Design Problem I

8/7/2019 Design Problem I

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Design Problem Title/No.: II Course Code

Course Instructor: Nishtha mam Course Tutor (if app

Date of Allotment: 29-01-2011 Date of submissi

Students Roll No.: RA7801B22 Section No.: A7801

Declaration:

I declare that this assignment is my individual work. I have not copied from any other

students work or from any other source except where due acknowledgment is made

explicitly in the text, nor has any part been written for me by another person.

Students Signature: Nitin Thukral

Evaluatorscomments:

________________________________________________________________

Marks obtained: ___________ out of ______________________

Content of Homework should start from this page only:

Design an experiment to express a eukaryotic protein in a prokaryotic system.

The experiment should involve the following steps:

i) Selection of suitable vector (Justify the reason to prefer one type of vector over

the other)

ii) Extraction of foreign DNA

iii) Method to screen recombinant vector/ transformed host cell

iv) Steps to overcome the limitations associated with use of prokaryotic system

v) Method to check the expression and function of desired protein.

Solution:

The central dogma ofmolecular biology deals with the detailed residue-by-residue

transfer ofsequential information. It states that information cannot be transferred back

from protein to either protein or nucleic acid.
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Sequentialhttp://en.wikipedia.org/wiki/Informationhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Sequentialhttp://en.wikipedia.org/wiki/Information


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Transcription of DNA to RNA to protein: This dogma forms the backbone of

molecular biology and is represented by four major stages.

1. The DNA replicates its information in a process that involves many enzymes:

replication.

2. The DNA codes for the production of messenger RNA (mRNA) du

transcription.

3. In eukaryotic cells, the mRNA is processed (essentially by splicing) and migrates

from the nucleus to the cytoplasm.

4. Messenger RNA carries coded information to ribosomes. The ribosomes "read" this

information and use it for protein synthesis. This process is called translation.

In this manner, a gene may be produced, which contains the. coding region from one

organism joined to regulatory sequences from another organism; such a gene is calledchimeric gene.
http://www.accessexcellence.org/RC/VL/GG/collaboration.phphttp://www.accessexcellence.org/RC/VL/GG/rna_synth.phphttp://www.accessexcellence.org/RC/VL/GG/rna_synth.phphttp://www.accessexcellence.org/RC/VL/GG/protein_synthesis.phphttp://www.accessexcellence.org/RC/VL/GG/collaboration.phphttp://www.accessexcellence.org/RC/VL/GG/rna_synth.phphttp://www.accessexcellence.org/RC/VL/GG/rna_synth.phphttp://www.accessexcellence.org/RC/VL/GG/protein_synthesis.php


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Recombinant DNA molecules are produced with one of the following thr

objectives:

(1) to obtain a large number of copies of specific DNA fragments,

(2) to recover large quantities of the protein produced by the concerned gene, or

(3) to integrate the gene in question into the chromosome of a target organism where it

expresses itself. Even for the latter two objectives, it is essential to first obtain a large

number of copies of the concerned genes.

To achieve this, the DNA segments are integrated into a self-replicating DNA

molecule called vector; most commonly used vectors are either bacterial plasmids or

DNA viruses. This is achieved by using specific enzymes for cutting the DNA(restriction enzymes) into suitable fragments and then for joining together the

appropriate fragments (ligation). In nature, gene transfers are rather imprecise, and

their range, in terms of species involved, is remarkably limited. The above problems

are circumvented by the recombinant DNA technology.

The entire procedure of cloning or recombinant DNA technology may be classified

into the following five steps for the convenience in description and on the basis of the

chief activity performed.

1.Identification and isolation of the desired gene or DNA fragment to be cloned.

2. Insertion of the isolated gene in a suitable vector.

3. Introduction of this vector into a suitable organism/cell called ho

(transformation).

4. Selection of the transformed host cells.

5. Multiplication/expression/integration followed by expression of the introduced

gene in the host.

The protein Taken is Somatostatin (also known as growth hormone-inhibiting

hormone (GHIH) orsomatotropin release-inhibiting factor (SRIF)) is a peptide

hormone that regulates the endocrine system and affects neurotransmission and cell

proliferation via interaction withG-protein-coupledsomatostatin receptorsandinhibition of the release of numerous secondary hormones.
http://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Endocrine_systemhttp://en.wikipedia.org/wiki/Neurotransmissionhttp://en.wikipedia.org/wiki/Cell_proliferationhttp://en.wikipedia.org/wiki/Cell_proliferationhttp://en.wikipedia.org/wiki/G-protein-coupledhttp://en.wikipedia.org/wiki/Somatostatin_receptorshttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Peptide_hormonehttp://en.wikipedia.org/wiki/Endocrine_systemhttp://en.wikipedia.org/wiki/Neurotransmissionhttp://en.wikipedia.org/wiki/Cell_proliferationhttp://en.wikipedia.org/wiki/Cell_proliferationhttp://en.wikipedia.org/wiki/G-protein-coupledhttp://en.wikipedia.org/wiki/Somatostatin_receptors


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Somatostatin has two active forms produced by alternative cleavage of a single

preproprotein: one of 14 amino acids, the other of 28 amino acids.

In all vertebrates, there exists six different somatostatin genes which have been named

SS1, SS2, SS3, SS4, SS5, and SS6. Tetrapods only possess SS1 and SS2, while teleost

fish possess SS1 - SS6. The six different genes along with the five different

somatostatin receptors allows somatostatin to possess a large range of functions.

GENERAL PRCOEDURE

hGH (human growth hormone) used to be obtained from dead people's pituitary gland,

which was unsafe; then it started being 'manufactured' by genetic recombination.

Suppose you want to 'manufacture' a human protein in large amounts using bacteria.

1. First, take into account the differences between bacterial and eukaryotic molecular

processes. In particular, most human genes contain introns that must be spliced out of

the mRNA before it gets translated into a protein, but that process does not occur in

bacteria. So if we just put the human gene into bacteria, they will produce a defectiveprotein (one with all the pieces that should be taken out still left in).
http://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Vertebrateshttp://en.wikipedia.org/wiki/Tetrapodshttp://en.wikipedia.org/wiki/Teleosthttp://en.wikipedia.org/wiki/Somatostatin_receptorshttp://annonc.oxfordjournals.org/content/17/12/1733/F1.large.jpghttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Vertebrateshttp://en.wikipedia.org/wiki/Tetrapodshttp://en.wikipedia.org/wiki/Teleosthttp://en.wikipedia.org/wiki/Somatostatin_receptors


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So what we can do is isolate the mRNA and use reverse transcriptase to produce a

cDNA copy of the gene (since it is made from mature mRNA, the introns will have

already been spliced out). You can amplify the amount of cDNA using PCR, if

needed.

2. Then you create an expression vector. Suppose we use a bacterial plasmid.

a. Choose a plasmid that has resistance to one or more antibiotics so that we can

screen them.

b. Use a single restriction endonuclease (restriction enzyme) to digest both your

cDNA and the plasmid. Splice onto the end of your cDNA the sequences needed for

bacteria to transcribe and translate your gene. If we don't, we will amplify your DNA,

but it won't be expressed - it won't produce proteins in the bacteria. Then mix the two

sets of DNA fragments together with DNA ligase. Different combinations of DNA

will result: plasmid-plasmid, plasmid-cDNA, and cDNA-cDNA. You isolate the

recombinant DNA -- the plasmid-cDNA combinations.

3. Then introduce the recombinant plasmids into the bacteria. One method of doing

this is simply transformation.

4. You grow the bacteria on a medium that has the antibiotic that the plasmid provides

resistance to. That way, only the bacteria that have taken up your recombinant DNA

will survive.

5. The bacteria produce your protein along with others.

a. Vectors - A Vector is a DNA molecule that has the ability to replicate in an

appropriate host cell, and into which the DNA insert is integrated for cloning.

Therefore, a vector must have an origin of DNA replication (denoted as ori) that

functions in the host cell. Any extrachromosomal small genome, e.g., plasmid,

phage and virus, may be used as a vector.

pBR322 is a plasmid and for a time was one of the most commonly used E.

colicloningvectors. Created in 1977, it was named eponymously after its

Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez.

pBR322 is 4361 base pairs in length and contains a replicon region (source

plasmid pMB1), the ampR gene, encoding the ampicillinresistance protein

(source plasmidRSF2124) and thetetR gene, encoding thetetracycline

resistance protein (source plasmid pSC101). The plasmid has unique restriction
http://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Vector_(molecular_biology)http://en.wikipedia.org/wiki/Ampicillinhttp://en.wikipedia.org/wiki/Antibiotic_resistancehttp://en.wikipedia.org/w/index.php?title=RSF2124&action=edit&redlink=1http://en.wikipedia.org/wiki/Tetracyclinehttp://en.wikipedia.org/wiki/PSC101http://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Vector_(molecular_biology)http://en.wikipedia.org/wiki/Ampicillinhttp://en.wikipedia.org/wiki/Antibiotic_resistancehttp://en.wikipedia.org/w/index.php?title=RSF2124&action=edit&redlink=1http://en.wikipedia.org/wiki/Tetracyclinehttp://en.wikipedia.org/wiki/PSC101


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sites for more than forty restriction enzymes. 11 of these 40 sites lie within the

tetR gene. There are 2 sites forrestriction enzymes HindIII and ClaI within the

promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene.

The origin of replication orori site in this plasmid is pMB1 (a close relative of

ColE1). The ori encodes two RNAs (RNAI and RNAII) and one protein (called

Rom or Rop).

The circular sequence is numbered such that 0 is the middle of the unique

EcoRI site and the count increases through the tet genes. The ampicillin

resistance gene is a penicillin beta-lactamase. Promoters P1 and P3 are for the

beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by

the ligation of two different DNA fragments to create pBR322. P2 is in the

same region as P1, but it is on the opposite strand and initiates transcription in

the direction of the tetracycline resistance gene. The molecular weight is 2.83 x

106 daltons.
http://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/HindIIIhttp://en.wikipedia.org/w/index.php?title=ClaI&action=edit&redlink=1http://en.wikipedia.org/wiki/Restriction_siteshttp://en.wikipedia.org/wiki/Ori_(genetics)http://en.wikipedia.org/wiki/Transcription_(genetics)http://en.wikipedia.org/wiki/Restriction_enzymeshttp://en.wikipedia.org/wiki/HindIIIhttp://en.wikipedia.org/w/index.php?title=ClaI&action=edit&redlink=1http://en.wikipedia.org/wiki/Restriction_siteshttp://en.wikipedia.org/wiki/Ori_(genetics)http://en.wikipedia.org/wiki/Transcription_(genetics)


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b.

The isolated DNA is then treated with the suitable Restriction enzyme to extract the

foreign DNA.


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The diagram below illustrates the DNA nucleotide sequence for the humansomatostatin gene insert. This diagram indicates where EcoRI and BamHIeach cut the human DNA sequence.

Recombinant somatostatin was made using EcoRI and BamHI to remove apiece of the pBR322 plasmid. The human gene insert that was cut with thesesame restriction enzymes was inserted into the plasmid. The diagram belowillustrates how the human somatostatin DNA and the pBR322 plasmidDNA fit together using the sticky ends produced by these two enzymes.

Human somatostatin gene in bold Plasmid DNA in not in bold


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c. Screening

The pBR322 plasmid possesses genes coding for ampicillin and tetracyclin

resistance. These gene markers facilitate the detection of bacteria that take up the

recombinant plasmid possessing the human gene for somatostatin. The transformed

bacteria are plated on agar containing one of the antibiotics. After growth,

replica plating is used to test the colonies for resistance to the other antibiotic.

a. The boxed colony represents a transformed colony of E. coli cells. Cellsthat were not transformed with the recombinant plasmid are able to grow inthe presence of either antibiotic.


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a. The boxed colony represents a transformed colony ofE. coli cells. Cells that were

not

transformed with the recombinant plasmid are able to grow in the presence of either

antibiotic.

b. The restriction enzyme PstI would cut the ampicillin gene in the plasmid, rendering

it

inactive.

c. Therefore, cells that were transformed with the recombinant plasmid would be able

to growin the presence of tetracycline, but not in the presence of ampicillin.

a. The boxed colony represents a transformed colony ofE. coli cells. Cells that were

not

transformed with the recombinant plasmid are able to grow in the presence of eitherantibiotic.

b. Either of the restriction enzymes BamHI and Sal1 would cut the tetracycline gene in

the

plasmid, rendering it inactive.

c. Therefore, cells that were transformed with the recombinant plasmid would be able

to grow

in the presence of ampicillin, but not in the presence of tetracycline.


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Prokaryotic Protein Expression Systems

Prokaryotic recombinant protein expression systems have several advantages. These

include ease of culture, and very rapid cell growth meaning you won't have to wait

long to get protein from bacterial systems once you clone your cDNA. Expression can

be induced easily in bacterial protein expression systems using IPTG. Alpurification is quite simple in prokaryotic expression systems and there are a plethora

of commercial kits available for recombinant protein expression.

In the 1970s, when recombinant DNA technology found its way into molecular

biology laboratories, the bacterial cell was prevalently presented as a universal host

for heterologous protein expression. However, due to their inability to adequately

process complex proteins and due to their insufficient protein secretion capabilities

prokaryotic expression systems nowadays are mainly used for the production of rather

uncomplex proteins and peptides. In order to produce complex human recombinant

proteins a reinforced development of eukaryotic expression systems has proceeded inthe last decades, which was mainly based on yeast cells and mammalian cells. As a

result, mammalian cell-based biopharmaceuticals account for almost 60% of today's

biopharmaceutical market.

Also if you need to use your proteins for functional or enzymatic studies prokaryotic

systems are a problem as most proteins become insoluble in inclusion bodies and are

very difficult to recover as functional proteins. Furthermore, most if not all post-

translational modifications are not added by bacteria and therefore your protein of

interest may not be functional. Enzymatic studies thus may be unfruitful.


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Various different expression systems which have been technologically used for

recombinant protein production were evaluated: an inducible prokaryotic expression

system (Escherichia coli) for intracellular human superoxide dismutase, an E. coli

secretion system for antibody Fab fragments, a constitutive eukaryotic secretion

system (Pichia pastoris) for human trypsinogen and antibody Fab fragments as wellas an intracellularP. pastoris system, and a constitutive eukaryotic expression system

(CHO) for an EPO/Fc fusion protein and human monoclonal antibodies. W

developed production scenarios for each expression system and compared specific

growth and productivity as well as product secretion rates to determine the full

potential in a bioprocess.

(e) When you want to characterise a gene or protein of interest, you must first study itsfunction. In this molecular era, obtaining a cDNA of your gene of interest is not

difficult.

To express the cDNA as a protein, ie. a recombinant protein, one can then easily

perform functional studies using the recombinant purified protein.

Once you have a purified protein you can conduct:

Protein-protein Interaction Experiments

Enzyme Kinetics

Functional Studies of the Protein

Structural Studies - including protein crystallization, protein structure and

NMR.

You can also use the protein to make antibodies for further experiments.

There are two main systems for the expression of recombinant protein. Once you get

your cDNA cloned, you must decide where you want to amplify your protein. This

will be either a prokaryotic (bacterial) or eukaryotic (usually yeast or mammalian cell)

system. The choice of your system will decide which vector you will need to clone

your cDNA into as there are different promoters which function in E.Coli and others

that work best with yeast or mammalian systems.

The northern blot is a technique used in molecular biology research to study gene

expression by detection ofRNA (or isolated mRNA) in a sample. A general blotting

procedure starts with extraction of total RNA from a homogenized tissue sample. The

mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to

maintain only those RNAs with a poly(A) tail. RNA samples are then separated by gel

electrophoresis. Since the gels are fragile and the probes are unable to enter the
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/MRNAhttp://en.wikipedia.org/wiki/Chromatographyhttp://en.wikipedia.org/wiki/Poly(A)_tailhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/MRNAhttp://en.wikipedia.org/wiki/Chromatographyhttp://en.wikipedia.org/wiki/Poly(A)_tail


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matrix, the RNA samples, now separated by size, are transferred to a nylon membrane

through a capillary or vacuum blotting system.

Hybridization is the technique in which the nucleic acid or protein immobilized on

the membrane is challenged with a probe or antibody known. Hybridization is widely

used to confirm the presence or absence of the DNA/RNA/protein in the unknown

sample. Hybridization depends on the function of the labelled base pair between

theProbe and the target sequences.

Gene expression levels are commonly determined using northern blot analysis.

However, this technique is time-consuming and requires a large quantity of RNA.

RT-PCRconverts RNA into first strand cDNA, which is then used as a template for

PCR. RT-PCR is more rapid and sensitive and can be more specific than northern blot

analysis.

For this purpose mRNA molecules are first converted into cDNA sequences by means

of enzymic reactions involving the use of Reverse transcriptase. The resulting cDNAs

are then subjected to ordinary PCRamplification reactions. This technique is called

also mRNA phenotyping orMessage amplification phenotyping since the use of

different primers specific for individual mRNAs can provide a snapshot picture of

gene expression within cells at a given time.

In all cases reaction mixtures obtained from amplification are subjected to gel

electrophoresis and amplified bands can then be visualized by appropriate staining of

the DNA.
http://www.copewithcytokines.org/cope.cgi?key=cDNAhttp://www.copewithcytokines.org/cope.cgi?key=PCRhttp://www.copewithcytokines.org/cope.cgi?key=mRNA%20phenotypinghttp://www.copewithcytokines.org/cope.cgi?key=message%20amplification%20phenotypinghttp://www.copewithcytokines.org/cope.cgi?key=Gene%20expressionhttp://www.copewithcytokines.org/cope.cgi?key=cell%20typeshttp://www.copewithcytokines.org/cope.cgi?key=cDNAhttp://www.copewithcytokines.org/cope.cgi?key=PCRhttp://www.copewithcytokines.org/cope.cgi?key=mRNA%20phenotypinghttp://www.copewithcytokines.org/cope.cgi?key=message%20amplification%20phenotypinghttp://www.copewithcytokines.org/cope.cgi?key=Gene%20expressionhttp://www.copewithcytokines.org/cope.cgi?key=cell%20types

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Design Problem I