TITLE: Serratia produces quit similar trypyrrole ring prodigiosin

 TITLE: TRANSFORMATION OF PRODIGIOSIN GENE INTO E. COLI FOR
ITS ENHANCED PRODUCTION

Summary:

Prodigiosin is secondary metabolite, red pigment.
synthesize by different strains of Serratia
Marcescens, Pseudomonas
and Streptomyces. The
molecular formula is C20H25N3O and the molecular weight is 323.4 gm/mol. In
recent years it has gained a lot of attention by scientist because of its new
found features. Such as, antibacterial, antifungal, antiprotozoal,
antimalarial, antiproliferative, immunosuppressive and anticancer properties Such as, antibacterial, antifungal,
antiprotozoal, antimalarial, antiproliferative, immunosuppressive and anticancer properties. The prodigiosin
pigment is also used in textile industries as dying agent. Out of these, immunosuppressive and anticancer
activities are the biggest reason to achieve such extreme attention since they
show clinical promise. As prodigiosin and its derivatives has many applications with some of them
are beneficial to human health, gives us the reason to produce it on a larger
scale with minimum cost so that everyone can be benefited from it, which is the
main aim of this project

 

Background:

Prodigiosin
is simply assessed secondary metabolite of S.
Marcescens and it can be used as a model system for study of secondary
metabolite production mechanism in microbe. As it illuminates red colour it is
easy to interpret the changes. Hence the isolation of the recombinatly produced
prodigiosin will give us information about biosynthetic pathways, gene
expression products, genetics and enzymology. In this research project, we are
isolating the genes responsible for the production of the prodigiosin and then
with the help of a suitable vector such as plasmid, cosmid introduce them into E.Coli and check the expression with
different screening methods. Some strains of S. Marcescens are cause of nosocomial infections. The red pigment,
prodigiosin is a member of the family prodiginines

Produced
by Serratia strains, actinomycetes
and some other bacteria. Prodigiosin is built with a trypyrrole ring and
produces in the last phase of the growth of the host. The other species except Serratia produces quit similar
trypyrrole ring prodigiosin and some of its derivatives.

 

A very
complex pathway is known for the production of prodigiosin, biosynthesis steps
involve dual pathway, 4-methoxy2,2-bipyrrole-5-carboxyaldehyde (MBC) and other
is monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP). The precursors of prodigiosin
are methionine, serine, proline, acetate and alanine. Recent reports show that,
the procedure of proline incorporation in a pyrrole complex, also a pathway for
biosynthesis of undecyl prodigiosin. Prodigiosins have been reported to show antibacterial,
antifungal, antiprotozoal, antimalarial, antiproliferative, immunosuppressive
and anticancer properties.
Because of the potential application in health industry it has gained interest.
Prodigiosin and its derivatives shows potential and specific target activity
which is separate from
other drugs. The reports on prodigiosin suggest that it induces the apoptosis
of the cancer cells by an unknown mechanism and the study is still going on in
this area and scientist are finding some results.

   

Prior
efforts to recombinenat production of prodigiosin has unsuccessful, some has
low level expression, or loss of vector and another reason are because of
complex pathway for it biosynthesis. With this project our aim is to produce a
recombinant strain with high level expression of prodigiosin in that
recombinant strain with reduced time period and reduced cost. Also understand
its regulation, what are factors affecting production in recombinant strain,
what are parameters required for highest growth in recombinant strain and
easier way to purify it from recombinant host strain.

 

Aims and hypotheses:

The first aim of this project is isolation of
prodigiosin producing gene from S. Marcescens strain. Then add this gene to a suitable
vector like plasmid, cosmid and contruct a recombinant vector having our gene
of interest. After that make copies of that vector or clone it and store it for
further use. Now by using different transformation methods insert this
recombinant vector into the E.Coli strain
Then check the transformation with controls, here our pigment shows red colour
so it is very easy to interpret the results. If the recombinant strain shows
red colour that means transformation is successful if not, we need to do it
again till we get recombinant strain producing the red pigment. After getting
the Transformed recombinant strain, our aim is to cultivate it so that it can
produce prodigiosin in large amount. After cultivation isolation and
quantification of prodigiosin molecule with the help of extraction and
evaporation methods. If the quantity is low change parameter and try different
protocol for best results. At last purification of the prdigiosin with the help
of different chromatographic methods. Characterisation assays are done with the
help of spectrometric methods. During this whole process our main aim is to
produce prodigiosin less time with reduced cost that the prodigiosin produced
from the natural strains.

 

Methodology and programme of work:

1.     
DNA
Isolation of Serratia Marcescens. and
isolation of prodigiosin genes: This part of experiment can be done in
molecular biology lab and requires simple equipment’s like incubator,
refrigerator, laminar, PCR, GELGOG, laminar, pipettes, different chemicals and
UV illuminator. For isolation experiment requires normal DNA isolation kit for
isolation of DNA from Serratia
Marcescens or we can manually isolate it by various DNA isolation protocols. From
that isolated DNA we will Isolate the gene which produces prodigiosin. The
whole gene sequence data is available in NCBI database. First isolated DNA is
treated with restriction enzyme; it will cut the DNA in fragment. These
fragments will then separate by Southern Blotting Technique, in blotting we
will attach the complementary probe of our gene of interest so that we can
easily isolate the gene of prodigiosin. After isolation make multiple copies of
it with PCR (PCR is normally present in a mol. Biology lab) and store it for
further use 2,3,4.

 

2.     
Insertion of that gene into suitable vector: This part
of experiment can be done in molecular biology lab and requires simple
equipment’s like incubator, refrigerator, Laminar, pipettes etc. Take one of
the copies of our gene of interest and insert it to the suitable vector to get
n recombinant vector containing our gene of interest. To achieve this goal we
will first get a vector such as Plsmid or cosmid (we can order it from any
biotech industry like
Thermo Fisher Scientific. Sigma etc.). By cleaving the vector
with the help of restriction enzyme, we will then introduce our gene to vector
and ligate the vector with ligation enzymes. Finally, we will get recombinant
vector with our gene of interest 2,3,4.

3.     Transfer
of transformed vector into host cell: This part of experiment can be done in
molecular biology lab and requires simple equipment’s like incubator,
refrigerator, laminar, pipettes, different chemicals etc. Now we have our
recombinant vector and

We are introducing it to the host cell that is E.
Coli (why E. 
Coli because it is a model organism which gives us feature like
easily available, not harmful, has very less doubling time, whole genome is
sequenced easy for genetic manipulation and etc.) with the help of different
transformation methods or simply we can make host cell as competent cell
(competent cell can be prepare by heat shock treatment, using chemicals etc.)
and then treat them with our vector in suitable solutions under suitable
physical conditions 2,3,4.

 

4.     
Check the transformation: This part of experiment can
be done in molecular biology lab for this experiment we can use different
methods of screening of transformation but our desired product shows red color
when it produces. So it is very to check the transformation, if bacterial
colony shows red color then the transformation is successful and if not then we
have to repeat the experiment with more cautions. Till we get the transformed E.
Coli cells

 

5.     Cultivate
the recombinant strain: This part of experiment can be done in molecular
biology lab and requires simple equipment’s like incubator, refrigerator,
laminar, pipettes, different chemicals, small lab fermenter etc. The
recombinant strain will need Luria–Bertani medium for its growth. All the growth
essential components and recombinant strain will be added to small lab
fermenter and maintained it under specific conditions 1.

 

6.     Extraction
of red pigment and its quantification: This part of experiment can be done in
molecular biology lab and requires simple equipment’s like centrifuge, rotating
evaporator, incubator, refrigerator, laminar, pipettes, different chemicals
etc. The red pigment can be isolated from host by extraction methods. We will
use organic solvent systems were use chloroform:methanol (1:1; 2:1 and 1:2
v/v), after this step we will again do extraction using pure methanol. The
extract swill be concentrated by rotating evaporator at 45°C, and raw
Prodigiosin can be obtained. The quantification of Prodigiosin will be assessed
using this equation.  Prodigiosin
unit/cell = OD499 – (1.381 x OD 620) x 1000/ OD620 OD499 – pigment absorbance
OD620 – bacterial cell absorbance 1.381 – constant 1.

 

7.     Purification
of red pigment using chromatographic methods: This part of experiment can be done
in molecular biology lab and requires simple equipment’s like centrifuge,
rotating evaporator, HPCL, Thin Layer Chromatography equipment’s, Liquid
exclusion chromatography equipment, incubator, refrigerator, laminar, pipettes,
different chemicals etc. The crude extract will introduce into the
chromatography equipment (exclusion chromatography column is filled with
Sephadex LH-20, as an adsorbent) with methanol solution. Then elution will be
done with help of solvent system (chloroform: methanol: ethyl acetate (5:30: 65
(v/v)) and eluted fractions will be collected and from that further
purification can be done. The pigment then separately concentrated by Thin
Layer Chromatography (silica gel coated on TLC aluminum foil.), her we can use
developing solvent system (methanol: chloroform: acetone (2: 3: 4 v/v)). The Rf
value of chromatogram tells us about pure form of pigment and shows different
red spots which are prodigiosin.

 

8.     
Characterizing
the red pigment: This part of experiment can be done in molecular biology
lab and requires simple equipment’s like spectrophotometer, GCMS, centrifuge,
refrigerator, pipettes, different chemicals etc. The filtered red pigment will be characterized by
spectrophotometry. The purified red pigment will be characterizing by spectrophotometry
scan at a wavelength of 200-700 nm using chloroform as solvent. The molecular
weight of our red pigmnent can be determined by mass spectrometry or GCMS and
we can compare our red pigment spectra and data with the standard prodigiosin
molecule data and report the that the prodigiosin is produced by recombinant
strain

 

Budget Table

 

Sr.
No.

Items

Cost

Source

1

DNA isolation kit

280

Chromatrap

2

DNA transformation kit (customise)

400

Thermofisher

3

Southern Blotting kit (customise)

250

Thermofisher

4

Strains (Serratia Marcescens and E.
coli)

350

NCTC

5

Taq PCR Master
Mix Kit (customise)

£400

QIAGEN

6

Gel Extraction Kit

£389

QIAGEN

7

PCR Purification Kit
 

£389

QIAGEN

8

DNA Ligation Kit (K1423)

£265
 

ThermoFisher

9

Eppendorf Thermal Cycler (PCR)
 

£5941

Cole-Parmer
 

10

Gel electrophoresis system 

£488

Cole-Parmer
 

11

Centrifuge

£754

Cole-Parmer

12

Digital Compact Incubator
 

£931

Cole-Parmer
 

13

Ultra-Low Temperature Freezers
 

£6125

ThermoFisher

14

Refrigerator

500

Camlab

15

Pipettes

£270

Cole-Parmer

16

Microwave

£49

Russell Hobbs

17

Laminar
air flow

£1710

Wolflabs

18

incubator

£1070

Camlab

19

Rotary evaporator

£1100

Wolflabs

20

Chromatographic equipment’s

£500

Labclub

21

Lab fermenter

£1500-2000

Brewuk

22

Chemicals

4£000-5000

Sigma

 

Total

£29161

 

 

Funding body:

Biotechnology and Biological
Sciences Research Council (BBSRC)
is a UK Research
Council and  Non-Departmental Public Body (NDPB) is the
largest UK public funding organisation for non-medical bioscience. BBSRC mainly funds scientific research
institutes and university research
departments in the UK. BBSRC’s main motive is promotion, maintenance and funding, to high quality
straightforward, planned and practical research and associated postgraduate
exercise involving to the considerate and utilisation of biological sciences.
They offer a variety of funding chances
to allow individuals and groups to chase world class bioscience research. Likewise, BBSRC’s motive our project also involves,
planned and practical research and utilisation of biological sciences. These
properties eligible us to fit in there funding beneficiary. As prodigiosin and
its derivatives has many
applications with some of them are beneficial to human health, gives us the
reason to produce it on a larger scale with minimum cost so that everyone can
be benefited from it, which is the main aim of this project. These properties eligible us to fit in
there funding beneficiary.

 

Ethical Issues

 

As the basis of this research is on microbial cells such as Serratia Marcescens and E. coli, therefore all the experiments,
manipulation, cloning and expression etc. occurred on these above microbes.
Which has no concern of harming to animals, humans and environment, also
because of this research or any of its purpose there will be no dangerous
epidemic will arise. Therefore, there are no ethical issues associated with
this project.

Impact

Prodigiosin has many aplications such as,
antibacterial, antifungal, antiprotozoal, antimalarial, antiproliferative,
immunosuppressive and anticancer.
Because of this in recent years it has gained a lot of attention. If we strat
this project it will benefit to human health by dealing with many diseases.
Like Malaria is a very
dangerous disease and which is responsible for many deaths also effecting a lot
of individuals around the world particularly those who cannot acquire the
medications with affordable prize which helps them to resist the malaria
disease which can spread very rapidly if appropriate treatment is not taken.

Another major disease is cancer, which is one of the prominent
reason of deaths around the globe and it is considered as the second main
reason of deaths afterwards cardiovascular diseases. The medicines which are
used to inhibit or cure the cell growth or proliferation of cancer (tumour
cells) tissues are called as anticancer drugs. Prodigiosin has tremendous
anticancer drugs activity compare to other drugs. These medicines are used for
curing of different types of cancer as well as used in the combination with
surgery, chemotherapy, or radiation therapy. The worldwide market of anticancer
medicines was around US$ 85,000 million in year 2016-2017,

Because of the great price for the medication of cancer, it is very
common for a lowly people to be loss their life. It is very important to
management of circulation of the medication at the national level. Maintaining
the price of medication lower means, will not just promising a controllable
resource of the raw material for medicine production, but also in addition not
putting pressure on manufacturers to retain the price of the medication; both
of this can be accomplished by using new and advanced technique for the
production of medicines There are many other creative involvements are going on
in field of science which is most likely to change many level of developing and
providing the drug at More quantity and at less prize. Our project also aimed
in that direction so that common people will be benefited from it