Question

How might you use molecular cloning and heterologous expression to identify a gene or genes responsible for a particular phenotype (e.g. pectin degradation)? What are some advantages and disadvantages?

Answer 1

Classical method: In classical method to identify a gene responsible for a particular phenotype are elucidated by constructing genomic library of a given microbe. First a given organisms total genomic DNA is isolated and restricted digested in to several thousand fragments and each fragment is cloned in to vectors/plasmids and maintained in hosts like E.coli and this gives a genomic library of that organism. Each plasmid is either maintained as such or expressed in E.coli, Bacillus subtilis or yeast (heterologous expression) and the gene product is studied for the characteristic phenotype for example pectin degradation enzymes.
Advantages: if we are elucidating new organisms genome we will have not only have data regarding new genes as well as the intended phenotype genes or charecteristics of that organism.
Disadvantages: this method is time consuming and library maintenance and studies may be costly, as we do not know where our intended characteristic gene is it will be labor intensive to do this type of work.

Modern methods or reverse genetics method: from the modern sequencing methods we know the protein sequence of the pectin degradation enzymes (purify the protein and sequence it); by reverse genetics we can construct mRNA sequence responsible for that enzyme or characteristic phenotype protein. From mRNA we can construct cDNA and will be cloned in to the heterologous expressing host and the produced protein can be studied. Other method is we can find consensus sequence of the intended gene from the DNA library of other organisms and we will construct DNA primers and using the genomic DNA of the organism we will do a polymerase chain reaction to amplify the gene responsible for that character and clone the amplified gene fragments in to the heterologous hosts, express the proteins and study them.
Advantages: Both the above methods are precise and rapid and we can screen faster.
Disadvantages: method requires modern laboratory with facilities like sequencer, PCR and purification systems and many other things. Failures may happen if any mistakes is done during right data identification and processing.