Question

pUC19 is a cloning vector, but cannot function as an expression vector.

a) Explain the difference in requirements for cloning and expression vectors.

b) For the purpose of expressing a eukaryotic protein in bacterial cells, will you rather use a clone from a genomic or a cDNA library? Motivate.

c) Assume you have created a genomic and cDNA library for the same cell type. You then screen both libraries with the same probe. You observe a signal from some of the genomic library colonies but no signal from any of the cDNA colonies. Explain this result.

Answer 1

Answer a)- The difference in requirements for cloning and expression vectors are-

• Cloning vectors are those vectors which are used for cloning purposes. in cloning vectors, gene of interest( foreign DNA) is inserted and maintained in a suitable host while expression vectors express the gene of interest in the suitable host i.e.they make the desired protein product.
• Cloning vectors mainly have selectable marker , reporter gene ( in some cloning vectors, reporter gene is required in order to screen the cloned gene) and multiple cloning sites (MCS) along with high copy number while expression vectors mainly have components required for expression of gene of interest which include , ribosomal binding site, promoter to start transcription and termination sequence ( stop codon) , translation machinery, etc. so that they can express the desired protein product.

Answer b) - To express a eukaryotic protein in bacterial cells, we can use clone from both genomic DNA library and cDNA library, the choice depende upon you. But cDNA serves better than genomic DNA library because cDNA library is made up of only processed mRNA i.e. which have exons and donot have introns and bacterial cells also donot express introns so using clones from cDNA  library is not a cumbersome procedure and we can easily express eukaryotic protein in a bacterial cells.

• We can also genomic DNA library as it is made up of toatl genome and there is always a high probability that genomic DNA library conatins a clone to express a eukaryotic protein in bacterial cells.

Answer c)-When screening for the same cell type is done for both genomic and cDNA library, a signal was observed from some of the genomic library colonies but no signal from any of the cDNA colonies ,the reason for this observation can be that cDNA library is made up of only processed mRNA (which have exons and can express protein ) while genomic DNA library is made up of total genome ( both exons and introns) so we can say that the signal observed is not from processed mRNA (exons) part of genome , it if from introns ( part of genome which is not transcribed). That's why we observed from signal from some of the genomic library colonies but not from any of the cDNA colonies.