BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

1. Transgenic. The genetically modified organism that contains a foreign gene/segment of DNA and expresses it is called a transgenic. 2. Gene cloning. Gene cloning refers to the technique of obtaining identical copies of a particular DNA segment or a gene. . Plas!i". Plasmid refers to the extra- chromosomal circular DNA molecule that !replicates autonomously "along #ith the chromosomal DNA$ in a bacterial cell. #. Reco!$inan% DNA &rDNA'. %t is the DNA formed by combining DNA from t#o different organisms. (. Recogni%ion si%e)se*+ence. The specific base sequence of DNA "of six base pairs$ #here the restriction en&yme cuts the DNA. ,. Palin"ro!es. Palindromes are groups of letters !that form the same #ord #hen read both for#ard and bac'#ard. -. S%ic./ en"s. (tic'y ends refer to the single stranded portions at the ends of DNA #hen cut by a restriction en&yme) 0. Elec%ro1ora%ion. *lectroporation is the process by #hich transient holes are produced in the plasma membrane of the "host$ cell to facilitate the entry of foreign DNA. 2. 3icroin4ec%ion. +icroin,ection is the process/ technique of introducing foreign genes into a host cell by in,ecting the DNA directly into the nucleus by using microneedle or micropipettes. 15. Gene g+n)Biolis%ics. %t is the technique of bombarding micropro,ectiles "gold or tungsten particles$ coated #ith the foreign DNA #ith great -elocity into the target cell. 11. Gene T6era1/. Gene therapy is the technique of replacement or alteration of a defecti-e gene responsible for a hereditary disease. 12. Reco!$inan% 1ro%ein. The protein produced by the expression of recombinant DNA in the transgenic organism is called recombinant protein. 1 . Biocon7ersion. .iocon-ersion refers to the process "es$ by #hich ra# materials are

biologically con-erted into specific products using microbial plant or animal cells or the en&ymes obtained from them. I3PORTANT NOTES 1. Bio%ec6nolog/. .iotechnology can be defined as the use of microorganisms plant or animal cells or their components to generate products and ser-ices "processes$ useful to human beings. +odem biotechnology is the term used in a restricted sense to refer to such processes/production technologies that in-ol-e genetic engineering. The *uropean /ederation of .iotechnology "*/.$ defines biotechnology as the integration of natural science and organisms cells parts there of and molecular analogues for products and ser-ices0 it encompasses both traditional -ie# and modem molecular biology. (ome of the biotechnological products and processes are) "i$ "ii$ "iii$ "i-$ "-$ 1ecombinant DNA -accines. (ynthesis of a gene and introduction of it into a target cell/organism. Gene therapy. %n -itro fertilisation for production of test-tube babies. Production of many biological compounds.

2. Princi1les o8 Bio%ec6nolog/. The t#o core2 techniques that enabled the birth of modern biotechnology are ) "i$ Genetic engineering - the technique of alterning the nature of genetic material and/or introduction of it into another host organism to change its phenotype. "ii$ Techniques to facilitate the gro#th and multiplication of only the desired microbes or cells in large number under sterile conditions for the manufacture of biotechnological products. The techniques of genetic engineering include) "i$ creation of recombinant DNA "r DNA$

"ii$ "iii$

use of gene cloning and gene transfer.

The first recombinant DNA #as constructed by (tanley 3ohen and 4erbert .oyer "5678$. They cut the piece of DNA from a plasmid carrying antibiotic-resistance gene in the bacterium Salmonella typhimureum and lin'ed it to the plasmid of *scherichia coli.

This rDNA #as introduced into * coli and made to multiply "gene cloning$ ma'ing a number of replicas. There are three steps in creating a genetically modified organism "G+9$ or transgenic0 they are) "i$ "ii$ "iii$ %dentification of DNA #ith desirable genes. %ntroduction of the identified DNA into a target/host cell. +aintenance of the introduced DNA in the host and transfer of the DNA to its progeny.

. Tools o8 Reco!$inan% DNA %ec6nolog/. The 'ey tools needed for the recombinant technology to be accomplished are) "i$ 3ell culture #ith desired DNA "iii$ DNA polymerase "-$ -ector "s$ #. Res%ric%ion en9/!es 1estriction en&ymes belong to a class of en&ymes called nucleases. They are of t#o 'inds ) "i$ "ii$ *xonucleases #hich remo-e nucleotides from the ends of DNA and *ndonucleases #hich cut the DNA at specific positions any#here in its length "#ithin$. (te#art :inn and ;erner Arber "56<=$ isolated t#o en&ymes from *coli that #ere responsible for restricting the gro#th of bacteriophage0 one of them added methyl groups to the DNA "modification en&yme$ and the other cut the DNA into segments and is called restriction endonuclease. "ii$ 1estriction en&ymes "i-$ :igases "-i$ 4ost organism/cell

49. (mith >; ;ilcox and T?. >elley "56<@$ isolated and characterised the first restriction endonuclease from Haemophilus influenzae bacterium and called it 4ind %%.

They obser-ed that 4ind %% al#ays cut the DNA molecule at a particular point by recognising a specific sequence of six base pairs called recognition sequence. The recognition sequence is a palindrome #here the sequence of base pairs reads the same on both the DNA strands #hen the orientation of reading is 'ept the same i.e. AB =B direction. or =B AB direction. e.g AB =B GAATT3 3TTAAG =B AB

*ach 1estriction endonuclease functions by inspecting the length of a DNA sequence and binds to the DNA at the recognition sequence. %t cuts the t#o strands of the double helix at specific points in their sugarphosphate bac' bones a little a#ay from the centre of the palindrome sites but bet#een the same t#o bases on both the strands.

As a result single-stranded portions called stic'y ends are produced at the ends of the DNA0 this stic'iness of the ends facilitates the action of en&yme DNA ligase.

;hen cut by the same restriction endonuclease the DNA fragments "of the donor as #ell as the host/recipient$ yield the same 'ind of !stic'y endsB #hich can be ,oined end-to-end by DNA ligases.

Naming of 1estriction en&ymes is as follo#s) The first letter of the name comes from the genus and the next t#o letters from the name of the species of the pro'aryotic cell from #hich they are isolated. The next letter comes from the strain of the pro'aryote. The roman numbers follo#ing these four letters indicate the order in #hich the en&ymes2 #ere isolated from that strain of the bacterium e.g. 5. *391 % is isolated from Escherichia coli, 1 C 5=. 8. 4ind %% is from Haemophilus influenzae

=. .am 4 % is from Bacillus amyloliquefaciens D. (al % is from Streptomyces albus A. Pst % is from Providencia Stuartii (. Cloning :ec%ors. Plasmids and bacteriophages are the commonly used -ectors. Presently genetically engineered/synthetic -ectors are also used for easily lin'ing the foreign DNA and selection of recombinants from non-recombinants. The follo#ing features are required to facilitate cloning in a -ector) "i$ 9rigin of replication "9ri$ "iii$ 3loning "1ecognition$ site "ii$ i(Electable mar'er : "i-$ (mall si&e of -ector.

Agrobacterium tumefaciens is the bacterium that infects a number of dicot plants and transfers a piece of its DNA 'no#n as T-DNA #hich transforms the normal plant-cells into tumour cells.

(imilarly retro-iruses also ma'e the normal animal cells into cancerous cells. The Ti plasmid "Tumour inducing Plasmid$ of Agrobacterium tumefaciens has been modified "does not cause tumour$ and used as a cloning -ector. The retro-iruses ha-e also been modified/disarmed and used as -ectors for transferring D animal cells.

,. Origin o8 Re1lica%ion &Ori'; This is a sequence of base pairs on DNA #here replication starts. Any piece of DNA lin'ed to this sequence can be made to replicate #ithin the host cells. This sequence is also responsible for controlling the copy number of the lin'ed DNA. -. Selec%a$le 3ar.er. A mar'er is a gene #hich helps in selecting those host cells #hich contain the -ector "transformant and eliminating the non-transformants. 3ommon selectable mar'ers for E.coli include the genes encoding resistance to antibiotics such as identified by a colour reaction. 0. Cloning Si%es.

The -ector should ha-e a fe# or at least one unique recognition site to lin' the foreign/ alien DNA. Presence of a particular recognition site enables the particular restriction en&yme to cut the -ector. %f a foreign DNA is ligated at the .arn 4 % site of tetracycline-resistance gene in the -ector p.1 =88 the recombinant plasmid loses the tetracycline resistance. %t can still be selected out from the non- recombinant ones by plating the transformants on ampicillin containing medium. Those transformants #hich gro# on ampicillin-containing medium are then transferred to a medium containing tetracycline. The recombinants #ill gro# on ampicillin- containing medium but not on tetracycline- containing medium but non-recombinants #ill gro# on both the media.

%n this case one antibiotic-resistance gene helps in selecting the transformants #hereas the other antibiotic resistance gene gets inacti-ated and helps in selection of recombinants.

Another method to differentiate bet#een recombinants and non-recombinants is on the basis of their ability to produce colour. %n this method a recombinant DNA is inserted #ithin the coding sequence of an en&yme galactosidase) This results into inacti-ation of the en&yme "insertional inacti-ation$. The bacterial colonies #hose plasmids do not ha-e an insert produce blue colour0 but those #ith an insert or the recombinants do not produce any colour.

2. Processes o8 Reco!$inan% DNA Tec6nolog/. 1ecombinant DNA technology in-ol-es the follo#ing steps )"i$ "ii$ "iii$ "i-$ "-$ %solation of DNA. /ragmentation of DNA by restriction endonucleases. %solation of the desired DNA fragment. Amplification of the gene of interest. :igation of the DNA fragment into a -ector using DNA ligase.

"-i$ "-ii$ "-iii$ "ix$ &i' -

Transfer of recombinant DNA into the host. 3ulturing the host cells on a suitable medium on a large scale. *xtraction of the desired product. Do#nstream processing of the product as a finished product ready for mar'eting. Isola%ion o8 DNA DNA has to be isolated in pure form for the action of restriction en&ymes. DNA can be released from the cells by digesting the cell en-elope by the use of en&ymes li'e lyso&yme for bacterial cells chitinase for fungal cells and cellulose for plant cells. (ince DNA is intert#ined #ith histone proteins and 1NAs proteins are remo-ed by treatment #ith proteases and 1NAs by ribonucleases. 9ther impurities are remo-ed by employing suitable treatments. The purified DNA is precipitated by the addition of chilled ethanol0 it is seen as fine threads in suspension.

&ii'

<rag!en%a%ion DNA. - /ragmentation of DNA is carried out by incubating the purified. DNA molecules #ith suitable restriction en&ymes at optimal conditions of temperature and p4.

&iii'

Isola%ion o8 DNA &gene' o8 In%eres%. The fragments of DNA are separated by a technique called gel electrophorosis. The DNA is cut into fragments by restriction endonucleases. These fragments are separated by a technique called gel electrophoresis. Agarose a natural polymer obtained from sea #eeds is used as the matrix. DNA fragments being negati-ely charged are separated by forcing them to mo-e through the matrix to#ards the anode under an electric field. The DNA fragments separate/resol-e according to their si&e. The separated molecules are stained by ethidium bromide and -isualised by exposure to FG -radiation as bright orange coloured bands. The separated bands of DNA "on the gel$ are cut from the gel and extracted

from the gel piece "elution$. &i7' (uch DNA fragments are purified and used for constructing recombinant DNA by ,oining them #ith cloning -ectors. A!1li8ica%ion o8 %6e DNA)gene o8 In%eres%. Amplification refers to the process of ma'ing multiple copies of the DNA segment in -itro. %t employs polymerase chain reaction "P31$. The process #as designed by >. +ullis. This technique in-ol-es three main steps) Denaturation Primer annealing and *xtension of primers. The double-stranded DNA is denatured by using high temperature. T#o sets of primers are used0 primers are the chemically synthesised short segments of DNA "oligonucleotides$ that are complementary to the segment of DNA "of interest$. &7' &7i' DNA polymerase is the en&yme used to ma'e copies of DNA ma'ing use of the genomic template DNA and the primer Liga%ion o8 %6e DNA 8rag!en% =i%6 %6e DNA o8 %6e 7ec%or. After cutting the source DNA segment and the -ector DNA "for ma'ing the space for source DNA$ the t#o are mixed and incubated #ith ligase under suitable conditions. This results in the formation of recombinant DNA "rDNA$ Trans8er o8 reco!$inan% DNA in%o %6e 6os%. The bacterial cells must be made competent to ta'e up DNA0 this is done by treating them #ith a specific concentration of calcium that increases the efficiency #ith #hich DNA enters the cell through the pores in its cell #all. 1ecombinant DNA can then be forced into such cells by incubating the cells #ith recombinant DNA on ice follo#ed by placing them at D8H3 and then putting them bac' on ice. +icroin,ection is a method in #hich the recombinant DNA is directly in,ected

into the nucleus of the animal cell #ith the help of microneedles or micropipettes. Gene gun or biolistics is a method suitable for plant cells #here cells are bombarded #ith high- -elocity microparticles of gold or tungsten coated #ith DNA. &7ii' &7iii' &i>' Disarmed pathogens are used as -ectors0 #hen they are allo#ed to infect the cell they transfer the recombinant DNA into the host. C+l%+ring %6e %ransgenic cell. The cell containing the foreign gene "trans gene$ is cultured on a suitable medium. The cells multiply and ma'e clones. E>%rac%ion o8 %6e "esire" 1ro"+c%. The transgene expresses itself in the form of protein "s$ under appropriate condition. The product "s$ can be extracted from the medium by employing suitable procedure. .ioreactors are used for processing large -olumes of culture for obtaining the product of interest in sufficient quantities. Do=ns%rea! 1rocessing The product obtained is sub,ected to a series of processes "collecti-ely called do#nstream processing$ before it is made into a finished product ready for mar'eting. The t#o main processes are "a$ separation and "b$ purification. The product is then formulated #ith suitable preser-ati-es. (uch formulations ha-e to undergo clinical trials in case of drugs. The bioreactors can be thought of as -essels in #hich ra# materials are biologically con-erted into specific products by microbes plant and animal cells and/or their en&ymes. The bioreactor pro-ides optimum gro#th conditions and facilitates achie-ing the desired product.

15. Bioreac%ors.

The most commonly used bioreactor is of stirring type. A stirred-tan' bioreactor is usually a cylindrical -essel or -essel #ith a cur-ed base to facilitate of the contents. %n the sparged stirred-tan' bioreactor sterile air bubbles are sparged.

:er/ S6or% Ans=er T/1e ?+es%ions 1.1 A technique used in ma'ing copies of a specific segment of DNA in-ol-es. "i$ "ii$ "iii$ "i-$ ligase chain reaction transcription polymerase chain reaction translation

1.2 Define biotechnology 1. Name the class of en&ymes to #hich restriction en&ymes belong. 1.# Define gene cloning.