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Week 13 - B-gal's

Week 13 - B-gal's - BIOL 519 Week 13 LacZ-galactosidase...

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BIOL 519 Week # 13- LacZ - β -galactosidase Assays 1 BACKGROUND This week, you will perform quantitative assays on the rhaB-lacZ fusions you have constructed. These assays will allow you to determine the amount of β -galactosidase produced from your fusions in the absence and presence of rhamnose, and will therefore give you a quantitative measure of the regulation of the fusions. Growth of cells for β -galactosidase assay - There are a variety of factors that were taken into account when designing the media and the steps that will be used to prepare the cultures to be assayed in this experiment. Some of these factors, and the reasoning behind them are described below: Growth in LB - On Tuesday, you will inoculate each of your strains into LB broth, and incubate the tubes at 37 o C (with shaking) overnight. These cultures will provide a large and consistent number of cells for an inoculum into the minimal media tubes on Wednesday. When using a colony to inoculate into liquid media, the amount of inoculum can be highly variable from one tube to another. This variability is not much of a problem when inoculating into rich media (like LB). However, it is much more difficult for the cells to grow in minimal media, so too small of an inoculum may result in no, or poor, cell growth. Using an LB broth culture to inoculate into the minimal media tubes avoids this problem. Growth in Minimal media in tubes - On Wednesday, you will inoculate from the LB cultures into Minimal media with glycerol plus or minus L -rhamnose. This step ensures that the cells that are assayed are fully adapted to the minimal media they are growing in by the time we assay them on Thursday. The physiology of E. coli grown in LB broth is very different than the physiology of E. coli grown in Minimal media with glycerol as a carbon source. If, on Thursday, we used an overnight culture in LB as an inoculum for the cultures you will assay, the cells would only have been growing in the minimal media for a relatively few generations by the time we assay them. It is likely that the cells would not have fully shifted their physiology from the LB grown physiology to the Minimal media grown physiology. Our results would therefore not be an accurate measure of the amount of expression from the fusions in Minimal media with glycerol. By adding this extra round of growth of the cells in Minimal media with glycerol plus or minus L -rhamnose, you can be sure that the cells you assay will be fully adapted to the media you assay them in. Growth in Minimal media in flasks - These cultures are grown in flasks with baffles so that they receive good aeration during growth. This will allow the cultures to grow fairly rapidly, even in this minimal media. Rapid, well aerated, growth is important both for the physiology of the cells, and for convenience of having the cells grown up in time for class.
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