School of Bio-Engineering and Biosciences

Name: Kavita Jaiswal

Reg No: 11915154
Roll No: A31
Section: BE053
Course Code: BTY733
Course Title: Bioprocess Calculations
Submitted to: Ajay Kumar
CA-II
SR TOPIC PAGE
NO. NO.
01. Question 1. a 02-3

02. Question 1. b 03-04

03. Question 1. c 05-06

Page 1 of 6
Q1. You wish to express a particular peptide in E. coli using a high-copy-
number plasmid. You have the amino acid sequence for the peptide.
a. Explain the experimental process for generating and selecting the
genetically engineered E. coli using restriction enzymes, ligase, E. coli,
a plasmid with neomycin resistance, and the known amino acid
sequence.
Proteins with known amino acid sequences can be cloned into E. coli
using plasmid vectors. By increasing the number of bacteria, you can produce
more of the desired protein.

The following steps are followed to clone the protein of interest:

I. Amplification of gene of interest (Using PCR):

When the amino acid sequence is known then the gene of interest is
selected using a hybridization probe. The probe is a fragment of DNA
(deoxyribonucleic acid) of 8-15 nucleotide length. The probe will have the
complementary sequence to isolate the gene of interest.
II. Insert into a cloning vector (plasmid) by ligating the plasmid with a
restriction enzyme, or by microporation:
The desired gene is cut from the host using restriction enzymes. The
most widely used restriction enzymes are Eco RI and Bam H1. The Eco
RI has the restriction sequence 5’ – GAATTC – 3’ and Bam HI has the
restriction sequence 5’ -GGATCC -3’. The same restriction enzyme must
be used to cut both donor and vector DNA.
III. Sub-cloning:
The E. coli DNA is cut at the restriction site by Eco RI and the desired
gene is obtained. The high copy number plasmid with neomycin resistant
gene is selected and the plasmid is cut with the same restriction enzyme to
insert the desired gene. The ligase enzyme is used to seal the ends of the
gene and plasmid. at its 5’ ends forming phosphodiester bonds.

Page 2 of 6
IV. Transformation into protein-expressing bacteria (E. coli):
The plasmid vector with the desired gene is introduced into the host by
transformation. The desired gene is transformed is screened by culturing
the cells in the medium containing neomycin. Neomycin-resistant gene is
present in the plasmid and is a selectable marker. Thus, it is easy to identify
that the transformed cells grow in presence of Neomycin.
V. Selection of clones by allowing the bacteria to grow in Neomycin
containing plates:
Finally, the desired gene expresses the peptide with the known amino
acid sequence and is screened by running SDS-Polyacrylamide Gel
Electrophoresis. The bands formed during the electrophoresis will give
the size and molecular weight of the peptide.
VI. Isolation and purification of the protein of interest:
Further, the amino acid sequence of the peptide is compared and
confirmed with the synthesis of complementary DNA to the
peptide (cDNA method).
b. What control elements would you place on the plasmid to regulate the
expression and to prevent read-through?
Regulatory elements placed on the plasmid act as promoters to regulate
the expression of cloned products. Promoters act as regulatory switches to
transcribe the cloned gene product on demand. When RNA (ribonucleic
acid) polymerase binds to the promoter, transcription is initiated and
multiple products are produced. When the product is not needed,
transcription is turned off by binding a repressor molecule to the promoter.
Thus, when a promoter is present on the vector molecule, it regulates
the expression of the cloned product by turning the transcription process
on and off.

Page 3 of 6
Restriction enzyme digestion followed by ligation for E. coli with the
neomycin resistance

Page 4 of 6
c. Design the bioreactor to express the particular peptide.
Recombinant DNA is replicated in the host and expressed as protein
under optimal conditions. This is now a recombinant protein. A small
amount of cell culture does not yield large amounts of recombinant protein.
Therefore, large-scale production is required to produce products that are
useful to people. A vessel called a bioreactor is used for this purpose.
A bioreactor is a large vessel with a continuous culture system that adds
new medium from one side and removes spent medium from the other side.
A bioreactor can process approximately 100 to 1000 liters of cell culture.
Bioreactors provide optimal conditions (temperature, oxygen, pH value,
vitamins, etc.) for the biological transformation of raw materials into
specific proteins, enzymes, etc.
A “stirred tank bioreactor” is the most common type of bioreactor to
express the particular peptide.
• Agitation system - for evenly agitating the contents
• Oxygenation system - for introducing air into the system
• Foam control system
• Temperature control system
• pH control system
• Sampling port - For bringing out small cultures

Page 5 of 6
 Stirred tank Bioreactor

References:

• https://www.frontiersin.org/articles/10.3389/fmicb.2014.00172/full
• https://www.ncbi.nlm.nih.gov/books/NBK26837/
• https://www.sigmaaldrich.com/IN/en/technical-documents/technical-
article/genomics/cloning-and-expression/restriction-enzyme-cloning-manual-
cloning

Page 6 of 6