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Operon Fusion

Methods & Materials
Data & Analysis


The phages used are derivatives of Mu, whose genome consists of structural gene C and a lytic gene. MuCts and MuD1 were used together in the experiment, acting as a dilysogen. The genome of Mu and MuCts are identical, except the temperature permissive nature of MuCts’s lytic gene. The genome of MuD1 consisted only of the bla and lac genes; MuD1 cannot initiate propagation of itself and must rely on the lystic capability MuCts. A lysate of these two phages were created for introduction into a series of dilutions of MC4100.

The specific strain of E. coli used in this experiment was MC4100. The strain of E. Coli used to create the lysate was Mal103. Escherichia coli, because it is well-understood and easily manipulated, was used as the target for the vectors used in this experiment.

The assay to examine insertion of the phage DNA involved the inability or ability of MC4100 to metabolize arabinose. The pathway of arabinose metabolism is show in figure 1. As figure 1 indicates, arabinose is first broken down into l-ribulose by the products of the A gene, then l-ribulose-5-P by the B gene and then into xyulose by the D gene. The sugar can be metabolized further without the arabinose genes.

The arabinose pathway consists of, in order, genes D, A, B, the promoter, and autoregulated C. The metabolism of arabinose cannot proceed if there is a disrupting insertion in structural genes D, A, or B.  Disruptions in gene A and B result in an inability to use arabinose in E. Coli. Disruptions in the D gene are lethal, resulting in a buildup of toxic ribulose-5-phosphate (R5P). In this experiment, only araD mutants were used because only mutants who carried a reverse mutation back to wild type and/or a mutation earlier in the pathway could survive on a plate containing arabinose.

Four types of plates were used in this experiment: LBC agar plates, Arabinose MacConkey with and without ampicillin plates, and Lactose MacConkey plates with ampicillin. MacConkey plates were chosen because they are designed to grow gram-negative bacteria like Escherichia coli and contain indicators that stain the bacterial colony when arabinose is metabolized.

Steric Hinderance - Sec 002 - BIOL 302L - FA09