Name : Ndumiso

Surname : Ndawonde
Student number : 60516364
Module code : MIB3702
Examination period : may/June 2020
ID number : 9704175437084
Question 1

Transformation and selection of bacteria are key steps in DNA cloning. DNA cloning is
the process of making many copies of a specific piece of DNA, such as a gene.
Researchers first insert a piece of DNA, such as a gene, into a circular piece of DNA
called a plasmid. This step uses restriction enzymes and DNA ligase and is called
a ligation. After a ligation, the next step is to transfer the DNA into bacteria in a process
called transformation. Then an antibiotic selection and DNA analysis methods are used
to identify bacteria that contain the plasmid that is going to be used.
PCR amplifies gene of interest in plasmid. Digestive reaction cuts gene of interest out of
the plasmid using specific restriction enzymes. Bacteria is then grown for a brief period
of time in lysogeny broth. Each restriction enzyme has its own recognition site and will
cut strand of recognition site. Gene of interest is then ligated into plasmid of choice,
using DNA ligase enzyme. Bacterial cells are the spread on an agar plate containing
antibiotics specific to the plasmid and then incubated overnight. We then check for
colonies on an agar plate because only cells that contain the plasmid will be able to
grow in the presence of the antibiotic, due to the antibiotic gene on the plasmid.
Question 2

2.1. The one type of PCR I would employ to amplify a desired gene from the RNA virus
is RT-PCR or qPCR. Here the amount of RNA template, which is converted to DNA with
reverse transcriptase prior to starting PCR, present in a given sample can be
determined. This is accomplished by adding a fluorescently labeled probe to the
reaction mixture and measuring its signal during the initial cycles. This is when the rate
of DNA amplification is logarithmic, but the PCR cycles continue, substrates are
consumed and polymerase efficiency declines. So although the amount of product
increases, its rate of synthesis is no longer exponential. Specially designed
thermocyclers record the amount of PCR product generated as it occurs, thus the term
real-time PCR. Gene expression studies often rely on real-time PCR because mRNA
transcripts can be copied by reverse transcriptase to cDNA, which is then quantified.
In AIDS patients, the viral load is determined by RT-PCR. The person’s viral load is
quantified to allow physicians to monitor the progression of the disease and the patient’s
response to therapy. Viral load assessment before, during and after therapy has
tremendous potential for improving the clinical management of diseases caused by viral
infection, including AIDS and hepatitis. These tests assist in narrowing the window
period, resulting in improved blood safety.
Real-time PCR such as TaqMan and Abbott RealTime, such as Retina Rainbow or
NucliSens EasyQ, might be useful for measuring viral load in resource-limited countries.
Real-time PCR detects amplicon production in real time with each PCR cycle, and thus
does not rely on post-amplification detection of amplicons, which helps reduce the
possibility of contamination and improves turnaround time. However, commercially
available real-time PCR assays are just as expensive as the more standard nucleic acid
viral load tests and also use expensive equipment. The use of in-house versions of
these assays can help to reduce kit costs, but laboratories must provide their own
reagents such as primers and probes, and optimize their methods. Progression of AIDS
is monitored by using viral load. When viral load increases and CD4 cell counts
decreases in a person on anti-HIV therapy are indications that adherence is not
adequate or that the drugs being used are not effective and may need to be changed. In
addition, today there are other tests that can help physicians determine whether current
therapy is working or whether a treatment option under consideration is likely to work.
Therapeutic drug monitoring (TDM) are used to help individualize anti-HIV therapy by
measuring the amount of drug in an individual's blood. This is important because
different people absorb, process, and eliminate drugs at different rates, and blood levels
may vary considerably among individuals taking the same doses of the same
medications.
2.2. Agarose gel electrophoresis separates DNA fragments according to their size. An
electric current is used to move the DNA molecules across an agarose gel, which is a
polysaccharide matrix that functions as a sieve to hold back the molecules as they are
transported by the electric current. Thus, from a mixture, smaller molecules will move
more rapidly through a gel compared to larger molecules. An electrophoresis buffer is
used to maintain the current in the gel. Agarose is made from purified agar. The
electrophoresis buffer and agarose are mixed and heated, and when it has cooled, it
forms a gel.
Question 3

3.1. Microbiology is the study of organisms that are usually too small to be seen with the
naked eye. These includes bacteria, archaea, viruses, fungi, prions, protozoa and
algae, collectively known as microbes. These microorganisms are extremely important
in our everyday lives. Some are responsible for a significant proportion of the disease
affecting not only humans but also plants and animals, while others are vitally important
in the maintenance and modification of our environment. Some are involved in water
purification. These microorganisms originate from waste, wastewater, or water stream
itself. Biota are an essential component of most sewage treatment processes and
many water purification systems. Most of the organisms involved are derived from the
waste, wastewater or water stream itself or from the atmosphere or soil water. However
some processes, especially those involved in removing very low concentrations of
contaminants, may use engineered eco-systems created by the introduction of specific
plants and sometimes animals. Some full scale sewage treatment plants also
use constructed wetlands to provide treatment.
Saprophytic bacteria and fungi can convert organic matter into living cell mass, carbon
dioxide, water and a range of metabolic by-products. These saprophytic organisms may
then be predated upon by protozoa, rotifers and, in cleaner waters, Bryozoa which
consume suspended organic particles including viruses and pathogenic bacteria. Clarity
of the water may begin to improve as the protozoa are subsequently consumed
by rotifers and cladocera . Purifying bacteria, protozoa, and rotifers must either be
mixed throughout the water or have the water circulated past them to be effective.
Sewage treatment plants mix these organisms as activated sludge or circulate water
past organisms living on trickling filters or rotating biological contactors. Rotifers are
microscopic complex organisms and are filter feeders removing fine particulate matter
from water. They occur naturally in aerobic lagoons, activated sludge processes, in
trickling filters and in final settlement tanks and are a significant factor in removing
suspended bacterial cells and algae from the water column. Annelid worms are
essential to the effective operation of trickling filters, helping to remove excess bio-mass
and enhancing natural sloughing of the bio-film. Supernumerary worms are very
commonly found in the drainage troughs around trickling filters and in the final
settlement sludge. Annelids also play a key role in lagoon treatment systems and in the
effective working or engineered wet-lands. In this environment worms are a principal
force in mixing in the upper few centimeters of the sediment layer exposing organic
material to both oxidative and anoxic environments aiding the complete breakdown of
most organics. They are also a key ingredient in the food-chain transferring energy
upwards to fish and aquatic birds.
3.2. Recombinant DNA technology or "genetic engineering," can benefit people in
several ways. For example, scientists made artificial human insulin with the help of
recombinant DNA technology. Since Diabetic people cannot produce their own
insulin, which they need in order to process sugar and Animal insulin is not a suitable
replacement because it causes severe allergic reactions in most people. Thus,
scientists used recombinant DNA technology to isolate the gene for human insulin
and insert it into plasmids. These plasmids were then inserted into bacterial cells,
which created insulin based on the human genetic code inside of them. The resulting
insulin was safe for humans to use. Thus, people with diabetes went from having a
life expectancy of around four years after diagnosis to having a normal human life
expectancy. Recombinant DNA technology helped improve food production. Fruits
and vegetables, which were prone to attacks from pests, now have genetic
modifications to be more resistant. These advancements greatly increased crop
yields, which means that more food is available to the public at the end of each
growing cycle.
The downside of recombinant DNA technology are ethical in nature. Some people
feel that recombinant DNA technology goes against the laws of nature, or against
their religious beliefs, due to how much control this technology gives humans over the
most basic buildings blocks of life. Some people worry that if companies can pay
scientists to patent, buy and sell genetic material, then genetic material could become
an expensive commodity. Such a system might lead to people having their genetic
information stolen and used without permission. Other people worry that humans may
begin tampering too much with their own genetic material and create societal
problems. What if people use recombinant DNA technology to live longer, become
stronger? Will societal division swell between genetically modified people and
"normal" people? These are questions that scientists and the public will likely
continue to consider as humanity moves toward a future where manipulating DNA is
easier than ever before.
Question 4

Wastewater includes sewage as well as industrial and agricultural effluent and street
runoff collected in storm sewers. These waters often contain high levels of organic
matter, heavy metals, nutrients, and particulates. Wastewater treatment involves at
least three spatially segregated steps: primary, secondary, and tertiary treatment.
Primary treatment prepares wastewater for treatment by physically removing much of
the solid material. This can be accomplished in several ways, including screening,
precipitation of small particulates, and settling in basins or tanks. The resulting solid
material is usually called sludge. The goal of primary treatment is to generate water
(effluent) that can be further purified by biological treatment and produce sludge that
can be treated before disposal.
Secondary treatment promotes the biological transformation of dissolved organic
matter (DOM) into microbial biomass and carbon dioxide. Several approaches can be
used in secondary treatment to remove DOM. When microbial growth is completed,
under ideal conditions the microorganisms will aggregate and form stable flocs, also
called granular sludge, that settle. Efficient flocculation is important because a sewage
treatment facility that generates stable granular sludge requires less space and reduced
cost. When these processes occur with lower oxygen levels or with a microbial
community that is too young or too old, unsatisfactory floc formation and settling can
occur. The result is a bulking sludge, caused by the massive development of
filamentous bacteria such as those in the genera Sphaerotilus and Thiothrix, together
with many poorly characterized filamentous microbes. An aerobic activated sludge
system involves the horizontal flow of materials with recycling of sludge the active
biomass that is formed when organic matter is oxidized and degraded by
microorganisms. Aerobic secondary treatment is often carried out with a trickling filter.
The waste effluent is passed over rocks or other solid materials upon which microbial
biofilms have developed, and the microbial community degrades the organic waste. A
sewage treatment plant can be operated to produce less sludge by employing the
extended aeration process. Microorganisms grow on the dissolved organic matter, and
the newly formed microbial biomass is eventually consumed to meet maintenance
energy requirements. This requires extremely large aeration basins and long aeration
times. All aerobic processes produce excess microbial biomass, or sewage sludge,
which contains many recalcitrant organics. Often the sludge from aerobic sewage
treatment, together with the materials that settled in primary treatment, are further
treated by anaerobic digestion, which occurs in large tanks designed to operate with
continuous input of untreated sludge and removal of the final, stabilized sludge product.
Within the anaerobic digester, at least three processes occur, firstly is the fermentation
of the sludge components to form organic acids, including acetate; secondly is the
production of the methanogenic substrates acetate, carbon dioxide and hydrogen; and
finally, methanogenesis by methane producers. Anaerobic digestion has its advantages.
Most of the microbial biomass produced in aerobic growth is degraded in the anaerobic
digester. Also, because the process of methanogenesis is energetically very inefficient,
methanogenic archaea consume about twice as many nutrients to produce an
equivalent biomass as that of aerobic systems.
Tertiary treatment further purifies wastewaters by removing nitrogen and phosphorus
compounds that can promote eutrophication. It also removes heavy metals,
biodegradable organics and viruses. To remove phosphorus, oxic and anoxic conditions
are used alternately in a series of treatments, and phosphorus accumulates in microbial
biomass as polyphosphate. Excess nitrogen may be removed by “stripping,”
volatilization of NH3 at high pH. Ammonia itself can be chlorinated to form dichloramine,
which is then converted to molecular nitrogen. In some cases nitrogen is removed by
the process of denitrification, whereby nitrate produced by microbes under aerobic
conditions, serves as an electron acceptor during anaerobic respiration. Nitrate
reduction yields nitrogen gas and nitrous oxide as the major products. In addition to
denitrification, the anammox process is also important, where ammonium ion reacts with
nitrite to yield nitrogen gas. The anammox process can convert up to 80% of the initial
ammonium ion to nitrogen gas.
The process of wastewater treatment must be monitored to ensure that waters released
into the environment do not pose environmental and health risks. Successfully treated
water should have very little organic carbon, this is accomplished by being measured as
Total Organic Carbon (TOC), as chemically oxidizable carbon by the Chemical Oxygen
Demand (COD) test, or as biologically usable carbon by the Biochemical Oxygen
Demand (BOD) test. The TOC includes all carbon, whether or not it can be used by
microorganisms. The carbon dioxide produced is measured by infrared or potentiometric
techniques. The BOD test measures only that portion of total carbon oxidized by
microorganisms in a 5-day period at 20 degrees Celsius. TOC is fastest, but it is less
informative with regard to biological processes. The COD involves the use of wet
chemicals with higher waste chemical disposal costs.
Question 5

Bioremediation, the process of stimulating the degradative activities of microorganisms

already present in contaminated waters or soils. These existing microbial communities
typically cannot carry out biodegradation processes at the desired rate due to limiting
physical or nutritional factors. Therefore, it is necessary to determine the limiting factors
and supply the needed materials or modify the environment. Often the addition of easily
metabolized organic matter such as glucose increases biodegradation of recalcitrant
compounds that otherwise would not be used as carbon and energy sources by
microorganisms, in a process called cometabolism. Bioremediation involves
stimulating hydrocarbon degradation in waters and soils, bioaugmentation, metal
bioleaching.
Stimulating hydrocarbon degradation in waters and soils = Hydrocarbons lack
phosphorus and nitrogen, so these elements limit their degradation. Another key
variable in promoting bioremediation is the degree of contact between microorganisms
and the hydrocarbon substrate. To increase hydrocarbon accessibility by microbes,
pellets containing nutrients and a hydrocarbon soluble preparation are often used.
Bioaugmentation = this is the acceleration of microbiological processes by the addition
of known active microorganisms to soils, waters, or other complex systems.
Microorganisms can be added directly to natural communities, where they create their
own microhabitats but in most cases microbes are added with other components to
ensure their viability. A blend of microorganisms can be purchased from commercial
bioremediation engineering companies to treat petrochemical contamination of soils and
water. These microbes are most often added in a slurry along with nutrients, enzymes,
surfactants, and chemicals that release oxygen to promote aerobiosis. In other cases,
microbes are introduced with protective inert microhabitats, this helps the foreign
microbes to establish an effective population in the face of competition from the natural,
endogenous microbial community . Thus the application of principles of microbial
physiology and ecology can facilitate the successful management of microbial
communities in nature.
Metal bioleaching = is the use of microorganisms that produce acids from reduced
sulfur compounds to create acidic environments that solubilize desired metals for
recovery. This approach is used to recover metals from ores and mining tailings with
metal levels too low for smelting. This involves the biological oxidation of copper present
in these ores to produce soluble copper sulfate. This process is now used for copper
mining.
References
Prescott, Harley & Klein, Microbiology, 10th edition. J, Willey, Sherwood L and
Woolverton C, 2014. McGraw-Hill New York, USA.
MIB3702 study guide, Advanced Microbial Genetics, Recombinant DNA Technology
and Industrial Microbiology, 2020.
UNAIDS Epidemic update 2007.
Vajpayee M, Mohan T. Current practices in laboratory monitoring of HIV
infection. Indian J Med Res. 2011.
Sciencing.John Brennan, 2017.