box-Behnken designs
- Rapid reservoir filling for optimization
- Multi-component pH control
Reduced need to swap microtips
Click
here for videos
All Oryx systems use multi-bore dispensing tips (microtips), which
have several independent channels to dispense small volumes. At the end
of e.g. a 3-bore tip there are three holes. Each channel dispenses a
different solution. The solutions do not mix in the tip - they mix in
the drop after they are dispensed. This means that there is no
dead-volume. Oryx8 has two chassis and can use 3, 4 and 7-bore microtips.
The microtip is on the left-hand arm.
• The microtip always touches the plate when liquids are dispensed.
This gives very reliable dispensing, especially when suspensions of e.g.
seed crystals are used, which makes the system ideal for
microseeding experiments.
• Stock solutions do not come into contact with the motorized syringes, which are filled with degassed pure water - this avoids the need for
flushing out the syringes when the stock solutions are changed.
• The manual syringes on the front panel
are used to refill the motorized syringes, to remove air bubbles, and to
load stock solutions for optimization experiments.
• Large volumes, including oil for
microbatch experiments, are dispensed by the right-hand arm using the
large (2 ml) motorized syringe. A 1 ml disposable tip is used for this.
Screening for sitting drop is carried out using plates that are
prefilled with reservoir solutions. (Reservoirs can be filled manually
with a 12-channel pipette, or large-volume dispenser such as the
Liquidator 96 by Rainin). During each dispensing cycle, the tip is
first cleaned in the reservoir by
moving it horizontally through the solution. The tip then picks up e.g.
100 nl of solution from a clean part of the reservoir and transfers it
to the drop, dispensing protein simultaneously (together with seed stock
for microseeding experiments). The level of contamination has been
tested and is very low.
A very simple user-interface is used to design experiments, as shown
below. The correct plate can be selected from a database of all
well-known crystallization plates (if your favorite isn't there, let us
know!), the volumes
of each ingredient of a drop are entered in a simple form.
Individual wells can be selected or skipped by clicking on the plate
with a mouse. (The form shown below allows seed stocks and additives
to be added to drops, as
explained below.)
Exactly the same mechanism is used to dispense drops up to 8+8 µl for
crystal harvesting and soaking experiments, and down to 100+100 nl for
screening experiments. Smaller drops can be used but will not give
reliable results with some proteins. Evaporation is reduced by covering
the plates with a sliding shield (see below). For soaking experiments,
microseeding is highly recommended, see below. For information about
dispensing reservoirs for optimization experiments, see below.
Sliding Evaporation Shield
Since all Oryx systems use contact dispensing (the tip always touches
the plate) they give very reliable dispensing even when suspensions of
solid particles are used. This makes them ideal for microseeding
experiments. The technique of adding crystal
seed-stock to random
screens (rMMS) is a significant breakthrough in protein crystallization
that is very effective. For example, one industrial group
used the
method to solve 38 out of 70 structures generated in a four year period,
finding particular success with antibody complexes. rMMS not only
produces more hits, it also typically generates better-diffracting
crystals – because crystals are more likely to grow in the metastable
zone of the protein’s phase diagram (see below).
Note also that in cases where only one or a few crystals are obtained in
screening experiments, the seed stock that can be made is very valuable
– often more valuable than the protein sample. It is therefore a great
advantage to be able to use the smallest possible sample of seed stock.
Using any robot from the Oryx range, seeding can be performed in a whole
96-well plate using only 1.5 µl of seed stock. This is particularly
helpful for membrane protein crystallization projects because membrane
protein crystals are often unstable and it is helpful to make seed
stocks without diluting the original mother liquor.
Videos about rMMS : (1)
Introduction and theory. (2)
Setting up
rMMS experiments with an Oryx system.
Contact dispensing has another advantage: almost no protein remains in
the tip at the end of the experiment. Moreover, 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. When they have
enough protein, most users set up 300 + 300 nl drops. For a 96-well
plate this requires only 29.4 µl of protein, i.e. only 0.6 µl is wasted.
If your pipette is accurate, there is no need to put more than the
specified amount into the tube!
Similarly if 10 nl of seed stock is added to each drop, only about 1.5
µl of seed stock is required for a whole 96-well crystallization plate.
(It is helpful to dispense around 5 µl of screen solution on top of the
seed stock.)
All Oryx systems can carry out simple optimization experiments using three different approaches.
The standard screening software allows users to define simple 2-d grids
for sitting drop experiments. Using three or four ingredients, the user
selects the volumes to be dispensed in two corners of a rectangular
grid. The software interpolates linearly between those conditions. (The
user interface does not show the concentrations in intermediate wells,
but the volumes dispensed to each well are shown in a log.) The script
works well with plates with small reservoir volumes (e.g. the SwissCI
3-drop) because the maximum volume of any ingredient that can be
dispensed is 1620 µl. (A more sophisticated approach to optimization
that includes 2-d Grid experiments is available with the Oryx8 – see
below.)
Click here for a video
showing how to set up 2-d grids
with an Oryx
system.
The systems' powerful “combinatorial optimization” approach allows a
different additive or seed-stock to be added to each row. Each additive
is picked up from the corresponding PCR tube on the right of the table
(A1, A2 etc. on the diagram below). By arranging e.g. precipitants in
columns (P1, P2 etc.), different combinations of precipitants and
additives can be tested very quickly. This is useful for reshuffling the
ingredients of several hits, so that ingredients that are not helpful
can be eliminated quickly, and trends can be identified. For example,
certain ingredients may encourage the formation of crystals with certain
morphologies.
The combinatorial approach can also be used to identify in a single
experiment the appropriate dilution of a seed stock to get around 5
crystals per drop. For example, 9 different concentrations of seed stock
could be placed in the rows A1 to A9 above. Six different conditions
could be place in the six columns labeled P1 to P6 above.
This is a very effective way to get a really reliable supply of crystals
for data collection and soaking experiments.
Click here for a video showing
Cross-Matrix (Combinatorial)
optimization.
Features of the Oryx4 and Oryx8 systems only:
Oryx4 and Oryx8 can set up experiments with Linbro plates and cover slides, with up to 5 drops on each
cover slide. Volumes can range from 100+100 nl up to 8+8 µl (assuming 24 drops). You can also add one additive
to each drop with Oryx4, and up to five additives with Oryx8. This is very useful for leads that
are picked up in random microseeding experiments, where it may be necessary to add e.g. diluted seed stock to get crystals.
For example, in the experiment shown (below left), one drop has seed-stock, protein, reservoir solution; the second drop has no seed but
a little extra protein, while the third drop has no seed but a little extra reservoir solution.
You have to transfer the cover slides onto the Linbro tray by hand. After a few drops the experiment can be paused to allow the transfer.
Oryx8 can set up the reservoirs for optimization experiments, including 2-d grids and multivariate experiments (see below). The hanging
drop capability is not available for the OryxNano.
Additive experiments are a well-known and effective approach to
optimization. Simply by adding a set of potential ligands or another
screen to a hit condition, crystals can often be optimized. Since they
have larger Plate Loaders, the Oryx4 and Oryx8 systems can accommodate
an extra 96-well plate containing additives to the right of the target
plate. The tip picks up e.g. 100 nl samples from the additive plate and
dispenses them along with protein and reservoir solution to the drops.
For initial screening relatively large volumes of additive are often
used, for example 300 nl (protein) + 100 nl (reservoir) + 200 nl
(additive). For example, these volumes could be used for an initial
screen with the Silver Bullet screen by Hampton Research. For final
optimization, smaller volumes of additive are often used, for example
300 nl (protein) + 200 nl (reservoir) + 100 nl (additive).
Microbatch is a very simple approach to crystallization. Small samples
of protein (100 nl to 5 µl) are mixed with stock solutions in small
drops, and covered with oil to prevent evaporation. For screening
experiments it helps to use a 50:50 mixture of paraffin oil and silicone
oil. The silicone allows slow evaporation over about a month, which
gives a scanning effect across the phase diagram of the protein. For
optimization, pure paraffin oil can be used, which reduces evaporation
to a minimum. Studies have shown that microbatch finds as many or
slightly more hits that vapor diffusion, but the main advantage is that
(for reasons that may not be well-understood) certain proteins
crystallize much better in microbatch than other methods. Microbatch can
help to protect sensitive proteins such as membrane proteins and
anaerobically-produced proteins because it reduces the oxidation and
gives thinner skins on the surfaces of drops.
A full set of scripts for microbatch is available, including screening,
additive experiments and microseeding. Oil is put onto the drop by the 1
ml plastic tip just after it is dispensed.
The Oryx4 system can easily be upgraded to Oryx8 simply by adding a
second Chassis and changing the colors of the connectors. However, the
OryxNano cannot be upgraded easily because it has a smaller Plate
Loader. Contact the company for more details.
Features of the Oryx8 system only:
The Oryx8 system has an application for optimization called XSTEP. This
provides a spreadsheet environment, where each drop or reservoir can
have up to seven ingredients (corresponding to a 7-channel microtip).
For example, 2-d grids can easily be set up by editing the two corner
wells of a rectangular grid, and clicking on “interpolate”.
However, 2-d grids are an inefficient approach to optimization because
it may be necessary to vary several parameters simultaneously to find
the best crystallization conditions (starting from a hit in a
crystallization screen). Putting it another way, crystallization
parameters may interact with each other. For example, the best
precipitant concentration to use at one pH may not be the best at
another pH. The same may be true when you try different protein
concentrations. The correct way to deal with complicated “multivariate”
problems like this is to vary all the important variables in each
experimental run.
Text books on experimental design recommend approaches such as the
Central Composite and Box-Behnken designs. One way to think of these
designs is to imagine that the experimental points are on the surface of
a multidimensional cube around your starting condition (shown as red
below - this could be a hit from a random screen). The Central Composite design comprises the
points at the corners of a multidimensional cube (blue points), together
with a set of “axial” points, where only one parameter varies (green).
The axial points “fill in the spaces” between the corner points. With the Box-Behnken design, only two
of the variables are varied at a time (yellow points).
These designs can easily be used without understanding the detailed
advantages of each approach. Simply adjust the parameters until
the desired number of wells are defined depending on the amount of
protein etc that you have - all experiments generated will be reasonably
well-balanced in the multidimensional crystallization space.
For more information and for an
explanation of the problems of conventional crystallization experiments,
see
the article published by Douglas Instruments in 1999
(the text also available on this web-site).
These “multivariate” designs can be extended to any number of dimensions,
and they usually give small enough numbers of experimental points to fit
onto a single plate. The XSTEP software allows you to select a center
point on a spreadsheet and design a multivariate experiment around it.
Many options are available, depending on the number of wells that you
want to set up. However all experiments set up with the Autodesign tool
will be reasonably evenly distributed in the multidimensional
crystallization space, and will help you to find the best direction to
go in to improve crystallization.
The stock solutions are shown below on the left. Each cell corresponds to one well on the target plate.
Separate spreadsheets show the concentrations in the drops and the
reservoir - just click on the well that you are interested in and edit
the numbers!
Oryx8 can quickly fill the reservoirs of sitting drop plates with
essentially unlimited volumes using the air-driven 1 ml tip in
combination with the 7-bore tip. The reservoirs are filled first, and
then the drops are dispensed independently.
As shown above our optimization software includes a sophisticated algorithm that can
calculate the pH of a mixture containing any number of buffers, using
the pHs, pKas and the concentrations of the buffers. This is useful in
pH experiments because it alerts the user when e.g. the buffer in the
protein stock is limiting the range of pH explored.
Oryx8 can be used for screening (with 3 or 4-channel microtips), or for
optimization (with a 7-channel microtip). If you are going to be using
screening and 2-d grids a lot, you can save time by leaving both the 3
and the 4-channel microtips on the system permanently. It takes less
that two minutes to swap them, but even this can be a “psychological”
barrier!
Click below for:
Oryx8 bench space
(This is a Visio file - Visio is part of Microsoft Office.) Follow this link to see how much space is required on your lab bench
using the on-screen ruler. Click on the tabs at the bottom of the screen for
possible configurations. If no ruler is visible in Visio of if the file
fails to load properly, click on the Oryx4/8 specification below.
Differences
between Oryx8, Oryx4 and OryxNano
Specification
of Oryx8
Specification of MCC control unit
Getting started with Oryx8
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