Biochemistry Assignment: DNA Technology, Fermentation, and GM Foods

This biochemistry assignment delves into various aspects of recombinant DNA technology, fermentation, and related topics. It addresses the role of ribosomes, enzymes like Hind III and T4 DNA ligase, and techniques for separating and visualizing DNA fragments. The assignment explores PCR technology, outlining its steps, components, and applications in forensic science and clinical diagnosis. It further discusses fermentation processes, focusing on metabolites produced by lactic acid bacteria and their use in food production. The electronic nose, buffers in enzymatic reactions, and advantages of insect-resistant GM crops are also examined. Cloning and expressing a plant gene in E. coli, along with blotting techniques like Southern, Northern, and Western blotting, are described. Finally, the principles of ion exchange, gel filtration, and affinity chromatography are explained, along with their use in purifying fermentation products, and the role of antibiotics and X-Gal in bacterial transformation. The document is contributed by a student and available on Desklib, a platform providing study tools for students.

Running head: BIOCHEMISTRY 1
Biochemistry
Student’s Name
Institutional Affiliation

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PART A
1. Ribosomes require Aminoacyl-transfer RNA to read and interpret the genetic code.
2. Consider the following, in which two DNA fragments are treated with T4 DNA ligase: -
the first is a plasmid digested with restriction enzymes BamHI (sticky ends) and HpaI
(blunt ends) and not treated with phosphatase; and- the second is a BamHI DNA fragment
treated with phosphatase. Identify the possible outcome(s) of the ligation reaction:
Answer: a circular plasmid containing the DNA fragment
3. An intron is a short mutation in a non-coding region.
4. Which of these fermenter types does really exist? Shaken fluid-bed fermenter.
5. In genomic studies, gene identification allows us to understand proteome
composition.
6. Identify actual membrane transporter proteins on the list below. Antiporters, Symporters
along with ATP-coupled active transporters.
7. Alpha-complementation is a common method to verify the successful insertion of cloned
DNA fragments into a plasmid.
PART B
1. What are the following tools used for in recombinant DNA technology:
1. a) Hind III (50 WORDS)
It is a type II site-specific deoxyribonuclease restriction enzyme separated from
Haemophilus influenza. The tool is used to cleave the DNA palindromic sequence AAGCTT in
the cofactor Mg 2+ presence through hydrolysis (Tayi, Maku, Patel & Sonti, 2016). Hind III is
used for genetic engineering and molecular biology.
1. b) T4 DNA Ligase (50 WORDS)
It is a ligation enzyme utilized to join fragments of DNA through catalyzing the
formation of phosphodiester bonds amidst 3’hydroxyl termini and juxtaposed 5’phosphate in

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double-stranded DNA by use of ATP as a coenzyme (Matsuzaki, Su, Walsh, Kennedy & Mei,
2018). Moreover, it seals nicks for the DNA substrates.
2. c) Calf intestine phosphatase? (50 WORDS)
Calf Intestine Phosphatase is utilized to catalyze the hydrolysis of 5’phosphate groups
from RNA, DNA and both ribo and deoxyribonucleoside triphosphates (Toro ET AL., 2018).
The removal of 5’phosphate is effective in avoiding self-ligation of the cleaved DNA vectors.
Furthermore, it dephosphorylates the DNA before kinase labeling reactions.
2. Answer the following with regard to DNA technology:
a) How and why you can separate and visualize DNA fragments by size? (50 WORDS)
DNA fragments are separated by size by a current run through a gel with molecules.
Depending on DNA size, the molecules move via the gel in opposed directions enabling them to
be segregated from each other. To visualize, the gel is discolored with a DNA binding dye and
put under UV light for the DNA fragments to glow.
b) Explain the principle behind the separation technique. (50 WORDS)
The principle behind the technique is to analyze DNA fragments produced by restriction
enzymes. It uses electromotive force to transfer molecules via a porous gel and separates them on
the basis of shape, charge, and size. However, the separation basis is grounded upon how the gel
and sample are prepared.
3. In recombinant DNA technology
a) What is a screenable marker gene used for? (50 WORDS)
A marker gene is utilized in molecular biology in ascertaining if a piece of DNA has been
effectively inserted into the host organism. A screenable marker gene is used to make the cells
carrying the gene be viewed differently (Bashir, Kim, Stahl & Cho, 2016).
b) Name two screenable marker genes. (50 WORDS)

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The green fluorescent protein (GFP) makes the cells glow green under UV light. Here, an
exclusive microscope is needed to view each cell (Sachsenhauser & Bardwell, 2018). On the
other hand, a GUS assay screenable marker gene detects a single cell by discoloring it blue
without using any complex apparatus.
4. With regard to PCR technology:
a) Outline the steps and main components of a PCR reaction; and (50 WORDS)
Steps in Polymerase Chain Reaction involve denaturing, annealing along with extending.
On the other hand, the basic elements of Polymerase Chain Reaction entail DNA template,
primer, DNA polymerase, thermal cycler, microfuge tube, Tris-HCl, MgCl2, Gelatin or bovine
serum, distilled water and deoxynucleotide triphosphates (Sochivko, Fedorov, Alekseev,
Kurochkin & Dubina, 2017).
b) Provide two examples of PCR applications. (50 WORDS)
PCR is used in forensic science for the recognition of lawbreakers along with the
gathering of organic crime scene proof like soil, semen, and blood. Moreover, PCR is applied in
clinical diagnosis to research into the diagnosis and help in treating various diseases. (Kim et al.,
2016).
5. Consider the fermentation process:
a) Name the main metabolites produced by lactic acid bacteria (LAB)(50 WORDS)
Lactic acid bacteria generate organic acids like propionic, acetic and lactic acids
generated as end products. Moreover, it produces antimicrobial metabolites like H2 O2 generated
during aerobic growth, bacteriocins which are ribosomally synthesized antimicrobial
compounds, ethanol produced through the heterofermentative pathway along with diacetyl
generated from excess citrate-derived pyruvate (Soro-Yao, Brou, Amani, Thonart & Djè, 2014).
b) Name five food products produced using LAB. (50 WORDS)

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Food products produced using LAB include; fermented vegetables like pickles and
sauerkraut, fermented cereals like sourdough, fermented fish products along with fermented meat
such as sausage. Furthermore, dairy products such as yogurt, kefir, butter and milk, cheeses with
small eyes, fermented probiotic milk along with Swiss-and Italian-type cheeses are produced
using LAB (Rattanachaikunsopon & Phumkhachorn, 2010).
6. What is an electronic nose and what it is it used for? (100 WORDS)
An electronic nose is equipment which recognizes the particular elements of smell and
evaluates its chemical composition to pinpoint it (Deshmukh, Bandyopadhyay, Bhattacharyya,
Pandey & Jana, 2015). It entails a technique for chemical detection like an array of electronic
sensors along with an approach of pattern identification like a neural network. Initially, the
electronic nose was utilized in the application of quality control in cosmetics, beverages and food
industries. Presently, it is utilized for recognition of pollutants along with gas leaks for the
purpose of protecting the environment along with detection of smell particular to disorders for
clinical diagnosis (Deshmukh et al., 2015).
7. What is a buffer and what is its role in enzymatic reactions? (100 WORDS)
A buffer is a solution which can resist pH change upon the inclusion of basic or acidic
elements. It contains a weak acid (proton donor, HA) along with its conjugate base (proton
acceptor, A-) and it is capable of neutralizing small amounts of added base or acid (Salvatore,
Filippelli, Salvatore & Andolfi, 2017). Buffer solutions are the most essential types of chemical
reagents utilized in industries, biological research along with chemical research. Their role stems
from their capability to resist changes in pH. It controls the concentration of hydrogen ions at a
steady value in a broad variety of chemical applications
8. Name some advantages of insect-resistant GM crops over conventional pest
control? (100 WORDS)

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They result in enhanced insect management where insect-protected GM plants give farmers
long protection from many destructive insects and eliminate or minimize the demand for
spraying of insecticide. Insect-resistant GM crops result in greater net return as there are
decreased pesticide costs but increased yields. Additionally, they lead to enhanced situations for
non-target organisms. Since the crops are able to protect themselves from insects, the chemical
insecticide utilization will substantially decrease hence enabling the proliferation of good
organisms. The good organisms then assist in controlling other insects which may be regularly
problematic when predators are reduced by conventional pest control.
9. Described the main steps involved in cloning and expressing a plant gene in E. coli. (100
WORDS)
The basic steps in coning include; cutting up the plasmid from the source utilizing
restriction enzymes and pasting in the gene of which the procedure depends on restriction
enzymes (that cut DNA) and DNA ligase (that joins DNA) (Brown, 2016). The next step is
inserting the plasmid into bacteria and using antibiotic selection to recognize the bacteria which
took up the plasmid. The last step is growing up lots of plasmids containing bacteria and utilizing
them as ‘industries’ to produce the protein. Here, the protein is isolated from the bacteria and
then purified (Brown, 2016).
10. Succinctly, describe what is blotted onto a membrane and what is used as a probe when
carrying out a (a) Southern, (b) Northern, or (c) Western Blot analysis. What are the
respective main uses of these techniques? (50 WORDS FOR EACH) = 200 WORDS
In Southern blotting analysis, a nylon membrane is utilized which normally has a binding
capacity of 500 μg/cm and it is mostly preferred since it binds more and is less fragile. What is
frequently used as a probe is 32P labeled ATP, bioluminescent or biotin/ streptavidin probe. In
northern blotting, it is similar to the southern blotting as a nylon membrane is utilized which is

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contributed to the factor that in nature, nucleic acids are negatively charged. A complementary ss
DNA sequence is the probe used and formamide is utilized as a blotting buffer since it minimizes
the annealing temperature.
In western blotting, a molecule which is a protein is blotted onto the membrane and the
probe which is normally an antibody raised against that specific protein allows the recognition of
the molecule of interest in a mixture of several other similar molecules. The Southern blotting
technique is used in detecting a sequence of DNA in a DNA sample. Northern blotting is utilized
in revealing the identity, size, number, and activity of a specific gene. On the other side, western
blotting is utilized in identifying particular proteins from a complex mixture of proteins obtained
from cells.
11. Briefly describe the principles of ion exchange, gel filtration and affinity
chromatography and how these are used to purify products derived from fermentation
processes. (100 WORDS)
In ion exchange chromatography, proteins are separated according to differences in their
charge (Coskun, 2016). It purifies by use of charged stationary phase to separate charged
compounds. In gel-filtration chromatography, proteins are separated according to their molecular
weight. The sponge-like gel beads separate the smaller molecules from the larger molecules. The
small molecules enter the gel beads and then stuck while the large molecules come out with the
mobile liquid. In affinity chromatography, proteins are separated according to differences in their
binding affinity (Coskun, 2016). It is used by binding the desired biological material reversibly
and specifically to a ligand that has been fixed to an inert carrier.
12. What are the essential components of a binary vector system and why is this a
preferred system for plant transformation? (100 WORDS)
The vital components of binary vectors include T-DNA along with vector bone. Binary
vector system is preferred for plant transformation as they introduce genes into plants using

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Agrobacterium-mediated genetic transformation. This Agrobacterium is a soil bacterium which
functions as a genetic engineer and it may attack crops via the roots or stem wounds. Therefore,
this enables the bacterium to proliferate until the plant expires.
13. Explain the role of antibiotics and X-Gal when plating bacteria transformed with
recombinant plasmids. (100 WORDS)
Cells are plated on LB agar with proper antibiotics for the purpose of identification along
with the recovery of successful transformants. X-gal is frequently used to test for the presence of
an enzyme called β-galactosidase. Furthermore, the role of the X-gal is to allow the distinction
between bacterial colonies produced from cells which have plasmid with insert from those which
have plasmid without an insert. Any colony which has the plasmid turns blue which is a result of
the β -galactosidase activity.
14. How was the cheese-coagulating enzyme produced in the past? How this enzyme is
currently produced? What are the old and the new enzymes called? (100 WORDS)
In the past, the milk-clotting enzyme was obtained from cleaned and air-dried stomachs.
In the enzyme, there contains chickpeas, white beans, figs, carob, salt, granulated sugar, yogurt,
raisins, and home-made rennet. The mixture was then left for five to six days at room
temperature. What followed was that the enzyme was filtered by use of cloth bag and then added
into milk for coagulation. Currently, lactic-acid bacteria are included in milk and the mixture is
then stirred at a constant temperature. Rennet is then added which hydrolyzes the kappa-casein in
the milk making the casein micelles to coagulate generating a gel.
15. Describe briefly what an enzyme is and how its amino acid composition determines its
structure and function. (100 WORDS)
An enzyme is a substance generated by a living organism that serves as a catalyst to
contribute to a certain biochemical reaction. The sequence of amino acids determines the
enzyme's structure and function in that the forces which attract the substrate to the surface of an

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enzyme may be of chemical or physical nature (Datta et al., 2017). Electrostatic bonds happen
amidst oppositely charged groups where the circles containing minus and plus signs on the
enzyme are attracted to their opposites in the substrate molecule. Moreover, the attractive forces
may involve hydrophobic bonds in which portions of the enzyme and the substrate are forced
together.
16. In the list below, name at least one industrially used enzyme per area of application
3. a) Pulp and paper industry: (10-15 WORDS)
Enzymes like laccases, xylanases along with rarely mannases are employed in the pulp
and paper industry.
4. b) Detergent production: (10-15 WORDS)
Protease and lipase are the used enzymes mostly contained in biological laundry
detergents.
5. c) Feed industry: (10-15 WORDS)
Glucoamylase, protease, α-Amylase, lipase, lactase along with phospholipase are
enzymes used in feed production.
6. d) Food and beverages: (10-15 WORDS)
Xylanases, peptidase, decarboxylase, Pectinases, and tannases are the enzymes employed
in food and beverages production.
17. Name and describe five points of control of gene expression located anywhere between
transcription and the final protein product. (100 WORDS)
The key control point is transcription which is the process where the DNA sequence of a
gene is copied into an RNA molecule. In the initiation point, the molecule of DNA disentangles
and then isolates to create a small open complex. Consequently, in elongation control point, the
RNA polymerase moves along the template strand integrating an mRNA molecule (Saldi,
Cortazar, Sheridan & Bentley, 2016). Termination is the fourth point where a protein called Rho
is accountable for interrupting the complex containing the RNA molecule, RNA polymerase, and

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the template strand. Finally, in processing point, introns are removed and the exons spliced
together to generate a mature mRNA molecule containing a single coding sequence.
18. Name five applications of genetically engineered crop plants and describe briefly the
type of genetic modification involved. (100 WORDS)
Genetically engineered crop plants applications include pollen control, delayed fruit
ripening, altered oil content, virus resistance, and herbicide resistance along with insect
resistance. The crossing is the type involved in pollen control since it happens when a plant
breeder takes pollen from a plant and brushes it onto the pistil of a sexually consistent plant.
Mutation breeding is involved in delayed fruit ripening where the fruit is exposed to mutagenic
of chemical agents. However, cell selection is involved in herbicide resistance, virus resistance
along with insect resistance (Ladics et al., 2015). Here, a cell is chosen and regenerated in an
entire plant and then tested to stabilize the phenotypic trait. The interspecies crossing is involved
in altered oil content as closely related cultivated oat and its weedy relative wild oat might cross-
pollinate for exchange of genetic data.
19. Describe the four basic steps involved in risk assessment and risk management
(applicable to novel foods, chemical, and biochemical compounds). (100 WORDS)
The first step is hazard identification. Risk assessment along with risk management is
done to identify what might be harmful or the biological hazards present in food compounds
(Gerba, 2019). The second step is hazard characterization where scientist study the effects such
hazards cause to calculate a safe level of exposure for customers. Exposure assessment is the
third step where scientists analyze the hazard present in the food and the amount of that food
consumed by persons of different ages. Lastly is the step of risk characterization where risk
evaluators come into conclusions on the degree of the risk (Gerba, 2019).
20. Describe briefly the main criteria used by FSANZ to approve GM crops (100 WORDS)

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GM foods along with ingredients are subjected to a compulsory pre-market safety
evaluation by FSANZ before they are allowed in a nation and before used as foods for
consumption as main criteria to approve them. All GM foods purposed for sale whether grown in
Australia or out are subject to a pre-market safety evaluation by FSANZ which is an independent
statutory authority in the health portfolio accountable for regulation of food. FSANZ would
never regulate a GM food for sale in case there is proof that the food could result in a public
health or safety issue (Preston, 2018).
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