Staphylococcal nuclease (Staph Nuc) is a heat-stable, potent secretory protein that cleaves dsDNA in vitro. You have a cloned the Staph Nuc gene on pBR322 plasmid and transferred into E. coli. You have named the chimeric plasmid pBR322 with the cloned Staph nuc gene as pNuc. In an experiment, it was necessary that you purify pNuc from an E. coli culture, but was unsuccessful. The cause for being unable to purify the pNuc plasmid was that the Staphylococcus nuclease enzyme hydrolyzing the plasmid along with the chromosomal DNA immediately after cell lysis and released in the salt buffer during purification process. How would solve this issue and protect the pNuc plasmid DNA so that you can proceed with the downstream molecular analyses of the gene?

Question

Staphylococcal nuclease (Staph Nuc) is a heat-stable, potent secretory protein that cleaves dsDNA in vitro. You have a cloned the Staph Nuc gene on pBR322 plasmid and transferred into E. coli. You have named the chimeric plasmid pBR322 with the cloned Staph nuc gene as pNuc. In an experiment, it was necessary that you purify pNuc from an E. coli culture, but was unsuccessful. The cause for being unable to purify the pNuc plasmid was that the Staphylococcus nuclease enzyme hydrolyzing the plasmid along with the chromosomal DNA immediately after cell lysis and released in the salt buffer during purification process. How would solve this issue and protect the pNuc plasmid DNA so that you can proceed with the downstream molecular analyses of the gene?

Answer 1

To solve the issue of Staphylococcus nuclease (Staph Nuc) hydrolyzing the pNuc plasmid DNA during the purification process, you can use a combination of several strategies:

1. Modify the lysis buffer: Staph Nuc is a heat-stable enzyme, so you can add a chelating agent like EDTA (ethylenediaminetetraacetic acid) to the lysis buffer. EDTA binds to divalent cations, such as Mg2+, which are essential for the activity of Staph Nuc. By sequestering these cations, you can inhibit the nuclease activity and protect the plasmid DNA from degradation during lysis.

2. Improve the speed and efficiency of cell lysis: One reason for immediate release of Staph Nuc and subsequent plasmid degradation could be slow or incomplete cell lysis. By using more effective cell lysis methods, such as sonication or enzymatic lysis with lysozyme, you can ensure the rapid release of cellular components, including Staph Nuc, while minimizing the exposure time that leads to DNA degradation.

3. Inhibit Staph Nuc activity during purification: To protect the pNuc plasmid DNA during the purification process, you can add an enzyme inhibitor specific for Staph Nuc. One such inhibitor is PMSF (phenylmethylsulfonyl fluoride), which irreversibly binds to the active site of the enzyme and blocks its activity. By including PMSF in the purification buffer, you can prevent Staph Nuc from hydrolyzing the plasmid DNA.

4. Optimize purification conditions: Adjusting the purification conditions can help minimize Staph Nuc activity. For example, lowering the temperature during the purification steps can reduce the activity of Staph Nuc. Additionally, choosing a purification method that minimizes exposure time, such as rapid size exclusion chromatography or affinity purification, can help protect the plasmid DNA.

By implementing these strategies, you can protect the pNuc plasmid DNA from degradation by Staph Nuc during the purification process. This will enable you to proceed with downstream molecular analyses of the gene without losing the intended DNA sequence.