This page contains a Flash digital edition of a book.
to break down RNA. Ulrich says, “These enzymes are on our hands and everywhere, so we use sterile methods in the incuba- tor, and we keep the tRNAs frozen.” She adds that tRNA mole- cules are “small, thin, and fragile,” and at first thawing them and returning them to the freezer seemed to damage them. “But we tweaked our procedures, and we’re getting better yields.” Once they have a big store of the tRNAs, they’ll get to the real acid test—or, in this case, an enzyme test. Their batches of Staph and Bdellovibrio tRNAs are of two kinds, specialized to bind with two amino acids: asparagine and aspartate, both used in the bacteria’s protein synthesis. While they normally contain both tRNAs, these bacteria are known to lack free asparagine—so how can their asparagine- binding tRNA do its job? It seems to do it with an enzyme that binds aspartate both to its matching tRNA and also to the as- paragine tRNA. (A separate enzyme reaction later converts that aspartate into asparagine.) Sheppard’s team will conduct assays to test whether that enzyme is in fact nondiscriminating, able to work on both tRNAs. They’ll add the enzyme to each type of

to the chromatograph they calibrated after it had sat idle fol- lowing repairs. Neither student had prior experience with Sree- nilayam’s research, so “their learning curve for the new termi- nology and processes was very steep,” she says, zooming her hand up over her head. She says their first week of the summer was “like a half-semester of biochem labs.” Zanetti admits it was “pretty overwhelming” until, as Hyde chimes in, “we did things so many times that they suddenly clicked: “Aha, this is what we’re doing!”


tRNA, wait a few minutes, and watch. If a binding reaction does take place, it will generate a byproduct that will change the color of a detector chemical. And if they see that color change in the assay with the tRNA for asparagine, that will demonstrate how bacteria can synthesize this crucial amino acid even without the “right” enzyme.

PURE AND UNADULTERATED “Unlike many lab exercises within a course,” says biochemist Brandy Sreenilayam, “actual research involves a lot of ‘hurry up and wait.’ My students learn detailed note-taking, collabora- tion, and plenty of patience.” Michael Hyde ’13 and Alex Za - netti ’13 are learning fast, from the centrifuge they got unstuck

In fact, what they’re doing is nine things, all preparation for solidifying the protein LGN into a 3D crystal so they can see and map its structure. Scientists are eager to know LGN’s shape, be- cause its role in cell division and its tendency to be more con- centrated in cancer cells make it a promising target for pharma- ceuticals. LGN has recently been identified as a regulator of proper polarities and alignments of chromosomes when cells di- vide. To do this work, an LGN molecule may unfold and refold to bring its binding sites into proximity with those of its partner proteins. “If we can see where protein X or protein Y tends to bind to LGN, that’ll help us use or block those receptor sites,” Sreenilayam says. Her students are working with full LGN mole- cules as well as two important segments of LGN, and they’re testing three different methods of purifying their three different versions. The full LGN is in Zanetti’s hands, while the segments are handled by Hyde and Liam Casey, a local high school student joining for four weeks through the “Grow- ing the Science Pipeline” program. For the supersaturation process of crys- tallizing protein molecules, their LGN sam- ple needs to be “about 95 percent pure,” says Sreenilayam, “and that’s not easy.” The LGNs must first be expressed in large quan- tities by E. coli cells, which means that at harvest time, when the researchers apply sound waves to break open the cells, the LGNs are suspended in a slurry of other molecules. Their investigations focus on how best to extract them.

One method is affinity binding. Zanetti

explains, “The LGNs have small clumps of histidine molecules attached as tags, which are known to have an affinity for bind- ing to nickel. When we run our sample through a column con- taining nickel resin, ideally it will pick out and bind the tagged LGNs.” In practice, he reports, it’s only partially effective. A second approach is an ion-exchange column, to separate molecules that have different electrical charges. With anion exchange, negatively charged segments of LGN adhere to a pos itively charged resin; with cation exchange, it’s positively charged LGN that sticks to a negative resin. Either way, the bound LGNs are then released in a saltwater buffer, where so - dium takes the place of the positively charged proteins, or chlo ride swaps places with negatively charged proteins,



Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72