University of New Hampshire
Mentor: Dr. Louis Tisa, Assistant Professor of Microbiology
Isolation and Identification of the calC Gene in Escherichia coli
In eukaryotic cells, calcium ions are involved in several cellular functions including cellular differentiation and cell motility. Calcium acts as a second messenger for muscle tissue contractions and to facilitate signal transduction. With prokaryatic cells, calcium participates in several cellular functions, but its exact role is less understood. These organisms have a common evolutionary origin, and it is possible that calcium has the same purpose in both eukaryotes and prokaryotes. Bacterial calcium concentrations are tightly regulated. Ca2+ efflux out of the cell is accomplished by Ca2+/PO42- symporter Ca2+/H+ antiporter activities. How calcium enters the cell is unknown. Four genes that are involved in calcium transport have been identified in Escherichia coli, (calA, calC, calD, and chaA). A defect in these genes (mutations) results in faulty transport of calcium ions. The focus of this study was to isolate and identify the calC gene. A calC mutation results in defective Ca2+ transport by Ca2+/PO42- symporter activity, and prohibits cell growth in the presence of calcium.
Bacteriophage lambda DNA (l #173) which contains the wild type calC gene was used to isolate this gene. The entire genome of E. coli is available as the Kohara ordered cosmid library. One of the cosmids (#173) complemented the the calC mutation. This means that the wild type calC is on the cosmid. Cosmid l #173 was propagated by infecting E. coli strain LE392. The phage particles were broken open, and the phage DNA was purified by use of a QIAGEN Lambda Kit. Purified DNA was cut by the use of restriction enzyme EcoRI. The cut fragments were separated by agarose gel electrophoresis. I am attempting to purify the DNA fragment of interest. After purification I will subclone this fragment into an expression vector.