Crister Lee - Academia.edu (original) (raw)

Papers by Crister Lee

Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zin... more Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber oYcinale were expressed in Escherichia coli under diVerent conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four diVerent bacterial strains; BL21 (DE3), BL21 (DE3) Tuner™, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner™ using diVerent inducing agents (IPTG, The Inducer™). The eVects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no signiWcant diVerence was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer™. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner™, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer™. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7-and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.

In ancient China and India, there was an effective, though highly dangerous, practice of introduc... more In ancient China and India, there was an effective, though highly dangerous, practice of introducing the fluid from the pustules of small pox (variola, variolae) patientsinto healthy individuals, through dermal incisions. This practice, called variolation, using live smallpox virus, was aimed at protecting the individual from contracting the disease.

When a doctor takes a swab from an infected wound and sends it to the lab, he needs to know what ... more When a doctor takes a swab from an infected wound and sends it to the lab, he needs to know what organisms are in the wound so he can select the proper antibiotic. There usually will be more than one organism present, so the lab will need to take the mixture of bacteria and separate them out into pure cultures before they can identify each organism. In this experiment, we will take a mixture of 3 organisms and separate them out into three pure cultures, one of each organism. Here are the three organisms we will use: E. coli (forms white colonies) Serratia marcescens (forms red colonies, but only at room temperature, 25°C) Chromobacterium violaceum (forms purple-blue colonies, but only at room temperature, 25°C). All of these organisms are Gram negative, so if you only did a Gram stain on the tissue swab, it would look like there is only one organism present. Therefore, you have to grow them on a plate so you can examine the colony morphology (color and shape) of the colonies, and then in real life, the lab would also run some tests on each pure culture to identify each organism. We already know the organisms in the mixture, so we will just use this experiment as practice for separating a mixed culture into pure cultures. You will need this skill when you get your unknown organisms in the next lab unit.