Question

Scientists have engineered bacteria to produce human proteins, such as factor VIII, a blood clotting factor used to treat hemophilia. Suppose the unmodified eukaryotic gene coding for factor VIII is inserted directly into a bacterial chromosome. Select the reasons why no expression would be seen in the bacterial cell for the factor VIII gene. Bacteria cannot remove intronic sequence from a gene, so if the gene for factor VIII were transcribed, it would translate to a nonfunctional protein. The bacterial nucleoid does not have the proper proteins to transcribe the eukaryotic gene. The gene will induce attenuation in the bacterial cell, which will cause a delay in the transcription and translation of the factor VIII gene. The bacteria will not recognize the eukaryotic promoter to transcribe the gene. Arrange the steps that would be used in a laboratory to engineer a bacterium that could express the human gene coding for factor VIII. Not all steps will be placed.

Answer 1

- Bacteria cannot remove intronic sequence from a gene, so if the gene for factor VIII were transcribed, it would translate to a nonfunctional protein.

- The bacteria will not recognize the eukaryotic promoter to transcribe the gene.

Genome engineering steps:

I. Isolate by PCR the mature mRNA of the target factor VIII gene

II. Synthesize a complementary DNA (cDNA) for this mRNA by using a reverse transcriptase enzyme

III. Insertion of the cDNA into a plasmid vector

IV. Expression of the protein in the host bacterial cell through transformation

Step-by-step explanation:

Bacteria lack the spliceosome (ie., the eukaryotic machinery required to remove introns from the primary mRNA), because bacterial genes do not have introns. Moreover, eukaryotic organisms require several proteins that bind to the promoter sequence and subsequently recruit the RNA polymerase to initiate this process. In bacteria, transcription is controlled by proteins that bind to cis-acting sequences which regulate the transcription of adjacent genes. In contrast to bacteria, eukaryotic genes have many classes of promoter and enhancer elements.

Genome engineering technologies can be used to produce bacteria expressing human proteins derived from synthetically introduced human genes (see steps above). For example, recombinant bacteria expressing the human insulin gene have already been designed by using E. coli as cell factories.