Crystal cookery - using high-throughput technologies and the grocery store as a teaching tool

Abstract

Crystallography is a multidisciplinary field that links divergent areas of mathematics, science and engineering to provide knowledge of life on an atomic scale. Crystal growth, a key component of the field, is an ideal vehicle for education. Crystallization has been used with a 'grocery store chemistry' approach and linked to high-throughput remote-access screening technologies. This approach provides an educational opportunity that can effectively teach the scientific method, readily accommodate different levels of educational experience, and reach any student with access to a grocery store, a post office and the internet. This paper describes the formation of the program through the students who helped develop and prototype the procedures. A summary is presented of the analysis and preliminary results and a description given of how the program could be linked with other aspects of crystallography. This approach has the potential to bridge the gap between students in remote locations and with limited funding, and access to scientific resources, providing students with an international-level research experience.

Figure 1

A view from the bottom of the initial 96-solution source or mother plate, showing the colorful nature of the PSBs chosen for the experiment.

Figure 2

The robotic plate-imaging system in operation. A total of 28 plates can be placed on the translation stage at any one time, with the images being made available via a secure ftp site as soon as an individual plate has been completed. The process is part of the high-throughput crystallization screening pipeline at HWI and operates completely automatically, requiring only placement of the 1536-well plate on the translation stage where the position is automatically indexed. Chemical data are linked to the images through a database. One of the students involved with the project, Nicholas Furlani, is shown placing a plate on the imaging robot. In a more accessible application of this program, students would not have to be physically present in the HWI laboratory, as the robotic processes and data dissemination would allow the students to gain access to the image data from remote locations.

Figure 3

DSF results for concanavalin A. The melting temperature is obtained from the inflection point of the initial rise in florescence. Three curves are shown, a control curve with a melting temperature determined to be 342.9 K and then curves for two PSBs, aftershave and Cherry Pez, with melting temperatures of 355.1 and 331.7 K, respectively.

Figure 4

A table of DSF results for the proteins and PSBs. The numbers represent temperature shifts in K. Only 11 of the 20 protein samples gave useful DSF signals. PSB 93, hand soap, was removed as it was hydrophobic and significantly interfered with the DSF assay by interacting with the dye and causing unacceptably high background fluorescence, making the data uninterpretable. We used this position as a control, with only the protein and dye diluted with water loaded in that well. The temperature shift was calculated by reference to this control. In the case of proteins H, R and S a melting temperature could not be calculated from the control data. Blank areas in the table indicate that the melting data were difficult to impossible to interpret. In cases H, R and S, where we could not calculate a melting temperature of the protein in the absence of a PSB, the values in the table were calculated as a shift from the average value of the T _m for that protein for all of the interpretable T _m values of the protein with a PSB. Green and pink shaded cells denote temparature shifts ≥±2.0 K, respectively.

Figure 5

Crystal hits from the PSB cocktails. The proteins are arranged (a) A–J and (b) K–T. There are eight columns for each protein, representing the cocktails 1–8 (Table 3 ▶), and 96 rows, representing the 96 PSBs. Crystallization hits are represented by filled blocks. Clear blocks represent null results.

Figure 6

The number of crystal hits for all 20 proteins, plotted against the PSBs given in Table 2 ▶. All PSBs had a minimum of five hits. For those with 29 hits or more the PSB is identified on the graph.

Figure 7

The number of hits shown as a function of protein. For some proteins there were a large number of hits, the largest being bovine liver catalase with 434. For others there were few, bovine hemoglobin showing only one. For this reason the data are presented on a logarithmic scale.

Figure 8

A plot of the number of crystallization hits against the conditions in which the experiments were set up, i.e. the original concentration of the PSB and 1:10 dilution, then the original and diluted PSB at three different pH values, 5.8, 6.8 and 7.8.

Figure 9

Crystallization results from proteinase K (a) from fish sauce with 12.5% PEG 3350 in HEPES buffer at pH 6.8, and (b) using Hampton Research Silver Bullet H12 with 12.5% PEG 3350 in HEPES buffer at pH 6.8. Silver Bullet H12 contains aspartame, gly-gly-gly, leu-gly-gly, pentaglycine, tyr-ala and tyr-phe, which are also likely to be found in fish sauce made from fermented anchovies. The well diameter is 0.9 mm.

Figure 10

Chemical diagrams for (a) tartrate used for crystallization of thaumatin and (b) ferrous fumarate found as an ingredient in prenatal vitamins, showing obvious chemical similarities.

Figure 11

A simple-to-construct low-cost vapor-diffusion crystallization experiment using a plastic Petri dish. A disposable polystyrene Petri dish is easily adapted for setting up a hanging-drop vapor-diffusion crystallization experiment. The lid of the plate is removed and Vaseline applied around the inside rim using a syringe, or simply painting it on with a cotton swab. Once the grease seal is applied to the perimeter, the protein and crystallization cocktail drops are sequentially added to the inside of the lid (the same side as the grease seal). It is important to mark the lid so that drops can be identified later. Recommended volumes are 5 µl each of protein and cocktail, and 1.0 µl of any additive. Any of the illustrated devices (e.g. adjustable pipette, Hamilton syringe, Wiretrol) can be used to dispense the drops. The volumes of these components, the volume ratios, the order of adding the components, and mixing or leaving the drops undisturbed after delivery are all variables the students can pursue if a more comprehensive investigation is undertaken. A volume of reservoir solution is added to the bottom of the Petri dish (10 ml), and the lid is inverted over the bottom of the plate and pressed to ensure the Vaseline will form a good seal.