Question

You have discovered an enzyme for turning lead into gold (Pb2Au) in the extinct bacterium Alchemistia imbecilis. You need to express the protein with the Mgk promoter. Outline the steps you would use to construct a single plasmid containing all of the following characteristics to create bacteria that might make gold from lead:

1) the Mgk promoter directly upstream of the Pb2Au gene (and in the same orientation),

2) the intact kanr selectable marker,

3) the Mgk::Pb2Au promoter::gene combination disrupting the LacZ gene, and

4) exactly 1 origin of replication.

Answer 1

To generate the plasmid construct with the above 4 characteristics a stepwise cloning is required to create a perfect clone that can generate the gene in the plasmid with the desired characteristics. The stepwise cloning procedure will have the following steps:

1. Isolate the gene of interest from the bacterium Alchemistia imbecilis.
2. Generate a forward primer with Mgk promoter within the primer then followed by 12 bases of the gene of interest.
3. The primer generation should also take into account the incorporation of the restriction enzymes that are necessary for the insert to get inserted into the lacZ gene.
4. Selection of a plasmid that has a kanr selectable marker and a lacZ gene with atleast two restriction sites in the lacZ gene.
5. Based on the orientation of the origin of replication with respect to the lacZ gene the forward and reverse primers should have the respective restriction sites incorporated into them.
6. By following these steps the plasmid that will be obtained will have sequence (which is being shown in a linear way) in the following way: ORI----lacZ---RE1---Mgk---Pb2Au----RE2---lacZ-----kanr------

The abbreviations ORI= origin of replication, RE= restriction endonucleases.

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