Chapters 1-4
Chapter 1
1. There are several drawbacks to the “mutation and selection” method, including the amount of time and
money it cost to select, screen, and test large numbers of microbial colonies. This method was also
limited biologically, where other important metabolic functions could be impaired by the increased
synthesis of a product. Also, only properties that were naturally occurring in the strain could be
amplified, nothing new could be introduced.
2. In 1973, Boyer and Cohen essentially established recombinant DNA technology. This was a major
step forward in the field of biotechnology. Instead of simply isolating desired strains, they could be
created. This cut down on the time required to implement genetic improvements and increased the
possible uses an organism could have. It was because of their work that a bacterial strain could be used to
produce insulin and other such proteins.
3. Karl Ereky was a Hungarian engineer who coined the term biotechnology to describe work where
products are derived from raw materials with the help of living organisms.
4. The first step in a bioengineered biotechnology process is the upstream processing of a raw material so
that it can be used as a food source. The second step is fermentation and transformation, where the
microorganism grows and produces the desired product. The final step is downstream processing to
purify the desired compound from the cells which produced it.
5. Biotechnology can be applied to any system where products come from living things. Ereky applied
the term to the raising of pigs using sugar beets as food. Molecular biotechnology, however, is more
specific, being the combination of biotechnology with recombinant DNA technology. Here, genetically
modified organisms can be created to produce proteins and other molecules that are desired.
6. One major concern about molecular biotechnology is the possibility that the use of genetically
engineered organisms will reduce the natural genetic diversity. In the past, evolution has progressed by
natural selection. Depending on the extent of genetic engineering, it could conceivably become human
selection. And what man may consider to be good, may not in fact, be in the best interest of the planet.
Some other concerns include the possibility of genetically engineering humans, privacy, and the chance
that traditional farming methods could be undermined.
7. Molecular biotechnology is a diverse science no matter how one looks at it. While considered a
specific field, it’s the merging of seven different scientific disciplines: molecular biology, microbiology,
biochemistry, immunology, genetics, chemical engineering, and cell biology. To be successful, one
would need to know a little about each of these separate fields. The products created by molecular
biotechnology are also very diverse, yet with the common thread of recombinant DNA technology.
Plants and animals can be modified, medicinal compounds can be created, and diagnostic techniques can
be developed. Many ideas go into the field and many different areas receive the benefits.
8. There are many potential benefits, including the diagnosis and prevention/curing of genetic diseases,
increased crop yields with the creation of hardier plants, microorganisms that produce such things as
drugs, polymers and enzymes, and the removal of pollutants from the environment.
9. 1960s
The genetic code was deciphered
Mutagenesis and selection as the means by which biotechnology was undertaken
1970s
The first restriction endonuclease was isolated
Recombinant DNA technology was established
Chapters 1-4
- research guidelines
- techniques to determine DNA sequence
Genetech produces human insulin in E. coli
1980s
Biotechnology goes commercial with various products available on the market for
research use and the start of patent applications
Biotechnology expanded to include plants and animals, not just microorganisms
1990s
An increased focus on humans with the Human Genome project, genetic maps of our
chromosomes, and human somatic cell gene therapy trials.
The FDA began approving various drugs and proteins.
An interest in nuclear cloning is aided by the successful cloning of a sheep
2000s
Several complete genomes are sequenced, including humans and Arabidopsis
Investments in biotechnology have risen to $33 billion
10.
Startup Chimerix gets federal funds to develop smallpox drug. New biotechnology
companies are still a viable business venture today. While a new company with only 9 employees
currently, Chimerix has been awarded $36.1 million dollars to develop a pill for the treatment of
smallpox. The compound being developed is called CMX-001. No details about it are released, since this
is a field where people are out to make money and have exclusive rights. It is quite likely that even once
Chimerix has patented the compound, they won’t release details.
EU Court Upholds Italy's Right To Ban Monsanto Corn. While Italy was allowed to continue
banning genetically modified corn, they will have to show that there are indeed suspected risks to health
and/or the environment in order for it to be sustained much further. This goes back to one of the major
concerns about molecular biotechnology: the introduction of modified plants into nature. Italy should be
allowed to refuse to import a company’s product if they want to.
Bugpower, the energy of the future. Using R. ferriducens, a bacterium that breaks sugar down
into carbon dioxide and electrons, researchers at the University of Massachusetts were able to construct a
battery prototype that ran for 25 days. The device doesn’t produce much current, but enough to operate a
calculator. The early stages of research are often promising, and while not much may be accomplished at
the present it gives an idea for the future.
Chapter 2.
1. While many organisms have some similarities, there are distinct differences between them. Therefore,
the biological system chosen to work with depends on the nature, yield, and quality of the desired
product. In other cases, such as corn, the genetically modified organism is the desired product.
2. A prokaryote is a cell that lacks a nucleus and other membrane-bound organelles. In addition, its cell
wall may contain peptidoglycan.
3. A eukaryote is a cell where the chromosomes are enclosed in a nucleus and there are membrane-bound
organelles in the cytoplasm. Also, the cell wall may contain chitin or cellulose.
4. E. coli is a gram-negative, nonpathogenic, rod shaped bacterium. Due to its quick and simple
reproduction, its one of the most studied organisms in the world.
5. Gram negative means that there is both an inner membrane and an outer membrane, with a periplasmic
space between them.
Chapters 1-4
6. S. cerevisiae is a nonpathogenic, single celled yeast. Since it is a eukaryote, it has been studied as a
model system where E. coli doesn’t apply. It has also been used to produce alcoholic drinks and bread for
centuries.
7. A simple liquid culture medium contains the ions Na+, K+, MG2+, Ca2+, NH4+, Cl-, HPO42-, and SO42-;
trace elements Fe2+, Co2+, Zn2+, Cu2+, and Mo2+; and a carbon source like glucose.
8. A signal recognition protein binds to the signal peptide of a secretory protein. This then binds to the
membrane bound Sec complex. In another pathway, the signal peptide itself binds to the Sec complex.
The Sec complex contains a channel through which the protein travels. Once outside the membrane, the
signal peptide is removed by signal peptidase(s).
9. A primary cell culture is a population of growing cells started directly from tissue of an organism.
10. An established cell line is a population of cells grown in vitro that can be subcultured indefinitely.
These cells have undergone genetic changes and may or may not retain the biochemical characteristics of
the original cells.
Chapter 3
1. Starting at an origin of replication, small segments of DNA are unwound (where DNA is double
stranded) and replicated, with each strand serving as a template to form the daughter strands. DNA
polymerase both binds nucleotides based on the template strand and creates the phosphodiester linkages
that join the 3’ OH group of one nucleotide to the 5’ phosphate of the next one. (YOU SHOULD ALSO
DISCUSS LEADING AND LAGGING STRANDS IN THIS ANSWER)
2. Both DNA and RNA are strands composed of nucleotides that can associate through hydrogen bonds
between complementary bases. They have the same 3’ to 5’ orientation. One main difference is that the
two molecules contain different sugars. Ribose in RNA has a hydroxyl group on 2’ carbon, while
deoxyribose does not. The other difference is in the bases used. Uracil is found in RNA instead of
thymine. DNA IS GENERALLY DOUBLE STRANDED, WHILE RNA IS USUALLY SINGLE
STRANDED)
3. In prokaryotes, a gene is encoded by a continuous strand of DNA. In eukaryotes, however, the DNA
strand contains both exons and introns, the latter of which is cut out from the final mRNA product. In
both cases, the binding of RNA polymerase to a promoter region initiates transcription.
POLYCISTRONIC VS MONOCISTRONIC
4. Once the initiation complex forms with the start codon, elongation starts. The next three nucleotides
in the mRNA pair with the anticodon of a tRNA. This transfer RNA carries a specific amino acid and
recognizes certain mRNA sequences. The first amino acid is cleaved from it tRNA and a peptide bond is
formed between the two amino acids. The mRNA shift sites, exposing the next codon so that it can pair
with the appropriate tRNA. (YES, MAKE USE OF THE HUMAN CODON BIAS IN TABLE 3.2)
5. The most likely coding sequence is 5’-AUG GCC GGC GGC ACC UGG UAC CAG CUG UUC
CCC AGG AAG AUG UGG AAC GAC AGC ACC CUG CAC CCC UUC AUC CUG CCC AUG AAC
GUG GCC GGC UGA-3’
6. The amino acid sequence would be Met Phe Leu Lys Val Ser His Arg Asn Glu Gly Ser
(LOOK FOR START AND STOP CODONS)
Chapters 1-4
7. A TATA box is a region of DNA rich in A and T that lies upstream from the site of initiation of
transcription. It is where RNA polymerase binds and ensures that transcription begins at a certain
nucleotide. (REMEMBER, IN EUKARYOTES RNA POLYMERASE BINDS INDIRECTLY TO THE
PROMOTER)
8. An operon is a series of genes that are continuous on the DNA which are under the control of a single
promoter. These genes are all part of the same metabolic pathway, and this is an efficient way to regulate
the expression of genes. Some gene products are only needed sometimes, and so activators and repressors
are used to turn these genes “on” and “off”. The use of a single promoter ensures that the entire pathway
is shut down by the presence of a single repressor, saving energy.
9. One mode of regulation involves the binding of a repressor protein to an operator region. This blocks
transcription unless another molecule binds to the repressor and changes its conformation so that it can no
longer bind to the DNA. In another mode, the repressor is normally inactive, unless an effector binds to it
and changes it so that it can bind to the DNA and therefore block transcription. The third mode involves
an activator, which binds to the DNA between the TATA box and the initiation site. The activator
increases the affinity for RNA polymerase, increasing transcription.
10. In eukaryotes, there are two regions, the CAAT and the TATA box which act as promoter regions.
Transcription factors will bind to these. There may also be enhancer regions of DNA further upstream
from the promoters. Activators, which are proteins with two domains, will bind to these. A co-activator
is another protein needed to bring these two regions into contact. RNA polymerase II then binds to this
large complex to start transcription.
Chapter 4
1. Type II restriction endonucleases are enzymes that cut nucleic acid molecules internally at specific
base pair sequences. These are important in recombinant DNA technology since the digestion of vectors
with these enzymes allows for the specific combination of two DNAs from completely different sources.
2.
Chapters 1-4
3. pBR322 is digested with either PstI or BamHI restriction endonucleases. The solution is then treated
with alkaline phosphatase to keep the plasmid from resealing itself. This is then combined with the target
DNA which has the same sticky ends and T4 DNA ligase. The vector DNA will then be inserted into the
plasmid. The restriction sites lie within genes that give resistance to ampicillin and tetracycline, enabling
one to find the bacteria into which the vector has been transformed by drug resistance.
4. The pUC cloning system is another plasmid. Like many, it has a gene for ampicillin resistance so that
the bacteria containing the plasmid may be easily found. The key features of this plasmid are its LacZ’
gene and its multiple cloning sequence. The LacZ’ gene enables the bacteria to break down sugars
containing galactose. When plated on X-gal, the resultant colonies turn blue. The multiple cloning
sequence is a segment of DNA containing a bunch of different restriction sites. If any of these are
cleaved and vectors inserted, then the LacZ’ gene won’t function and the desired colonies will be white.
5. a) It’s possible that after digestion with one restriction endonuclease some of the DNA fragments will
still be too large to clone, resulting in an incomplete library. To fix this, another library is made with a
second enzyme that cuts at a different site, resulting in different sizes of fragments. (Sau 3A is a 4bp
cutter and can cut more randomly in a partial digest compared to BamHI)
b) A partial digestion is when the DNA is treated with the restriction endonuclease under conditions
(time or amount of enzyme) that result in fragments that haven’t been cut at every restriction site. This
helps to ensure that all possible lengths will be created.
c) A genomic library is a collection of all the genomic DNA of an organism. This DNA has all been
turned into clonable entities. In order for a segment of DNA to be clonable, it can’t be too big and so it
must be digested. There are also a variety of different genes/segments that might be of interest, so partial
digestion ensures that any possible segment has been prepared.
6. If cleaved plasmid DNA is left on its own, it would simply join back together before the introduction
of the vector. Therefore, cloning efficiency would be very low. Alkaline phosphatase removes the 5’
phosphate groups from the newly created sticky ends. Since both the 3’ and 5’ ends of the plasmid’s
strands have hydroxyl groups, they can join back together.
7. In order for the plasmids to be introduced into E. coli, the outer membrane must be permeated. This
can be accomplished using either cold CaCl2 followed by heat shock, or by applying a current to the
solution containing the cells and plasmids. Conjugation can also allow bacteria to naturally transfer
plasmid DNA to another bacteria cell.
8. There are several ways to screen for a gene once it’s in an E. coli library. The first method is DNA
hybridization. Here, a specific DNA probe forms base pairs with the targeted DNA sequence which has
been denatured and bound to a matrix. The second method is by immunological assay. The clones are
attached to a matrix along with their protein products. Primary and secondary antibodies are washed over
the matrix. The primary antibody will bind to the appropriate protein, and the secondary antibody will
bind to the first antibody to form a colored product. The third method screens by protein activity which is
used if the target genes produces an enzyme not normally found in the host cell. There are a variety of
ways to do this, but they all involve a direct plate assay. One example is to plate the cells onto a medium
that contains a specific substrate. Those that contain the gene will hydrolyze the substrate to form a
colored product or a precipitate. The fourth method is functional (genetic) complementation. Host cells
with a particular genetic defect are transformed with the plasmids containing normal DNA. The cells
which contain the target gene will then function normally and will grow on a minimal medium.
9. A cDNA library is a collection of cDNA clones that were generated in vitro from the mRNA
sequences of a single tissue or cell population using reverse transcriptase and Klenow fragments.
Chapters 1-4
10. One would use a plasmid if one only wanted to transform a small sized DNA, no more than 10 kb.
Bacteriophage λ would be used to contain a 15 to 20 kb insert of DNA. Cosmids would be used as
cloning vectors for DNA segments that are 45 kb long. Fewer clones of a genetic library would need to
be screened to locate a single gene and its easier to clone a large gene than in a plasmid. BACs would be
used in the analysis of complex eukaryotic genomes since it can carry inserts greater than 100 kb.