Genetic Engineering and Biotechnology

Midterm I (Semester I 2020)

Name: Evelyn Gabriela González Simbaña

Group: ____________
Date: 30-03-2020 Professor: Nelson Santiago Vispo

The exam are prepared for 90 minutes

Books and notes will not be allowed in the exam room.
The exam will focus on all topics covered in lecture and any previous material.
Please, use only blue or black ink to answer the exam. Any response in pencil will not be
revised.
Read the instructions carefully and budget your time.

ANSWER FORM

Student's signature:__________________________

QUIZ

1. DNA cloning vectors can be developed from which of the following?

a) Plasmids

b) bacteriophage

c) bacterial chromosomes
d)

e) yeast chromosomes

f) any of the above

2. Explain the main characteristics to take into account to select a host system

Selecting a host system for expressing protein there are many factors to consider. These
include:

 Post translational modifications

- Phosphorylation
- Methylation
- Acetylation
- Ubiquitination
- Nitrosylation
- Glycosylation
- Llipidation
 Equipment availability
  Time constraints
- One week or less (bacterial, transient mammalian)
- One month or less (in addition to bacterial and transient mammalian, yeast &
insect systems can be used)
- More than one month (in addition to the systems above, you can use stable
mammalian systems)  

3. What are the essential components of a DNA extraction Procedure?

 Maximize DNA recovery

 Remove inhibitors

 Remove or inhibit nucleases

 Maximize the quality of DNA

4. What can we do with a linker or DNA adaptor?

Linker or DNA adaptor is used for various cloning strategies to introduce restriction sites in
the DNA after ligation. Linkers or DNA adaptors are simple or double-stranded synthetic
DNA fragments that allow us to complete sequences that were removed by restriction
enzymes. DNA ligation is one of the most crucial steps in the development of the DNA
vector with the gene of interest. For ligating the gene of interest to the vector, we
use DNA ligase and T4 ligase. However, in some conditions, it is possible that the gene of
interest may have blunt ends instead of sticky ends after the treatment with the
restriction endonucleases. The ligation of the blunt end fragment of the gene with the
vector having sticky ends is difficult. In such condition, we use the linkers and adapters to
generate the sticky ends.

5. What does the detergent do in DNA purification?

• The detergent breaks apart membranes by attaching to the lipids (fats) & proteins in the
membranes and nuclei, they allow the cell membrane to dissolve, as well as inhibitors to
inactivate enzymes that break down DNA.

6. Describe the DNA manipulative Enzymes. The four groups

The range of DNA manipulative enzymes DNA manipulative enzymes can be grouped into
four broad classes, depending on the type of reaction that they catalyze:

 Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules.
 Ligases join nucleic acid molecules together
 Polymerases make copies of molecules
 Modifying enzymes remove or add chemical groups
 7. Give practical examples of
uses of the phosphorylation
and dephosphorylating
reaction

 Phosphorylation and
dephosphorylation of
protein tyrosine residues
, catalyzed by protein
tyrosine kinases (PTKs)
and protein tyrosine
phosphatases (PTPs),
respectively, are crucial
events in many cell
signaling pathways, including insulin signaling.

 Mechanism of action of phosphorylation, with particular attention to the

importance of phosphorylation under physiological and pathological conditions,
phosphorylation on serine/threonine-protein kinase (Ser350) residue of the death-
associated protein (DAP) leads to the translocation from the cytoplasm to the
nucleus of apoptosis-inducing kinase 2 (DRAK2) which is able to induce apoptosis
in T and B cells.

The mechanism of phosphorylation regulation consists of kinases, phosphatases and their

substrates phospho-binding proteins. For example, phosphorylation is activated by stimuli
such as epigenetic modifications, cytogenetic alterations, genetic mutations or the tumor
micro-environment. Consequently, the protein receives a phosphate group by adenosine
triphosphate (ATP) hydrolysis and due to enzymatic activity of kinase. This is the
mechanism for the basis of post-translational modification (PTM) formation. In addition,
phosphorylation is a reversible process due to activity of phosphatase. Phosphorylation
and dephosphorylation are a molecular switch and, in particular, a PTM can cause
oncogenic pathway activation by a phospho-binding protein that bind to the phosphate
group of a phosphoprotein.

8. In a DNA ligation reaction and transformation in a host cells define the

components, including de controls +/--

Control 1. Any colonies formed from transformation of this control will be the result of undigested
or re-ligated vector, and subtracting the number of colonies formed from this control from the
number of colonies formed from the ligation will give an idea of whether the ligation reaction has
been successful. The ideal scenario is that there are few or no colonies on the control plate and
tens, hundreds, or thousands of colonies on the ligation plate. If your ligations are going well,
control 1 is sufficient. However, if you are experiencing problems, the controls described below
will help you pinpoint the source. 

Control 2 . Colonies formed from transformation of control 2 can only be the result of undigested
vector, not vector re-ligation, since there is no ligase present. If you are obtaining a high number
of background colonies, comparing controls 1 and 2 will allow you to determine whether the
problem is due to undigested vector or vector re-ligation.

Control 3 uses a known amount (about 0.1 ng works well) of the undigested parent vector. If
transformation of E.coli with the undigested parent vector leads to no colonies on the plate, the
problem is either in the transformation procedure, the competent cells, or antibiotic selection.
Thus, the next step would be to try new cells, or review your transformation procedures.

On the other hand, if the transformation is a success, you can calculate the competency of the
cells by counting the number of colonies obtained from the transformation. Realistically, the
minimum competency of cells for cloning should be 1×10^6 colony forming units (CFU) per
microgram of DNA. If your cells are below this, this is likely to be the problem and you should re-
do the transformation with a fresh batch of competent cells.

For control 4, you need to prepare ahead. Take a reasonable amount (1 microgram) of any
vector, digest with a single enzyme, then run on a gel, and isolate the band. This can now be used
to test whether your ligase is working properly. Run a ligation reaction with this vector plus and
minus ligase, then transform your competent cells. Compare the number of colonies on the two
plates. If the ligase-containing reaction has significantly more colonies (at least 5-10x) than the
ligase minus plate, then the ligase is likely working. If not, the ligase may be a problem

9. Why is glycosylation important in biotechnology?

Glycosylation is a post-translational modification that is of paramount importance in the

production of recombinant pharmaceuticals as most recombinantly produced therapeutics
are N- and/or O-glycosylated. Being a cell-system-dependent process, it also varies with
expression systems and growth conditions, which result in glycan microheterogeneity and
microheterogeneity. Glycans have an effect on drug stability, serum half-life, and
immunogenicity; it is therefore important to analyze and optimize the glycan decoration of
pharmaceuticals.
• For example: glycosylation in biotech industry
- Glycosylation patterns can affect product quality
- Too much variation in glycosylation leads to discarding of product
- Regulatory agencies (eg FDA, Health Canada) have regulations for amount of
acceptable variability in glycosylation –deviations can lead to redoing clinical
trials
- Change in glycosylation can lead to another company claiming a new patent
- Adverse reactions in patients to non-human glycosylation

10. What are the basic requirements for a Successful Cell Culture?

1. The first necessity is a well-established and properly equipped cell culture facility. All
facilities should be equipped with the following:
• A certified biological safety cabinet
• protects both the cells in culture and the worker from biological
contaminants
 • A centrifuge, preferably capable of refrigeration
• A microscope for examination of cell cultures and for counting cells
• And a humidified incubator set at 37°C with 5% CO 2 in air
• A 37°C water bath filled with water containing inhibitors of bacterial and fungal
growth can also be useful if warming of media prior to use is desired

2. The second requirement for successful cell culture is the practice of sterile technique
• Prior to beginning any work, the biological safety cabinet should be turned on and
allowed to run for at least 15 min to purge the contaminated air
• All work surfaces within the cabinet should be decontaminated with an appropriate
solution;
• 70% ethanol or isopropanol are routinely used for this purpose
• Any materials required for the procedure should be similarly decontaminated and
placed in or near the cabinet
• This is especially important if solutions have been warmed in a water bath prior to
use
• The worker should put on appropriate personnel protective equipment for the cell
type in question
• Gloved hands should be sprayed with decontaminant prior to putting them into the
cabinet and gloves should be changed regularly if something outside the cabinet is
touched
• Care should be taken to ensure that anything coming in contact with the cells of
interest, or the reagents needed to culture and passage them, is sterile (either
autoclaved or filter-sterilized)

3. A third necessity for successful cell culture is appropriate, quality-controlled reagents and
supplies
• There are numerous suppliers of tissue culture media and supplements
• Examples include:
• Invitrogen (www.invitrogen.com),
• Sigma–Aldrich (www.sigmaaldrich.com),
• BioWhittaker (www.cambrex.com),
• and StemCell Technologies Inc. (www.stemcell.com).
• Similarly, there are numerous suppliers of the plasticware needed for most cell
culture applications (i.e., culture dishes and/or flasks, tubes, disposable pipets)

4. The final necessity for successful cell culture is the knowledge and practice of the
fundamental techniques involved in the growth of the cell type of interest
• The majority of cell culture carried out by investigators involves the use of various
non-adherent or adherent continuously growing cell lines
• These cell lines can be obtained from reputable suppliers such as:
• the American Tissue Type Collection (ATCC; www.atcc.org)
• or DSMZ (the German Collection of Microorganisms and Cell Cultures)
• Alternatively, they can be obtained from collaborators
• Regardless of the source of the cells, it is advisable to verify the identity of the cell
line and to ensure that it is free of mycoplasma contamination