A researcher wants to express a human protein in E. coli. She isolates the gene directly from human genomic DNA, inserts it into a bacterial expression vector with a strong promoter, and transforms E. coli cells. The bacteria grow and are antibiotic-resistant, confirming the vector was taken up — but no human protein is detected. What is the most likely explanation?
AThe antibiotic resistance gene interferes with expression of the human gene
BThe human genomic DNA contains introns that E. coli cannot splice, so the ribosome cannot produce a functional protein from the transcript
CE. coli ribosomes cannot recognize human codon sequences
E. coli lacks the RNA splicing machinery (spliceosome) found in eukaryotic cells. When a eukaryotic genomic sequence is transcribed in bacteria, the resulting mRNA still contains intron sequences that interrupt the coding region. The bacterial ribosome attempts to translate this interrupted message and produces either a truncated or frameshifted, nonfunctional protein. The solution is to use a cDNA clone derived from processed mRNA, which has already had introns removed. Option D (glycosylation) is a real concern but a separate issue that would produce misfolded protein, not zero protein.
Question 2 Multiple Choice
A laboratory wants to identify which genes are actively expressed in liver cells during fasting versus fed states. Which library strategy is most appropriate, and why?
AA genomic library from any cell type, because all cells share the same genome
BA cDNA library constructed from liver mRNA isolated under each condition, because it captures only expressed genes and reflects tissue- and state-specific expression
CA cDNA library from any tissue, because mRNA sequences are identical across all cell types
DA genomic library from liver cells, because it preserves regulatory sequences that control expression
A cDNA library is made from mRNA — the population of transcripts actually being produced in a given cell type under specific conditions. Two cDNA libraries made from liver under fasting vs. fed conditions will contain different sets of clones reflecting differential gene expression. A genomic library from any cell contains the same DNA (all genes, expressed or not) and cannot distinguish which genes are active. This selectivity is the cDNA library's primary advantage when the question is about expression patterns.
Question 3 True / False
A cDNA library made from muscle cell mRNA will contain different clones than a cDNA library made from liver cell mRNA, even though both libraries come from the same organism.
TTrue
FFalse
Answer: True
cDNA libraries are made from mRNA, which represents only the genes being actively expressed in a specific tissue under specific conditions. Muscle cells express high levels of myosin and actin mRNAs but low levels of albumin mRNA; liver cells show the reverse. Because gene expression is tissue-specific, cDNA libraries faithfully capture this specificity. This is precisely what makes cDNA libraries useful for studying differential gene expression — and why they contrast with genomic libraries, which contain the full genome regardless of tissue source.
Question 4 True / False
An expression vector can produce protein from any inserted gene, as long as the gene is correctly inserted into the multiple cloning site of the vector.
TTrue
FFalse
Answer: False
Correct insertion into the multiple cloning site is necessary but not sufficient. An expression vector must also contain host-appropriate regulatory elements: a strong promoter recognized by the host's RNA polymerase, a ribosome-binding site matched to the host (Shine-Dalgarno sequence for bacteria, Kozak sequence for eukaryotes), and a transcription terminator. A gene inserted without these elements, or with elements from the wrong host system, will not be transcribed or translated even if the DNA is present. A bacterial expression vector won't work in mammalian cells, and vice versa.
Question 5 Short Answer
Why is a cDNA library rather than a genomic library typically used when the goal is to express a eukaryotic protein in bacteria, and what specific molecular feature of cDNA makes it functional in this context?
Think about your answer, then reveal below.
Model answer: cDNA is reverse-transcribed from mature, processed mRNA, which means introns have already been spliced out. The coding sequence in cDNA runs continuously from start to stop codon without interruption. Bacteria lack the spliceosome machinery needed to remove introns, so a genomic clone placed in a bacterial expression system would produce an unspliceable transcript. cDNA's intron-free structure allows bacterial ribosomes to read a continuous, uninterrupted open reading frame and produce the correct protein.
This distinction — between genomic DNA (intronic) and cDNA (intron-free) — is one of the most fundamental concepts in molecular cloning strategy. It explains why the first step in expressing eukaryotic proteins in bacteria is always 'clone from mRNA,' not 'clone from genomic DNA.' It also illustrates that knowing a gene's genomic sequence is not enough — you need to understand how its information is processed in the source organism before deciding how to clone it.