Chapter 13 Homework
1. For isolating the gene for restriction endonuclease Eco RI, I would take E. coli DNA, and
digest it with HindIII. Now, I would take a plasmid (let’s say pBR322), and ligate the different
digested pieces of E. coli DNA into the same HindIII site of the plasmids, and form a library. I
would then transform them into E. coli and grow them in liquid media. Now, infection with
bacteriophage lambda would result in only a few colonies to grow which should be lysis
resistant. These lysis resistant colonies contain the Ecor RI gene because, they would not allow
the bacteriophage lambda to cut the plasmid and degrade it. Since, it recognizes the foreign DNA
of bacteriophage lambda and degrades it, the colony having Eco RI gene can then be recognized.
4. Indigo is a secondary metabolite which can be produced in E. coli. There are certain enzymes
that need to be introduced because E. coli can produce indole but not indigo. The tryptophan can
be converted to indole by the enzyme tryptophanase in the E.coli host cell. Two different genes
which code for naphthalene dioxygenease, and xylene oxidase if introduced into E. coli can help
it to synthesize indigo. There are two pathways, and one relies on the addition of napthalene
dioxygenease and the other pathway on xylene oxidase. Napthalene dioxygenease oxidizes
indole to cis-indole-2,3 dihydrodiol, and spontaneous dehydration, and oxidation results in the
formation of blue colored dye indigo.
9. Usually erythromycin is synthesized by Saccharopolyspora erythraea. Ketoreduction,
dehydration, enoylreduction of the β-group of the growing polyketide chain helps in the
production of polyketides. Now, in order to modify the polyketide antibiotic erythromycin, I can
alter the polyketide synthase by deleting a DNA region that encodes β-ketoreductase activity.
This deletion then causes an accumulation of carbonyl moiety rather than hydroxyl group at the
C-5 carbon ring. Hence, if I can alter particular DNA fragments, the structure of the antibiotic
can be modified in a predictable manner.
10. Since it was not possible to sequence the DNA for finding the nucleic acid sequences of the
adhesive, the intracellular precursor form of the adhesive protein namely 130-kilodalton
precursor protein was isolated. It is rich in serine, threonine, lysine, proline and tyrosine. I can
clone the complete or partial cDNA of this protein into E. coli, and this can be used to construct
a 600 base pair synthetic gene encoding a protein of 25 kDa. T7 promoter expresses this gene at
very high levels. However, for the hydroxylation of the amino acid the bacteria tyrosinase was
used, because this step is essential for the crosslinking of the adhesive protein.
12. Whey is a by-product of the cheese making process that is rich in lactose. It has been used to
help produce important industrial compounds because it is an industrial waste that can serve as a
carbon source for the synthesis of compounds such as polyhydroxyalkanoates (plastics), or
xanthum gum (emulsifier). The idea is to add the genes for the lac operon that produce betagalactosidase and the permease gene necessary for the digestion of lactose. Once this
transformation has occurred the cells can be grown on whey and will thrive because they can
digest the lactose into glucose and use that as fuel for producing whatever product we have
designed them for.
17. E. coli have been engineered to produce lycopene (C40H56) by using the 2-C-methyl-Derythritol 4-phosphate pathway, which provides the precursors needed to produce lycopene.
Expression of these precursors can be dramatically increased if Saccharomyces cerevisiae genes
that encode the mevalonate pathway are added under the control of E. coli transcriptional
promoters. Next, genes from Pantoea agglomerans, a gram negative bacterium, are added that
will allow the E. coli cells to produce functional lycopene from the precursors. So far this
process has shown promise but more work must be done before genetically engineered lycopene
becomes commercially available.
Alternatively, the following are good answers as well.
1. To isolate the gene for EcoRI, first the chromosomal DNA from a bacterium containing the gene
should be digested with HindIII and ligated into the HindIII site on plasmid pBR322. Next, the clone
bank should be introduced into E. coli and grown in liquid medium. Bacteriophage λ should then be
used to infect the cells in order to test for the production of the restriction enzyme. The
transformants that are resistant to lysis by bacteriophage λ are then grown and samples can be
osmotically shocked to release the periplasmic proteins. These proteins should then be assayed for
EcoRI rectriction enzyme activity. Positive clones should also be assayed for EcoRI methylase
activity.
4. To produce indigo in E. coli, first the NAH7 plasmid from Pseudomonas must be digested with
HindIII and the fragments ligated with linear HindIII-digested plasmid pBR322. The clone bank is
then introduced into E. coli cells, and transformants are selected on the basis of their resistance to
ampicillin and sensitivity to tetracycline. The presence of tryptophan in the minimal growth
medium is the main cause for the synthesis of indigo. First, tryptophan in the growth medium is
converted to indole by the enzyme tryptophanase, which is produced by the E. coli host cell. Next,
indole is oxidized to cis-indole-2,3,-dihydrodiol by naphthalene dioxygenase, which is encoded by
the DNA that was clone from the NAH7 plamsind. Next, water is spontaneously eliminated,
followed by air oxidation to form indigo.
9. To produce modified versions of polyketide antibiotics, the catalytic domain of the polyketide
synthase should be altered. It can be altered in two ways: either a DNA region that encodes βketoreductase activity can be deleted or a DNA region encoding enoylreductase can be mutated.
Furthermore, new aromatic polyketide antibiotics can be created by interchanging polyketide
synthase genes between clusters.
10. The adhesive protein may be synthesized in E. coli by expressing a chemically synthesized
adhesive protein gene sequence in the E. coli. Repeating DNA units that encode the consensus
decapeptide repeat of the adhesive protein are used to construct the synthetic gene. The 30-bp
repeat consisted of codons optimized for E. coli expression. Furthermore, the T7 promoter is used
to obtain expression of the synthetic gene at very high levels. A hydroxylation system using a
bacterial tryosinase is also needed to hydroxylate to tyrosine residues to DOPA. Finally, ascorbic
acid must be included in the reaction mixture to prevent the premature oxidation of the DOPA
residues to o-quinone.
12. Whey is a by-product of the cheese-making process that consists of water, lactose (3-4%), and
small amounts of protein, minerals, and low-molecular-weight organic compounds. It can be used
as a carbon source to produce industrially important microorganisms.
17. E. coli can be engineered to produce lycopene by introducing Saccharomyces cerevisiae genes
encoding the mevalonate pathway under the control of E. coli transcriptional promoters. By
subsequently introducing Pantoea agglomerans genes encoding the biosynthesis of lycopene from
precursor metabolites, a relatively high level of lycopene can be produced.