Prof. Alex Tzagoloff
Office Hours: TBA
Experiments assigned to the entire class
1. Purification of BamH1 endonuclease from B. amiloliquefaciens - The final enzyme is expected to be of commercial grade free of other nucleases and of phosphatase. Each group is expected to purify the enzyme starting from cells grown in 4 liters of medium. The students are exposed to the following techniques.
-cultivation of bacteria
-lysis of cells by sonic disruption and use of French Press
-salt fractionation of proteins
-separation of proteins on ion exchange and hydroxylapatite columns
-assay of endonuclease activity
-determination of specific activity
-electrophoretic separation of DNA fragments on agarose
-test for extraneous nuclease
-test for ligatability of fragments generated by the purified enzyme
2. Purification of plasmid DNA from E. coli. Each group is expected to purify plasmid DNA to be used for the preparation of DNA size standards. Transformants harboring different plasmids are grown on a 2 liter scale and plasmid DNA is isolated and ultimately purified on CsCl. During the course of this exercise students learn the following techniques.
-conversion of cells to protoplasts
-extraction of nucleic acids free of protein
-precitation of nucleic acids
-purification of DNA on CsCl gradients
-quantitation of DNA by absorbance at 260 nm
-large-scale digestion and recovery of DNA
Projects assigned to individual groups
1. Preparation of DNA size standards. One group of students is assigned to collected the different plasmid digested to yield the desired fragment sizes. The students are expected to test the concentrationsof the different plasmid digests and to mix them in proportion that will achieve a well balanced mixture with fragment sizes ranging from 15 kb to 0.1 kb.
2. Construction of plasmids for expression of yeast genes from GAL4 promoter. Each of two groups are constructing two plasmids. One set of plasmids will have the LEU2 gene as a selectable marker in yeast, the second set of plasmids will have URA3 as the marker. The plasmids will also differ in the orientation of the GAL4 promoter region with respect to the multiple cloning region derived from pUC18. The students will learn the following methods.
-design of primers and PCR amplification DNA with new restriction
-separation and recovery of DNA fragments from preparative agarose gels
-ligation of DNA fragments
-transformation of E. coli with ligation mixtures
-plasmid miniprep screening of transformants
-partial digestion of plasmids
-destruction of restriction sites by Klenow filling-in of protruding ends
-galacatose induction in yeast of test gene cloned into the different GAL4 plasmids
3. Cloning and expression of Taq polymerase. This project is assigned to four groups. Two groups will be cloning the amino-terminal coding region of the polymerase by PCR amplification such that the fragment can be inserted into a high-expression E. coli plasmid. The other two groups will be cloning the rest of the coding region by preparing a recombinant library and screening it by colony hybridization. This will minimize the chances of introducing mutations into the active region of the enzyme. In addition to some of the methods and concepts described above this project will expose the students to the following methods.
-construction of libraries from genomic DNA
-screening of libraries by colony hybridization
-construction of recombinant plasmids capable of directing expression of foreign proteins
-analysis of proteins by gel electrophoresis
-assay of chain elongation activity of DNA polymerase