EquipmentThe COMPPÅ has automation and high throughput capabilities for accelerating membrane protein research.
Beckman Coulter Biomek FX Liquid Handling Robot
COMPPÅ utilizes automation heavy high throughput methodologies to enable us to screen thousands of integral membrane proteins. The liquid handling robot is employed to set up PCR reactions to amplify genes of interest at the 384 well scale. Downstream DNA purifications are handled using magnetic bead technologies adapted for the Biomek that enable rapid and economical processing. Protein purifications are also conducted on the 96 well scale using this robot. A plate carousel has been integrated with the robot to increase capacity.
The GNF fermenter from GNF Systems enables parallel culture of 96 independent expression growths to high density, thus removing the need for 96 shake flasks. The system holds 96 tubes (each with ~70ml of media) and feeds air/oxygen mixtures into the culture at define rates and therefore achieves very high cell densities. The protein produced from these cultures are used in the detergent selection procedures carried out using the HPLC systems.
Robotic Probe Sonicator
COMPPÅ has established a high-throughput pipeline for determination of novel integral membrane protein structures. In order to optimize the efficiency and productivity of this pipeline, steps have been automated and robots are employed. When over-expressing and purifying membrane proteins, sonication is the method of choice for lysing the cells. To accomplish this task, COMPPÅ designed and built a robot which consists of a 5-axis robotic arm, a controller, sonicator tip, sample holder, and thermally-controlled platform. The sonicator tip is attached at the end of the robotic arm and can enter each sample-containing well on a 96-well deep-well block. To remove liquid from the tip and prevent cross-contamination, the tip remains energized for a short time before moving to the next well. The block sits on a metal thermally-controlled platform that is held at 2 degrees Celsius to minimize heating of the samples. One round of sonication for the plate takes 13 minutes; one to 3 rounds are usually performed, so the maximum time of completion for this step is 39 minutes. This robotic sonicator can be used in conjunction with an 96-sample ultracentrifuge rotor when isolating membrane proteins and this additional purification step is desired.
LEX High Throughput Bioreactor System from Harbinger Biotechnology and Engineering
The LEX 48 system is an airlift fermenter that grows bacterial cultures in 48, one liter bottles or 24 two liter bottles. This system is very convenient as it enables many cultures to be grown in parallel at production scale, without the need for large number of shaker flasks.
Agilent Technologies Series 1200 High-Pressure-Liquid-Chromatography (HPLC)
COMPPÅ uses size exclusion chromatography over microbore silica matrix columns to determine membrane protein stability and detergent preference. HPLC systems from Agilent are used to determine detergent preference in mobile phases with up to 12 different detergents. The readout of the elution profiles of the columns is via UV absorbance which may be coupled to static light scattering and refractive index detection using Wyatt detector on one of the HPLC systems. Each size exclusion analytical run is ~20 minutes with a 5 microliter injection of protein onto the column.
Mosquito and Mosquito LCP by TTP Labtech
COMPPÅ uses Mosquito crystallization robots to set up our crystallization experiments. The positive displacement technology of the the dispensing tips are ideal for the dispensing of small volumes of very precious membrane proteins, which due to the presence of detergents, may be prone to foaming using other systems. The Mosquito LCP robot was purchased in the summer of 2011 and will be used to set up lipidic cubic phase crystallization experiments. The LCP method has proven to be useful for many GPCR proteins, and we will investigate it general utility for other membrane proteins.
Lipidic cubic phase-dispensing robot
COMPPÅ has constructed a small lipidic cubic phase-dispensing robot out of existing components: a fluid dispensing robot (typically used for dispensing glue on products or grease into bearings) and a Hamilton syringe pump (not shown). The robot is made by Fisnar (Wayne, NJ) and costs approximately $5,000 US. The robot has a 0.02 mm tolerance in each axis. A small aluminum plate connects the syringe pump to the robot. The Micro4 pump controller (World Precision Instruments, Inc.) is triggered by an output signal from the robot, relayed to the controller via a relay and power supply (~$20, Radioshack). The glass plate onto which the LCP is dispensed is held on a standard 96-well plate holder that was salvaged off another robot. The whole unit is in a Perspex [poly(methyl methacrylate) or PMMA] cabinet together with a humidifier. The robot has a very simple programming interface. It takes about 3 minutes to dispense 96 wads of LCP (30 nL each). The precipitant is placed on top with a 12-channel pipette while in the humidity box- or COMPPÅ uses two other robots with 96-channel heads that can dispense all at once.