Chapter 14 Flashcards
How would you genetically engineer a bacterium to degrade trichloroethylene?
Degrading trichloroethylene requires toluene dioxygenase gene. Express gene in E. coli with IPTG as inducer for tac promoter or create a hybrid strain with elements of two degradative pathways
Outline a protocol that you would use to clone fungal cellulose genes
mRNA isolated from cells grown without cellulose and from cells grown in the presence of cellulose
each mRNA population fractionated on sucrose gradient, translated in cell-free system. Identify cellulose-induced proteins
mRNA sucrose gradient fractions from cells that direct synthesis of cellulose-induced proteins and comparable fractions from non induced cells are used separately to program synthesis of cDNA bank isolate cDNA clones that hybridize only to cDNA from induced cells
fig 14.30
Delineate the role of alpha amylase and glucoamylase in the industrial production of alcohol. How might genetic manipulation of genes encoding enzymes be used to improve the process?
• Each of the enzymes could be overproduced in a fast-growing recombinant microorganism that utilizes an inexpensive substrate, thereby lowering the cost compared with production from native organisms.
• Variants of α-amylase, either naturally occurring or genetically manipulated, that function efficiently at 80 to 90°C could be used to allow the liquefaction step to be performed at this temperature.
Heat-resistant α-amylase would speed the hydrolysis of gelatinized starch while decreasing the amount of energy that is required to cool the gelatinized starch to a temperature suitable for starch
hydrolysis.
• The α-amylase and glucoamylase genes could be altered so that each enzyme would have the same temperature and pH optimum, thereby enabling the liquefaction and saccharification steps to be performed under the same conditions.
What is glucose isomerase? Why is it important? How and why would you modify the gene encoding this enzyme
Glucose isomerase catalyzes the conversion of 5-carbon D-xylose to D-xylulose and converts glucose to fructose as a side reaction. In the production of fructose, the higher then temperature the more fructose is produced.
To improve the glucose isomerase gene, isolate the enzyme from a thermophilic bacteria but express in E.coli or B. brevis under the control of various promoter and ribosome binding sites.
Substrate binding sites can also be modified to improve binding. Change tryptophane at 139 to phenylalanine and valine 186 to threonine.
Elaborate some of the advantages and disadvantages of using Z. mobilis instead of S. cerevisiae for alcohol production? How would you improve the industrial performance of Z. mobilis
Yields more ethanol the S. cerevisiae but can only use a limited number of carbon substrates, broad host cevtors are difficult to maintain in the organism, naturally resistant to many antibiotics. Include pathways to use other carbon substrates such as lactose. Instead of maintaining on plasmid, incorporate genes for ethanol directly on chromosomal DNA.
How can Z. mobilis be engineered to produce ethanol from xylose and arabinose?
Transform the bacteria with plasmid containing two synthetic operons, one with arabinose assimilation genes and one with pentose metabolism genes.
Starting with a pseudomonas strain that can utilize phenol as its sole carbon source at 0C, a pseudomonas strain that can degrade anthracene to catechol at 35C and a pseudomonas strain that can degrade p-toluene to protocatechuate at 35C, suggest a strategy for developing a strain that can utilize phenol, anthracene or p-toluene as its sole carbon source at 0C
Transfer by conjugation the genes containing anthracene degredative and p-toluene genes into psychrophile.
Explain how a pseudomonas strain that carries plasmid pWWO does not normally degrade 4-ethylbenzoate can be genetically manipulated to hydrolyze this compound?
Two problems: overcome inactivation of important exyme in degradative process by 4-ethylbenzoate and how to induce transcription of genes in this pathway with4-ethylbenzoate as the inducer.
Subclone pm promoter on pWWO onto another plasmid with tet and amp resistance while subcloning the xylS gene into another plasmid with Kan resistance. Transform E. coli with the two plasmid, mutagenize and grow with 4-ethylbenzoate and tetracycline. Transformed cells have a mutated xylS gene that allows it to be activated by 4-ethylbenzoate.
Transform the p. putida strain with broad host range plasmid carrying mutated xylS* gene which can activate the pm promoter on pWWO plasmid. The transformants are mutagenized and grown on minimal growth medium containing 4-ethylbenzoate. Cells that grow contain the mutated 2,3 dioxygenase gene .
Suggest schemes for the isolation of prokaryotic endoglucanas and beta glucosidase genes
Create a clone bank of DNA from cellulolytic prokaryote constructed on E.coli. Grow on media with selective antibiotic. Overlay agar with CMC and incubate. Flood plate with congo red and wash with sodium chloride. Yellow halo indicates that the colony secretes endoglucanase.
Transform a clone bank from beta glucosidase producing microorganism into E.coli and select for transformants that can grow on minimal media with cellulobiose as carbon source or detect beta-glucosidase activity with a chromogenic substrate such as BCIP.
What is a superbug?
Bacterial strains with expanded degradative capabilities, can degrade more than one type of compound.
How can pesticide-degrading enzymes be expressed on the surface of the bacterium?
Express enzyme as part of a fusion protein that contained the E.coli lipoprotein signal peptide, the N-terminal portion of the lipoprotein and outer membrane protein A.
(Gene: lec promoter, lpp signal sequence, lpp gragment, ompA transmembrane domain, opd signal sequence, opd gene)
How would you engineer yeast strains to more efficiently convert glucose into ethanol?
Yeast Strains must tolerate higher concentrations of glucose and ethanol. Generate a large number of randomly mutated yeast transcription factor genes and grow on high concentrations of glucose and ethanol.
How would you expand the substrate range of a strain of Burkholderia sp. That normally degrades 2,4-dinitrotoluene?
Isolate gene for 4-methyl-5-nitrocatechol monooxygenase gene and subject it to error prone PRC, clone into plasmid vector and transformed into E. coli, plate onto agar with 4-nitrophenol. Transformants that are dark brown can breakdown more similar nitroaromatics.
How would you engineer glucoamylase to be more efficient in degrading starch
Gene: GAL promoter-secretion domain-catalytic domain – starch binding domain
Starch binding domain increases ability for enzyme to hydrolyze insoluble starch
How would you engineer E.coli to produce isopropanol?
Engineer E. coli with genes encoding isopropanol biosynthesis pathway in clostridium along with an additional 4 genes from other bacteria
