Department of Technology and Biotechnology of Drugs – Biology for beginners

Virtual cloning - an In Silico DNA Cloning Experiment

Short background – recombinant insulin

Insulin is a hormone that regulates the amount of glucose (sugar) in the blood and is required for the body
to function normally. Insulin is synthesised by the β-cells of the islets of Langerhans in the pancreas. It is a
relatively small protein, comprising two polypeptides, one of 21 amino acids (the A chain) and one of 30 amino
acids (the B chain). In humans these chains are synthesized as a precursor called preproinsulin, which contains the
A and B segments linked by a third chain (C) and preceded by a leader sequence. After translation, the leader
sequence is removed which results in the C chain being disconnected. In effect, A and B polypeptides are linked to
each other by two disulphide bonds.

Ryc. 1 The structure of insulin molecule and a summary of its synthesis by processing from preproinsulin.
Source: “Gene Cloning and DNA Analysis” T.A. Brown

Introduction

 DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA
 In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid
 The plasmid is introduced into bacteria via process called transformation, and bacteria carrying the plasmid
are selected using antibiotics.
 Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express
the gene and make protein.

Most cloning experiments are carried out with Escherichia coli (E. coli) as the host. E. coli is particularly popular
when the aim of the cloning experiment is to study the basic features of molecular biology such as gene structure
and function. Human insulin is produced now by recombinant DNA technology and various companies differ in
their methodology but the basic principle is the introduction of human insulin or proinsulin gene into
microorganisms like E. coli or Yeast. Insulin displays two features that facilitate its production by recombinant
DNA techniques. The first is that the human protein is not modified after translation by the addition of sugar
molecules - glycosylation (many recombinant proteins require this kind of modification, that is available only in
eukaryotic cells). The second advantage concerns the size of the molecule (insulin is a relatively small protein).
Recombinant insulin synthesized by bacteria should therefore be active.
 Department of Technology and Biotechnology of Drugs – Biology for beginners

AIM OF THE LABORATORY EXCERCISE

Construction of a recombinant vector for in silico cloning the insulin gene. For this purpose, the free software
Serial Cloner will be used, which allows you to design a cloning vector, prepare a DNA sequence for cloning in
this vector, primers design for PCR and subcloning.

Software Serial Cloner => http://serialbasics.free.fr/Serial_Cloner-Download.html

Moreover, the following websites will be used:

NCBI http://www.ncbi.nlm.nih.gov/
Expasy to access Reverse Translate http://www.expasy.ch/

I PART: PREPARATION OF THE INSULINE SEQUENCE TO BE CLONED

1. Open the NCBI website (http://www.ncbi.nlm.nih.gov/) to obtain insulin protein sequence by typing ‘homo
sapiens insulin’ in the search box and searching ‘Protein’. Click on protein with accession number:
GI: 386828. Scroll to the bottom of the page to the world ORIGIN to obtain protein sequence (the one letter
code protein sequence). Now click on FASTA to obtain this sequence in FASTA format.
2. Open the Expasy website (http://www.expasy.ch/). The Expasy website is used to reverse transcribe the
protein to cDNA. From the ‘Categories’ on the left choose ‘proteomics’ and then ‘protein sequence and
identification’. From ‘Tools’ menu choose ‘Reverse Translate’. Paste the protein sequence in the box in
FASTA format, add ‘*’ at the end (to generate codon stop) and click ‘submit’. Leave this website with results
opened (for future use of nucleotide sequence).

3. Open Serial Cloner. On the ‘Toolbar’ choose ‘New’ and paste nucleotide sequence of insulin into ‘Sequence
Window’ and save on the pulpit as ‘Human insulin’.

II PART: PREPARATION OF THE CLONING VECTOR

1. In the new tab on the NCBI page, find the complete sequence of the cloning vector pUC19 (accession
number: GI: 6691170. Then copy the sequence of this plasmid to the Serial Cloner. For this purpose in the
program, open a new sequence window (in the upper toolbar, the ‘New’ button) and paste the sequence of
pUC19. On the toolbar in the ‘Sequence’ tab, click ‘Circularize’ to perform a linear plasmid sequence into a
circular form. Save the plasmid sequence on the desktop as "pUC19_circular".

2. Analyze the plasmid: on the upper toolbar click ‘Scan’ to locate all the sequences in the pUC19 plasmid. Now
features (sequences) are all visible in sequence windows and on graphic map. For a graphical plasmid map,
click ‘Graphic Map’ (useful tip! Uncheck the box ‘Unique Sites’ and check the box ‘Features’ – graphic map
will be easily readable).
TASK: Familiarize yourself with the construction of the plasmid and indicate the most important sequence
in this plasmid.
III PART: CLONING AND ANALYSIS OF RECOMBINANT VECTOR
1. On the toolbar, click ‘Function’ button and then select ‘Build construct’. In the ‘Select DNA 1’ field, enter
the sequence of the pUC19 vector (selecting the ‘pUC19_circular’ file) and enter the restriction sites for the
HindIII (on 3’ end) and EcoRI (on 5’ end) enzymes (in the window check ‘Particular sites’ and select the
enzymes: by clicking once you choose the restriction enzyme on 5’, by clicking double time you choose the
restriction enzyme on 3’). Check ‘Blunt (Remove)’ to get rid of blunt ends.
2. In the ‘Select DNA 2’ field, enter the sequence of the insulin (selecting ‘human insulin’ file) and check
‘Select all’ to clone the entire DNA fragment.
 Department of Technology and Biotechnology of Drugs – Biology for beginners

3. Click ‘Ligate’ to perform virtual cloning. In this way you received the recombinant vector (2964 bp). Save
the record as ‘pUC19_insulin’.
4. To verify your cloning experiment, locate insulin sequence in the recombinant plasmid. For this purpose, on
the toolbar in the ‘Features’ tab, click ‘Manage Features’. Select the ‘Generic’ tab and enter a new feature by
clicking "+". In the window on the right, enter the name "human insulin" and paste the insulin sequence in the
‘sequence’ site (choose the bright purple colour for this sequence). Close the window by clicking ‘ok’. To
view cloned insulin sequence, once again ‘Scan’ the recombinant plasmid and display ‘Graphic Map’.

IV PART: DESIGNING PRIMERS FOR PCR CLONING

Short explanation: The gene of interest usually has to be amplified from vector DNA by PCR (polymerase
chain reaction) before it can be cloned into an expression vector. The first step is the design of the necessary
primers.

1. On the upper toolbar click ‘PCR’. In the window with the sequence of the recombinant vector, locate the
sequence of the insulin gene. Note that the insulin gene sequence has been cloned between vector sequence
primers. Select the M13-fwd starter and left-click the mouse to select ‘Use as PCR reverse primer’ (in the
‘Edit’ tab). Proceed in the same way with the second M13-rev starter by clicking ‘Use as PCR forward
primer’.
2. Add the restriction enzyme recognition sequences for NdeI (to reverse primer) and BamHI (to forward
primer) (see the sequences below) so that the foreign DNA and plasmid can be digested with the two
restriction enzymes and the foreign DNA can be covalently added into the multiple cloning site by ligation in
the correct orientation using DNA ligase. Click on ‘Run PCR’. You should receive the PCR product length
394 nt. Save it as ‘PCR(pUC19_insulin)’. Why we use NdeI aand BamHI enzymes? See the explanation in
point 4.

NdeI BamHI

3. Open the website with pET-15B sequence https://www.addgene.org/browse/sequence_vdb/2543/ and copy

the sequence of this plasmid to the Serial Cloner and save as ‘pET-15B’.
This is expression vector. It is the most powerful system developed for the cloning and expression of
recombinant proteins in E. coli. pET-15B contains asymmetric site of cloning [NdeI / BamHI) which allows
you to enter a sequence encoding the gene into a suitable reading frame. E. coli cells infected with this
plasmid by transformation can be induced to express the protein.

V PART: PCR BASED CLONING

Short explanation: PCR based cloning is about making a copy of a piece of DNA and at the same time
adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of
interest. In this part DNA insert (insulin gene) is taken out of a donor plasmid (pUC19) and inserted into a
recipient plasmid (pET-15B). You have already obtain PCR product with introduced sites of restriction
enzymes. Now this PCR product will be cloned into expression vector.

1. On the toolbar, click ‘Function’ button and then select ‘Build construct’. In the ‘Select DNA 1’ field, enter
the sequence of the expression vector (selecting the ‘pET-15B’ file). Enter the restriction sites for the NdeI
 Department of Technology and Biotechnology of Drugs – Biology for beginners

(5’ end) and BamHI (3’ end) enzymes (in the window check ‘Particular sites’ and select the enzymes: by
clicking once you choose the restriction enzyme on 5’, by clicking double time you choose the restriction
enzyme on 3’).
2. In the ‘Select DNA 2’ field, enter the sequence of the PCR product (selecting ‘PCR(pUC19_insulin)’ file)
and check ‘Select all’ to clone the entire DNA fragment. Because in this case terminal end are not sticky,
check ‘Blunt (Fill-In)’ in both field ‘Select DNA 1’ and ‘Select DNA 2’.

3. Click ‘Ligate’ to perform virtual cloning. In this way you received the recombinant vector (total length 6096
nt). Save the record as ‘pET-15B_insulin’.