MATERIALS AND METHODS

    Supplemented with varying NaCl, KCl, sucrose, and temperature, aerated lactose L-broth-grown E.coli K-12 were grown up to abs600nm ≤ 0.01 and sampled. (1) Biochemical: [Lactase] was measured by the classic ONPG protocol on SDS-chloroform permeablized cells, and normalized on a per-biomass basis. (2) Physical: After NaBr-equilibrium gradient centrifugation (50K g-min), the cell bandıs specific gravity was read refractometrically. As our super-fast method for making gradients has not heretofore been described, it is presented here (Fig. 2, below). To be sure, cesium and rubidium salts would also work but would be very expensive. KI, while soluble enough in water to make the density needed (1.5 g/cc), is unstable such that the iodine reacts with the cells clumping them as well as adding significantly to their density. Thus NaBr is the solute of choice.

Rolling to Make Density Gradients

Figure 2: The Roll Method for making linear gradients. In brief, glass centrifuge tubes were first filled to the 45% level with a suspension of about 100,000 cfu in 7% NaBr. This was underlayered with an equal volume of 50% NaBr (i.e.: a saturated solution at room temperature). The tube was then stoppered in such a way that no air was trapped in the tube. Upon a platform that was tilted so the the interface between the top and bottom layers formed the diagonal in the tube, the tube was rapidly rolled by the palm of the hand for about two feet and immediately brought upright and the stopper removed before immediately centrifuging. The total time from start to finish is less than one minute; approximately 100 gradients can be made in an hour's time. (A HREF="http://www.science-projects.com/RollGrad.htm">www.science-projects.com/RollGrad.htm)