IGPS to ivePRAI: Selection

Having obtained a structure similar to PRAI, the next step was to engineer PRAI activity. The fact that PRAI is involved in the tryptophan synthesis pathway provided a convenient mechanism through which to select for PRAI activity.

A strain of Escherichia coli (JA300 Trp-) was found that could not synthesize Trp, a biochemical defect that results from a mutation in the PRAI gene (Figure 12). Trp- therefore requires an external source of tryptophan for growth. Thus, if the gene for a newly constructed PRAI protein is inserted via plasmid into JA300 Trp- DNA, and these bacteria are then plated on minimal media lacking tryptophan, the bacteria can only grow if they acquire the ability to synthesize tryptophan. Only recombinant cells with DNA coding for PRAI activity can grow in the absence of tryptophan.

The cells containing the initial DNA libraries (IGPS49L1, IGPS49L1RGD, and IGPS49LSV) were plated on media in the absence of tryptophan, but since they still lacked PRAI activity, none of them could survive. However, when the recombinant library, IGPSL1L6, was introduced to the Trp- cells, surviving colonies were obtained in the absence of tryptophan. IGPSL1L6 recombinants were plated on media containing a range of tryptophan concentrations, and colonies surviving on the lowest tryptophan concentrations were selected. DNA from these cells was shuffled with DNA from the original libraries to produce recombinant bacteria.

This cycle was repeated, and one strain containing the IGPSL1L6-2 library yielded a large colony in the absence of tryptophan. Controls showed that their survival was due to the inclusion of the reshuffled DNA (IGPSL1L6-2cycle), and the strain was dubbed in vitro evolved PRAI, or ivePRAI.

[Overview] [Background] [Structural Modification] [Selection] [ivePRAI] [Implications] [References] [Links]

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Nikki Jarrett, 11/20/00
Biochemistry462bH, Dr. D. Bourque