Chapter 14
2. In order to clone fungal cellulase genes, we can use the technique of differential hybridization.
The mRNA that encodes cellulolytic enzymes is rare, and has to be enriched or increased for
efficiency. We can do that by growing fungal cells with and without cellulose, and isolating
mRNA from both set of cells. Then, mRNA fractions are separated on a sucrose gradient, and
after a cell-free translation, the cellulose-induced mRNA fractions can be identified which
contain messengers induced by the addition of cellulose. The cDNA samples from the induced
cells are cloned into plasmid vectors and introduced into E. coli. After replica plating, they are
made to hybridize with the cDNA from both induced and non-induced fractions. The clones
hybridizing with the cDNA from induced cells, and not the non-induced ones are the ones
carrying cellulose-induced mRNAs. Such cDNAs that encode cellulose-induced genes, are
expressed in E. coli and tested for reactivity to antibodies generated from enzymes in the
cellulose complex to conclusively identify cellulase cDNAs.
5. S. cerevisiae has the disadvantages of being unable to tolerate high levels of alcohol,
insuffiecient expression of necessary enzymes, and high potential for loss of plasmids.
However, these yeast have been modified with gene constructs to become more suitable for
alcohol production. Z. mobilis is a bacterial system so it has the advantage of growing more
quickly. In addition, it produces a higher yield of ethanol, produces less biomass, has a
stronger alcohol tolerance, does not require additional oxygen (generally easier to handle).
The problem with Z. mobilis is that its substrate range is limited so it cannot use all forms
of sugar. Efforts are underway to engineer strains that overcome this limitation but so far
this seems to negatively effect overall yield
12. Pseudomonas synthesize a pesticide degrading enzyme called organophosphorus hydrolase
that can be used to degrade organophosphorus (a pesticide affecting the nervous system).
However, this degradation procedure is very slow. Therefore, what can be done is E. coli cells
can be engineered to express organophosphorus hydrolase as a part of fusion protein. This fusion
protein contains an E. coli lipoprotein signal peptide, N-terminal portion of lipoprotein, and outer
membrane protein A (with transmembrane regions). This fusion protein was localized on the
plasma membrane with the organophosphorus hydrolase directed to the outer surface of the
bacteria making it easier for the availability of the organophosphorus degradation. .
13. Usually, in the presence of high levels of radioactivity, the DNA of any microorganism is
affected causing irreversible DNA damage. However, the non-pathogenic soil bacterium
Deinococcus radiodurans is resistant to the ionizing radiation. The DNA repair processes are
highly efficient in repairing the DNA damage caused by the ionizing radiation. The
bioremediating genes encoding for such proteins can be expressed in the bacteria, since they are
also protected against the effects of radiation, and can be used for the degradation of organic
environmental pollutants even in the presence of radioactivity.
Chapter 16
3. The B. thuringiensis toxin doesn’t affect the human due to the fact that it requires alkaline
environment and the action of certain proteases. In contrast, the human gut is acidic and also it
lacks the presence of the required proteases which prevents the action of the toxin, and makes it
harmless to humans.
4. The Bt subspecies israelensis is effective because it carries a low risk for the
development of resistance. To isolate the protoxin gene from the bacterium one could use
the proteins that the translated Bt protein will bind in the guts of insects to bind the Bt
protein. The protein probe could be tagged so that the complex could be isolated and the
sequence for the protoxin could be determined using amino acid mass spectroscopy. This
gene would be useful because it carries less risk of insects developing resistance so it will
be better for the long term. It can be inserted into other bacteria such as E. coli.
9. To lessen the risk of resistance I would use the israelensis strain of Bt because this
subspecies has a low level of resistance because they not only synthesize three different
Cry proteins but also produce a toxic protein that is not homologous to the Cry proteins
which decreases the chances that insects will spontaneously develop resistance to toxins so
different in structure. In addition one should consider rotating different strains of the toxin
annually, alternating use of Bt toxin with the use of other environmental toxins, or mixtures
of Bt strains to decrease risk of resistance development.
15. To improve the insecticidal properties of baculoviruses I would suggest engineering a
baculovirus that synthesizes juvenile hormone esterase, which will prematurely cause the
larvae to stop feeding. This will slow destruction by the larvae. In addition, one could
insert a toxin gene such as the Bt toxin gene into the viral genome so that infection also
carries the toxin and the virus can more easily kill the insects. Many other genetic
engineering techniques can be used to enhance the insecticidal properties of baculoviruses.