Protein Streak Seeding

The goal of the Protein Streak Seeding project is the creation of an innovative high-throughput (HTP) microrobotic system for a protein crystallography task called streak seeding. The system uses visual feedback from a camera mounted on a microscope to control a micromanipulator which has the mounting tool attached as its end-effector. For use with our robotic system, we have developed unique new tools, called miscroshovels, which are designed to address certain limitations of the traditionally used by crystallographers whiskers, bristles, or other kinds of hair.

The motivation for this project, along with some background information, is described separately.

Streak seeding is useful when the initial crystallization experiments yield crystals which are too small (less than 40um in size) and/or of low quality and can not be used for structure determination. To obtain higher quality crystals, a new reaction is setup like the original one, however, before incubation, small fragments of the initially obtained crystals are transferred to the new protein-reagent mixture to bootstrap the crystallization process. This crystal fragment transfer process is called streak seeding.

The task of streak seeding consists of three steps (Fig.2). First, the tool to be used is washed in clean water to remove any residue. Second, the tool is used to touch and probe the existing crystals thus breaking them up into fragments and picking some up. Third, the tool is streaked through the fresh mixture, which deposits some of the fragments in it. For this to work, the tool has to have the necessary properties to be able to break up, retain and release crystal fragments, thus the material it is made of is very important factor in for the efficiency of the procedure. Traditionally, various types of hair, bristles, whiskers or horse tail have been used.

Based on earlier development efforts using our generic micro-robotic system for protein crystal manipulation, we have built a specialized streak seeding robot called CARESS. CARESS --- an acronym for Columbia [University's] Automated Robotic Environment for Streak Seeding, --- employs an innovative approach to protein streak seeding which utilizes our own custom tools, called microshovels, designed and fabricated using MEMS technology.

CARESS uses visual feedback from a camera mounted on a microscope to control a micromanipulator which has the mounting tool attached as its end-effector. All sensors and actuators are connected to a personal computer and controlled by an application running on it. The software is responsible for processing the visual stream from the camera using computer vision techniques to pinpoint the location of the objects of interest in the workspace, and for controlling the actuators accordingly. The user interacts with the application via a graphical interface to set the system up, execute tasks and view data.

The robot is designed to work with the hanging drop crystallization method, seeding from source crystals on a 21mm square coverslip to destination drops on a coversheet for a 96-well plate. The user sets up the system by placing on the stage the coverslip with the protein crystals, the coversheet of the 96-well plate with the target protein droplets, and a microbridge with water used for cleaning the seeding tool. Then the system is started and it performs the seeding autonomously. At top speed, one 96-well plate can be seeded in 5-6 minutes. The video in the side panel shows CARESS in operation.