\"The mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury r

Question

"The mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via the mer operator/promoter. An Escherichia coli cell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date."

Question

Briefly explain how GFP is used to screen for merR mutants

Explanation / Answer

GFP is Green Fluorescent Protein which is used to visulize protein in bacterial cells or any cells. A plasmid is constructed which contains a target gene of interest, an antibiotic resistance gene (ampicillin), GFP. GFP is introduced between promoter region and target gene of interest. This plasmid is introduced in gram negetive bacteria contains mercury regulating operons. The plasmid will get introduced in the host genome and upon transcription of the genome of the bacteria, GFP will also transcribe and fluoresce. The cells were also treated with antibiotic which will kill all the bacteria which do not have GFP integrated into the genome, however, all the bacteria containing anti-biotic resistance gene will survive and bacterial genome upon transcribing and translating will produce GFP which one can visualize through excitation at 395 nm.

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