You have found a new streptomycin resistance allele in Streptococcus (the same b

Question

You have found a new streptomycin resistance allele in Streptococcus (the same bacteria that Avery and Griffiths were working with). A wild type version of this allele is normally found within the strain you are working in (hence it’s normally streptomycin sensitive). When you clone this gene onto a plasmid in E. coli and then transform the original Streptococcus strain with this plasmid, you can isolate resistant Streptococcus colonies even though the plasmid does not replicate in Streptococcus. When you PCR up this same gene and transform Streptococcus, you can NEVER select for resistant colonies. Describe one potential explanation for these experimental results. 

Explanation / Answer

When you clone this gene onto a plasmid in E. coli and then transform the original Streptococcus strain with this plasmid, you can isolate resistant Streptococcus colonies even though the plasmid does not replicate in Streptococcus. When you PCR up this same gene and transform Streptococcus, you can NEVER select for resistant colonies.

The original strain of streptococcus is transformed as it is inserted with the plasmid. When the gene is inserted with the help of plasmid, the association with the plasmid causes addition of methyl groups in the gene, I.e. methylation. As a result of this methylation, the native restriction enzymes present in bacterial cells will not be degrading the gene.

When the gene is amplified using PCR , there is no methylation of the gene. As a result, restriction enzymes present in the bacterial cell will degrade the amplified gene. Hence, it could never be used for isolation of resistant cells.

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