Question

If you had to "adopt" a random gfp mutant, what might have gone wrong in the cloning step of random mutagenesis. Be sure your hypothesis is logical

Answer 1

The jelly fish Aequorea victoria and the sea pansy Renilla reniforms emit green light from a green fluorescent protein ( GFP) The characteristics of the in vitro emission match the in vivo bioluminescence emission. The Fluorophore responsible for the green fluorescence is an integeral part of the GFP peptide chain and is not a prosthetic group. This protein is made up of 238 amino acids. In A. victoria GFP, fluorescence occurs when aequorin , a separate luminescent protein interacts with Ca²⁺ions inducing a blue glow.Some of this luminescent energy is transferred to to GFP shifting the overall colour to green.

Later on,molecular biologists produced a GFP molecule that folded and was fluorescent at room temperature without the need of exogenous factors that were specific for jelly fish. But it did have drawbacks it showed two peak emissions, poor photostability and pH sensitivity. but, eventually through random mutagenesis mutants with highly improved GFPs, were produced  due to the introduction of point mutations(F64L) many mutants have been cloned in vitro.

Random mutagenesis is a tool for generating enzymes, proteins and entire metabolic pathways or entire genomes with improved properties.This is achieved by treating DNA or whole bacteria with various chemical mutagens by passing cloned genes through mutator strains by error prone PCR mutagenesis, by rolling circle error prone PCR,or by saturation mutagenesis.

Error prone PCR is a method that generates random mutations throughout a gene or gene region without being limited by a size constraint which is a limitation of conventional PCR. In this method,Taq or a similar polymerase l

that lacks proofreading activity and DNA repair mechanisms encourages the preservation of mismatched bases. The mutation rate is increased by the usage of Mn²⁺ ions or changing the concentration of Mg²⁺ ions and an unbalance nucleotide pool. Amplification of genes under these conditions leads to a population of molecules having point random mutations. Maybe, instead of subjecting the DNA to a conventional PCR process the gene was apmlified under error prone conditions.

In the cloning process E.coli cells are used for their plasmids which possess 2 genes amp^R which confers ampicillin resistance and lacZ that codes for beta- galactosidase which hydrolysis lactose. These properties are tested during the screening process. But, accidentally, the E.coli XL1- Red strain(Stratagene) which generally are responsible for random mutagenesis of the GFP cDNA was utilised. which resulted in the formation of gfp mutants.