The following answers below detail responses based on individual knowledge of

chemistry and biology to the questions asked.

1 (i)

List four differences between prokaryotes and eukaryotes.

Although prokaryotes and eukaryotes both contain cell membranes, which are
lipid bilayers (non-polar) surrounded around two phospholipids and DNA, which
contains instructions for making every protein used by the cell and ribosome use
for protein production, there are vast differences. The first difference is
prokaryotes or bacteria have no nucleus or membrane-bound organelles.
Secondly unlike larger eukaryotes, prokaryotes were also firstly evolved as
single-celled organisms, which also meant reproduction was initiated by asexual
reproduction (binary fission). This was not the case for multi-cellular eukaryotic
cells, as they had extensive distinction of cells and tissues [1]. Thirdly, in terms
of genome characteristics, unlike prokaryotes, eukaryotes possessed mitotic and
meiotic division of the nucleus, DNA observed as chromosomes, and DNA in
organelles [1]. However, prokaryotes possessed the formation of partial diploid
[1]. Lastly, in terms of organelles, prokaryotes do not possess mitochondria,
endoplasmic reticulum, Golgi apparatus, and flagella which are complex with
microtubules [1].
1 (ii)
Place the following under either prokaryotes or eukaryotes: Escherichia
coli; bakers yeast (Saccharomyces cerevisiae); fungi such Aspergillus flavus and
mushrooms; cyanobacteria; Streptococcus lactis and plants.
The following table below identifies whether the list above are prokaryotes or
eukaryotes.
PROKARYOTES
Escherichia coli
Cyanobacteria
Streptococcus lactis

EUKARYOTES
Saccharomyces cerevisiae (bakers
yeast)
Aspergillus flavus
Mushrooms
Plants

2. Describe the steps required to develop a complete industrial bioprocess for the
production of a recombinant-DNA-derived biopharmaceutical such as insulin

Recombinant DNA-derived biopharmaceutical industrial bioprocesses are extensive

in the production of different products such as insulin, interferon, or growth
hormones. Particularly in insulin production, there are multiple bioprocessing steps
required for production. Before detailing the steps, it is important to understand the
insulin chemically. It is a simple protein which consists of 51 amino acid with 30 of
which consist of one polypeptide chain and the other consisting of a second chain
[2]. With a disulfide bond (R-S-S-R), there are 153 nitrogenous bases [2]. The

synthesis of insulin is dependent upon its four different nitrogen bases (guanine,
thymine, cytosine, adenine). Understanding the chemistry of the protein allows the
steps described below to sound more linked. The first step/stages in the following
bioprocess is genetic manipulation of the host organism, which, for example,
considering an animal cell. With the addition of biochemical, part of the animal
chromosome is cut from the animal tissue and the gene is cut from the
chromosome. The gene is a double helix structure of the DNA, where in this
eukaryotic cell, the introns are removed. Next, a plasmid within a microorganism
such as Escherichia coli is removed and cut and the gene from the animal DNA is
cloned into the cut plasmid. This is considered genetic engineering and is performed
with the use of petri dishes, small quantities of restriction enzymes, electrophoresis
gels for DNA, and other equipments [2]. On the micro level, the genetic code of
insulin on the eleventh chromosome divides into two, separating nitrogen bases
which were held by hydrogen bonding. Messenger RNA is formed in the process
known as transcription by using the DNA strand as a template [2]. The m-RNA
strand where thymine has been replaced by uracil carries genetic information from
the nucleus to the cytoplasm [2]. It then latches to a ribosome where the m-RNA is
grouped into codons (threes) and transfer RNA molecules which have ungrouped
nitrogen bases bind as anti-codons to the bases of m-RNA [2]. This process is known
as translation, which invokes the amino acid sequence that forms proteins such as
insulin. This process can be considered as the production of recombinant DNA and is
formed within the cut plasmid. After the cloning process, the recombinant plasmid is
inserted back into the microorganism [2]. The recombinant cells within the
microorganism must be functionally measured with the culture environment. The
small-scale culture is allowed optimum growth on the basis of pH, composition,
temperature, and environmental factors [3]. This will allow optimum growth in
plasmid multiplication and gene expression, which will eventually promote cell
division. Parameters are also calculated at this time for the cell including cell growth
rate, yield, and specific productivity [3]. After successful cell division, the culture
conditions are scaled-up for production, particularly in a bench-top bioreactor.
Multiple process variables in the bioreactor including instruments for adjusting
temperature, pH, oxygen gradients, and speed of the agitator allow better control of
the culture [3]. In this step, ideal activity of the cells is necessary to identify reasons
for possible cell damage, nutrient exposure to the cells, and other shear sensitive
limitations. Different calculated parameters such as mass transfer coefficient,
energy dissipation rate, and mixing time must also help decide whether the culture
is best functioned for batch, semi-batch, or continuous processes [3]. If the benchtop bioreactor is successful, pilot-scale bioreactor is initiated, where the increase in
size of the equipment, will determine if the culture will have a substantial loss in
productivity (other factors are also considered). If the pilot-scale is successful,
initiation of industrial scale operation begins. This process will start the installation
of service facilities, which include sterilization equipment, steam generator, cooling
water, and medium preparation [3]. Product recovery of recombinant DNA-derived
insulin is necessary. Different type of extraction processes are used to increase

product recovery. The most economically viable process is induced in terms of

commercial procedure to ultimately package and market the biopharmaceutical
recombinant-DNA-derived insulin [3]. The insulin is typically used on animals before
humans.

References
[1] Millis N.F. Comprehensive Biotechnology, Vol. 1 , Elsevier Science , 1985
[2] Willson, Rhonda. "Recombinant DNA Technology in the Synthesis of Human
Insulin." Insulin

BioProcessing. Rajpreet Singh-Khaira, 2014. Web. 19 Feb. 2015.

[3] Doran, Pauline M. "Recombinant-DNA-derived Industrial Processing."Bioprocess

Engineering
Print.

Principles. 2nd ed. Vol. 1. London: Academic, 1995. 153-58.