Chapter 13 Flashcards
Describe the strategy for isolating the gene for the restriction endonuclease EcoRI
Digest the chromosomal DNA of E. coli with HindIII and ligated to HindIII site on plasmid pBR322. Then transform another organism such as P. stuartii with E. coli clone bank. Must ensure that the P. stuartii has a defective EcoRI enzyme. Grow the transformants on liquid medium and the infect with DNA bacteriophages. If the cell is expressing the restriction enzyme, the host cell is resistant to lysis because the RE degrades infecting bacteriophage DNA. Transformants that are resistant to lysis are grown and osmotically shocked to release periplasmic proteins and assayed for EcoRI RE activity. Following, positive clones are assayed for EcoRI methylase activity.
An alternative method would be to create a clone bank from E. coli with known EcoRI activity with pBR322 plasmid containing EcoRI recognition site. Then transform clone bank into P. stuartti and grow in medium that selects for presence of the plasmid. The plasmid DNA preparation is then treated with EcoRI and cells that transform and grow contain the genes for EcoRI methylation enzymes. The transformants are then assayed for EcoRI activity.
Outline a strategy for cloning the gene for 2,5 diketo-D-gluconic acid reductase from corynebacterium into erwinia. Why is this useful?
Purify the 2,5-DKG enzyme from Corynebacterium and sequence the first 40AA from the N-terminal.
Based on the AA sequence, two 43-NT DNA hybridization probes (each corresponding to a different position) is synthesized. Probes were synthesized to contain G or C at every third position so minimize mismatches between probe and target DNA.
Corynebacterium DNA clone back was screened with these two probes and clones that did not interact with both probes were discarded. Clones that were not discarded were isolated and sequenced as it contained the 2,5-DKG reductase gene.
Delete sequences upstream of ATG and replace with transcriptional and translational signals that function in E.coli because Corynebacterium is gram positive but Erwinia is negative.
Screen for 2,5-DKG reductase activity then subclone into broad-host-range vector which will be subsequently transformed into Erwinia.
This recombinant organism should be useful as a
source of 2-KLG for the production of l-ascorbic acid, thereby replacing the first three steps of the currently used process.
Suggest a strategy for improving the commercial utility of a cloned 2,5-diketo-D-glucuronic acid reductase gene
The 2,5-DKG reductase can be improved by replacing certain amino acids of the enzyme to create mutants with increased catalytic activity and enhanced thermal stability. Changing the glutamine 192 to arginine creates more activity because it improves the substrate binding sites.
Also change the co-factor to NADH because it is cheaper and more abundant than NADPH that is used by the native protein by changing arginine 238 to histidine and lysine at 232 to glycine.
How can indigo be produced in E. coli?
Conversion of tryptophan in the growth medium to indole is through trptophanase produced by E.coli host cell. Indole is oxidized by naphthalene dioxygenase to cis-indole-2,3-dihydrodiol from the NAH7 plasmid. Then there is the spontaneous elimination of water and oxidation for form indigo.
Outline a strategy for increasing the production of the amino acid tryptophan by C. glutamicum
Library of Brevibacterium flavum chromosomal DNA is cloned into C. glutamicum-E.coli shuttle vector and introduced into a mutant strain of C. glutamicum producing no active anthranilic acid synthetase. Mutant strain cannot grow without anthranilic acid -> select transformants that grow without anthranili acid. Transfer vector with anthranilic acid synthetase gene is transferred to wild-type strain of C. glutamicum. This way increases the amount of anthranilate that can be used to convert into trptophan.
Suggest a strategy for isolating some of the genes that are involved in the biosynthesis of the antibiotic undecylprodigiosin, which is normally synthesized by S. coelicolor?
Chromosomal DNA from wild-type antibiotic producing cells is spliced into a streptomyces cloning vector. The clone bank is then transformed into a non-coloured (non antibiotic producing) mutant of the wild type. When plated, transformants that are red have functionally complemented the defective mutant and contain the plasmid with the gene to produce the antibiotic.
Why is it difficult to genetically transform various streptomyces spp? How can this difficulty be overcome?
Streptomyces strains do not exist as individual cells but form mycelial filaments. Thus the cell wall must be removed to release individual cells (protopplast) before transformation. Otherwise, it is impossible to distinguish transformed cells from nontransformed cells because colonies would have formed from cell aggregates and not individual cells. Uptake of DNA by streptomyces can be enhanced by polyethylene glycol.
FYI: after transformation, protoplasts are plated onto a solid medium to regenerate cell walls and then covered in selective medium to act as selecting agent.
Suggest an approach for producing modified versions of polyketide antibiotics such as erythromycin
Modify the eryhtromycin polyketide syntase by deleting the DNA region encoding the beta-ketoreductase activity or by mutating the DNA region encoding endoylreductase.
Deleting the beta-ketoredctuase changes the hydroxyl group to carbonyl at C5. Mutating enoylreductase changes single bond to double bond at C6-C7.
How can an adhesive protein biopolymer that is normally produced by the blue mussel M. edulis be synthesized in E. coli?
Construct a synthetic gene consisting of consensus decapeptide repeat of the adhesive protein for a total of 600bp. The 30bp repeat was customized for codon usage in E. coli with a T7 promoter. To overcome the problem of underhydroxylated protein, create an in vito hydroxylation system using tyrosinase in the presence of ascorbic acid. Th ascorbic acid prevents the premature oxidation of DOPA to o-quinone. A his tag can also be added to facilitate purification if necessary.
Suggest a scheme for producing poly(3-hydroxybutyric acid) in E. coli
Transfer genes for the biosynthesis of the compound and its polymer from A. eutrophus to E.coli. To maintain plasmid stability, insert the parB genetic locus into the construct. The gene postsegregationally kills plasmid-free cells. To ensure the production of the copolymer, mutate the E. coli cells at fadR and atoC loci which together increase the amount of propionate uptake neede for the formation of the copolymer.
What is whey? How can it be used to produce industrially important compounds?
Whey is a waste by-product from the cheese-making process consisting of mostly of water and lactose. Whey could be used as a carbon source for growing industrially important microorganisms.
How can the very large DNA fragments encoding antibiotic biosynthesis genes be introduced into host bacteria?
One strategy for isolating the complete set of
antibiotic biosynthesis genes consists of transforming one or more mutant strains that are unable to synthesize the antibiotic with DNA from a clone bank constructed from wild-type chromosomal DNA. Following the introduction
of the clone bank DNA into mutant cells, transformants are screened for the ability to produce the antibiotic. Then, the plasmid DNA from the clone that supplies a functional gene and gene product is used as a DNA hybridization probe to screen another clone bank of wild-type chromosomal DNA to isolate clones with regions that
overlap the probe sequence. In this way, DNA segments that are adjacent to and usually bigger than the initial complementing DNA can be identified and cloned. A complete gene cluster can be reconstructed from the
overlapping clones.
How can E. coli be engineered to produce lycopene?
Add the acetyl-CoA -> isopentenyl diphosphate pathway from S. cerevisiae and additional enzyme to convert isopentenyldiphosphate to dimethylallyl diphosphate to E. coli. Also add the pathway from P. agglomerans that convert Farnesyl dipghosphate -> Lycopene
How can the level of succinic acid produced by bacterium M. succiniproducens be increased?
Excretion of acetic, formic and lactic acids by bacteria that produce succinic acid reduces the yield of succinic acid and makes purification difficult. Disrupt the genes involved in the synthesis of acetic, formic and lactic acids from pyruvic acid. Each substrate gene was disrupted individually and mutants were tested for its ability to synthesize succinate. Thus making purification easier and increasing the yield of succinic acid.
Why is hyaluronic acid? How can it be produced in B. subtilis?
Hyaluronic acid is a glycosaminoglycan and a component of articular cartilage.
First the gene for typa A hyaluronic acid synthase from Streptococcus pyogenes by immunologically screening clone bank. The PCR is used to amplify the gene for type C hyaluronic acid synthase (hasA) from S. equisimilis based on gene from S. pyogenes. Then a synthetic operon with hasA gene and B. subtilis analogs of has B and has C under control of B. amuloliquifaciens amyQ promoter. Introduce synthetic operon on plasmid into B. subtilis. Integrate operon into genome.
