A Practical Textbook of Genetic Engineering in Bacteria P.V.G.K. Sarma

A Practical Textbook of Genetic Engineering in Bacteria

A Practical Textbook of Genetic Engineering in Bacteria

Dr.P.V.G.K.Sarma Head, Department of Biotechnology ^ƌŝsĞŶŬĂƚĞƐǁĂƌĂ/ŶƐƟƚƵƚĞŽĨDĞĚŝĐĂů^ĐŝĞŶĐĞƐĂŶĚhŶŝǀĞƌƐŝƚLJ dŝƌƵƉĂƟϱϭϳϱϬϳŶĚŚƌĂWƌĂĚĞƐŚ͕/ŶĚŝĂ

Chennai

Trichy

NewDelhi

right r Copy & cy e Pira Exclud ġ ţź ŵŦŤŵŦť Ū Ŵ ġ ű ų Ű ŤŵŪŰůġ Űŧ ġ Ŭ Ű Ű ŕ ũ Ū Ŵ ġ ţ ŵ įġ œ Ŧ ű ų Ű ť Ŷ ŭŶťŪůŨġ Ũũ ůŤ Ť Ű ű ź ų Ū ŪůġŢůźġŧŰųŮġŪ ŦġťŰůŦġ ŵġţ ųŵġ ŢůźġűŢ źŪůŨġŴũŢŭŭġůŰ ůġŧųŰŮġ Űű ŵŪŰ Ť Ţ ŵŰ ŪŻ ų Ű Ű ũ ű ŪŵũġŢŶŵũ Ÿ ŵġ ű Ŧ ŦŹŤ ŭŪŴũŦųį ŵũŦġűŶţ

Honou

ISBN 978-93-88694-41-4

MJP Publishers

All rights reserved No. 44, Nallathambi Street, Printed and bound in India Triplicane, Chennai 600 005 MJP 763

© Publishers, 2020

Publisher: C. Janarthanan Project Editor: C. Ambica

PREFACE Genetic manipulation of bacteria is exploited largely in the laboratory because of their simple genetics and as they are easy to grow. These bacteria are predominantly important in producing large amounts of pure ”‡…‘„‹ƒ– Š—ƒ ’”‘–‡‹• ™Š‹…Š …ƒ „‡ —•‡† ‹ ‡†‹…‹‡Ǥ Š‡ ϐ‹”•– example of this kind was shown in 1978 by Herbert Boyer working in a laboratory in University of California. He expressed human insulin gene in Escherichia coli. This product was approved by U.S. Food and Drug Administration for treating diabetes. The ease with which the bacteria could be manipulated has helped researchers in obtaining important molecules ™Š‹…Š ‘–Š‡”™‹•‡ ƒ”‡ ˜‡”› †‹ˆϐ‹…—Ž– –‘ ’—”‹ˆ› ˆ”‘ –Š‡ ‘”‰ƒ‹• ‹ Žƒ”‰‡ amounts to be used in therapeutic purposes. The genetic sequences from a broad range of organisms can be easily ligated to a plasmid and transformed into bacteria for both storage and ‘†‹ϐ‹…ƒ–‹‘Ǥ ƒ…–‡”‹ƒ ƒ”‡ •‹’Ž‡ –‘ ‰”‘™ǡ †‹˜‹†‡ “—‹…Ž›ǡ ‡ƒ•› –‘ –”ƒ•ˆ‘”ǡƒ†…ƒ„‡•–‘”‡†ƒ–Ǧͺ͸鏑”‡‘”Ž‡••‹†‡ϐ‹‹–‡Ž›ǤŠ‡‘‡–ƒ gene is isolated it is very easy to store in bacteria and this provides limitless supply of active material for research. This has led to the development of huge number of recombinant plasmids for manipulating DNA/genes of different prokaryotic and eukaryotic organisms. The speed with which genetic tools were developed by scientists for —†‡”•–ƒ†‹‰‰‡‡ˆ—…–‹‘ǡ‹–•‡˜‘Ž—–‹‘ǡ˜ƒ”‹ƒ–‹‘‹–Š‡‰‡‡•‡“—‡…‡ is the causative reason for the disease or its role in the pathogenesis or its role in the development of therapeutic products. The majority of DNA exploitation takes place within bacterial plasmids/phages before transfer to another host. In this quest Escherichia coli became the organism which Š‡Ž’‡†•…‹‡–‹•–•–‘‡š’Ž‘‹–ǡ‡ƒ•‹Ž›ƒ‹’—Žƒ–‡ƒ†…‘„‹‡‰‡‡•–‘…”‡ate novel or disrupted proteins and follow the effect on various molec-

Preface

—Žƒ” •›•–‡•Ǥ ‡•‡ƒ”…Š‡”• Šƒ˜‡ Œ‘‹‡† ‰‡‡• ˆ”‘ „ƒ…–‡”‹ƒ ƒ† ƒ”…Šƒ‡ƒǡ paving way to understand the importance of the gene(s) of that organism ‹ †‹ˆˆ‡”‡– …‘†‹–‹‘•Ǥ  –Š‹• …‘–‡š–ǡ –Š‡ ‡š’‡”‹‡–• –Šƒ– Š‡Ž’ –‘ —derstand the DNA/gene sequence(s) are explained in this book. The experiments mentioned in this book are easy to perform and one can get answers quickly and can purify recombinant proteins rapidly so that they can be used readily for obtaining solutions to various problems. I hope that by following these procedures students can enjoy doing experiments in genetic engineering and learn the beauty of biology.

Dr. P.V.G. K. SARMA

– vi –

CONTENTS Chapter 1 Chapter 2

Cloning of DNA Fragments into Plasmid Vectors

1

Cloning of DNA fragments into Phage Vectors

51

Chapter 3

Cloning in cosmid vectors

103

Chapter 4

Techniques to understand the functionality of a gene

117

Glossary

147

Index

157

Chapter 1

CLONING OF DNA FRAGMENTS INTO PLASMID VECTORS BACKGROUND Vectors are described as double-stranded DNA molecules having origin of ”‡’Ž‹…ƒ–‹‘‘ˆƒ’ƒ”–‹…—Žƒ”„ƒ…–‡”‹ƒŽ‘”‰ƒ‹•ǡ•‡Ž‡…–‹‘ƒ”‡”ˆ‘”‹†‡tifying the vector in the host upon transformation and multiple cloning •‹–‡•…‘’”‹•‹‰‘ˆ•‡˜‡”ƒŽ—‹“—‡–›’‡ ”‡•–”‹…–‹‘‡†‘—…Ž‡ƒ•‡•‹–‡•ǡ into which on ligating a DNA insert having same ends can result in the inactivation of the marker helping us to identify recombinants and non-recombinants. In bacteria these properties are served by: plasmid molecules - an ext”ƒ…Š”‘‘•‘ƒŽ‡Ž‡‡–ǡbacteriophages and transposons.

PLASMIDS All bacteria possess extrachromosmal elements which are covalently …Ž‘•‡†…‹”…—Žƒ”‘Ž‡…—Ž‡•”ƒ‰‹‰ˆ”‘ǡηͳ„–‘ͷͲͲ„‹•‹œ‡ǡƒ† genes that makes the bacteria to survive in adverse conditions which otherwise would have killed the bacteria. These characters are encoded on the plasmids and they can be: ‡œ›‡•ǡ–‘š‹•ǡ˜‹”—Ž‡…‡ˆƒ…–‘”•ƒ†’”‘teins; these structural genes are expressed in the bacteria in those particular environmental conditions. These plasmid molecules also move from ‘‡„ƒ…–‡”‹—–‘ƒ‘–Š‡”„ƒ…–‡”‹—–Š”‘—‰ŠŠ‘”‹œ‘–ƒŽ–”ƒ•ˆ‡”‘ˆ‰‡‡–‹… material from host to the recipient and thus spreading these characters. ‘”‡šƒ’Ž‡ǡ”‡•‹•–ƒ…‡–‘ȾǦŽƒ…–ƒƒ–‹„‹‘–‹…•‹•’”‡•‡–‘’Žƒ•‹†ǡ•‹-

A Practical Textbook of Genetic Engineering in Bacteria

ilarly vancomycin resistance genes VanA and VanB are encoded on plas‹†•ǤŠ‹•Š‘”‹œ‘–ƒŽ–”ƒ•ˆ‡”‹•‡†‹ƒ–‡†„›…‘Œ—‰ƒ–‹‘ǡ–”ƒ•ˆ‘”ƒ–‹‘ and transduction.

Conjugation Plasmid possess an unique genetic transfer mechanism referred as conŒ—‰ƒ–‹‘ǡ‹™Š‹…Š‘‡…‘’›‘ˆ–Š‡plasmid DNA from donor is transferred to the recipient through conjugation tube as in Gram-negative bacteria or through surface contacts between bacteria in case of Gram-positive bacteria. Plasmids which transfer genetic material through conjugation phenomenon are referred as conjugative plasmids while plasmids that do not possess this feature are referred as non-conjugative plasmids. Conjugative plasmids contain two exclusive regions on the plasmid which helps the „ƒ…–‡”‹ƒ –‘ ƒ†ƒ’– –Š‡ …‘Œ—‰ƒ–‹˜‡ ’Š‡‘‡‘ǡ –Š‡•‡ ƒ”‡ǣ ‹Ǥ –”ƒ•ˆ‡” genes comprising of a cluster of 12 genes and ii. nic/bom regions which facilitate the transfer of DNA through conjugation tube in Gram-negative bacteria while through surface contacts in Gram-positive bacteria. Mechanism of Conjugation This is a process in which the expressed ’”‘–‡‹•ˆ”‘–”ƒ•ˆ‡”‰‡‡•”‡…‘‰‹œ‡•–Š‡†‘‘”„ƒ…–‡”‹ƒƒ†•›–Š‡•‹œes conjugation tube and allows the plasmid to divide into two copies and one copy passes through the conjugation tube from donor to the recipient and this process is facilitated by the proteins formed from nic/bom regions. This phenomenon is called as conjugationǤŠ‹•Š‘”‹œ‘–ƒŽ‰‡‡–”ƒ•ˆ‡” is a critical phenomenon in which drug resistant plasmids are transferred among „ƒ…–‡”‹ƒ–Š—•ǡ•’”‡ƒ†‹‰†”—‰”‡•‹•–ƒ…‡ƒ…”‘••–Š‡‰Ž‘„‡ǤŠ‹•’Š‡nomenon is usually observed in Gram negative bacteria while in Gram positive bacteria there is no …‘Œ—‰ƒ–‹‘–—„‡ˆ‘”ƒ–‹‘ǡŠ‘™‡˜‡”–Š‡–”ƒ•ˆ‡” is primarily through surface contact between recipient and donor. Here –”ƒ•ˆ‡”‰‡‡•–”ƒǡ–”ƒǡ–”ƒǡ–”ƒ ǡ–”ƒ ǡ–”ƒƒ†–”ƒˆƒ…‹Ž‹–ƒ–‡–Š‡…Ž‡ƒ˜age of one strand of donor plasmid molecule which forms complex with –”ƒ ƒ†–”ƒ…”‡ƒ–‡•™ƒ›ˆ‘”–Š‡plasmid DNA to move from the pores created by traG (muramidase) and thus pushes the plasmid DNA to move with –Š‡Š‡Ž’‘ˆ–”ƒƒ†–”ƒ–‘”‡…‹’‹‡–bacteria.

–2–

Cloning of DNA Fragments into Plasmid Vectors

Transduction Transduction is the genetic transfer of bacterial DNA from one bacterium (the donor) to another bacterium (the recipient) by a bacteriophage. Transducing bacteriophages are formed in donor bacterial cells during phage development. Transduction is of two kinds: a) Generalized Transduction: In this phenomenon the transducing phage carries a random fragment of host chromosomal DNA approximately the same length as that of phage DNA. b) Specialized Transductionǣ •’‡…‹ƒŽ‹œ‡†–”ƒ•†—…–‹‘ǡ ’Šƒ‰‡••—…Šƒ•ƒ„†ƒ„ƒ…–‡”‹‘’Šƒ‰‡ƒ”‡ƒ„Ž‡–‘–”ƒ•ˆ‡”‘Ž›…‡”–ƒ‹„ƒ…terial genes which are near the prophage (when a bacteriophage genome is inserted and integrated into the circular bacterial DNA chromosome and stays as a part of bacterial chromosome; and is also referred as lysogen) insertion site in the bacterial genome. These may be included as a single hybrid DNA molecule of phage genome. These hybrid molecules which are formed by rare aberrant excision of an integrated prophage from a lysogen can transfer the DNA once the phage genome gets integrated into the host „ƒ…–‡”‹ƒŽ ‰‡‘‡Ǥ Š—•ǡ •’‡…‹ƒŽ‹œ‡† –”ƒ•†—…–‹‘ …ƒ „‡ …ƒ””‹‡† ‘Ž› „› temperate phages whose lysogens involve reversible integration of the phage genome with that of the host bacterium. This is routinely used to construct mutant „ƒ…–‡”‹ƒ‡š’”‡••‹‰—‹“—‡‡œ›‡ȋ•Ȍǡˆ‘”‡šƒ’Ž‡—•ing T7 RNA polymerase.

TRANSFORMATION OF PLASMID DNA INTO E.COLI DH5α CELLS Transformation is a natural phenomenon when the genetic material (DNA) passes through the prevailing medium from donor into recipient organ‹•ǡƒ†Š‡”‡–”ƒ•ˆ‡”‹•‡–‹”‡Ž›†‡’‡†‡–‘–Š‡”‡…‹’‹‡–‘”‰ƒ‹•ǤŠ‡ƒ…“—‹”‡†‡š’”‡••‡•Š‡”‹–ƒ„Ž›‹•‹†‡–Š‡”‡…‹’‹‡–‘”‰ƒ‹•ǡ this is then referred as transformation.

–3–

A Practical Textbook of Genetic Engineering in Bacteria

Calcium Chloride Mediated Transformation Principle ‡†‡Žƒ†‹‰ƒ‘”‹‰‹ƒŽŽ›†‡˜‡Ž‘’‡†–Š‡’”‘…‡†—”‡ǡ™Š‹…Š‹˜‘Ž˜‡•‡š’‘sure of the growing bacteria to a hypotonic solution of calcium chloride at Ͳͼǡ…ƒ—•‹‰–Š‡bacteria to swell (spheroplast formation). DNA added to the transformation mixture forms a DNase resistant complex of hydroxyl calcium phosphate that adheres to the cell surface. The cell during brief ‡š’‘•—”‡–‘ͶʹͼŠ‡ƒ–’—Ž•‡…ƒ–ƒ‡–Š‹•…‘’Ž‡šǤˆ–‡”‰”‘™‹‰ˆ‘”ˆ‡™ Š‘—”•‹‡†‹—‹‘”†‡”–‘”‡˜‹˜‡‹–•…‡ŽŽ™ƒŽŽǡ‡–…Ǥǡ’Žƒ–‹‰‘•‡Ž‡…–‹˜‡ medium screens the transformants. In order to get proper transformation the E. coli‹•‰”‘™‹ƒ‡†‹—‡”‹…Š‡†™‹–Š‰2+ǡƒ†‰Ž—…‘•‡ǡˆ‡™‡” ƒ‘—–• ‘ˆ Ž ƒ† ƒŽǤ ‘—„Ž‹‰ –Š‡ –”›’–‘‡ …‘…‡–”ƒ–‹‘ ‡Šƒ…es the amino acid metabolism. Transformants are screened based on the controls used: the negative control in which the revived competent cells are plated without any selection pressure. This ensures two things one about the contamination of other microorganisms and the other revival of cell wall of the bacteria. The second control is known as positive control in which revived competent cells are plated with selection pressure. This gives us the information that the revived competent cells lack the genes whose expression will help in the removal of selection pressure. The transformants are screened on the plates containing the selection pressure which ensures entry of plasmid DNA in to the host bacteria. In case of recombinant plasmids (carrying a foreign DNA) the transformants are screened based on the following controls

Table 1പŝīĞƌĞŶƚĐŽŶƚƌŽůƐƵƐĞĚƚŽƐĐƌĞĞŶƌĞĐŽŵďŝŶĂŶƚĐůŽŶĞƐ Control

Selection pressure

Growth

Insert control

’‹…‹ŽŽ‹Ϊ˜‡ǡ  ƒ†Ǧ‰ƒŽ

-ve

Vector control

’‹…‹ŽŽ‹ژ‡ǡ  ƒ†Ǧ‰ƒŽ

+ve

‹‰ƒ–‹‘…‘–”‘Ž

’‹…‹ŽŽ‹ژ‡ǡ  ƒ†Ǧ‰ƒŽ

+ve

–4–

Cloning of DNA Fragments into Plasmid Vectors Control

Selection pressure

Growth

Positive cell control Ž›…‘’‡–‡–E. coliͷȽ bacteria were plated

’‹…‹ŽŽ‹ژ‡ǡ  ƒ†Ǧ‰ƒŽ

-ve

Negative cell control Ž›…‘’‡–‡–E. coliͷȽ bacteria were plated

No selection pressure

+ve

Based upon these controls whether the cloning is successful or not can be ascertained in the test plate. The number of colonies in the test plate —•–„‡ƒ–Ž‡ƒ•–ϐ‹˜‡–‘•‹šˆ‘Ž†•Š‹‰Š‡”–Šƒ’”‡•‡–‹–Š‡Ž‹‰ƒ–‹‘…‘trol to accentuate successful formation of recombinants. We calculate the –”ƒ•ˆ‘”ƒ–‹‘‡ˆϐ‹…‹‡…›ƒ•ˆ‘ŽŽ‘™•ǣ Transformationefficiency =

(

)

( ) eq.1

No.of transformants colonies x Final volume at recovery ml

( )

μg of plasmid DNA x volume plated ml

Materials ͳƒŽ2. 2 H2‘”

a) 

”ƒ•ˆ‘”ƒ–‹‘ „—ˆˆ‡” ȋȌǣ  ȏʹǦȋǦ‘”’Š‘Ž‹‘Ȍ ethane•—Žˆ‘‹… ƒ…‹†Ȑ „—ˆˆ‡” …‘–ƒ‹•ǣ ͳͲ  ’ ͸Ǥ͵ǡ Ͷͷ Cl2.4H2ǡ ͳͲ ƒŽ2.2H2ǡ ͳͲͲ Žǡ ͵ ‡šƒ‹‡…‘„ƒŽ– …ŠŽ‘”‹†‡ǡ’”‡’ƒ”‡‹ƒ—–‘…Žƒ˜‡††‹•–‹ŽŽ‡†™ƒ–‡”Ǥˆ–‡”ƒŽŽ–Š‡•ƒŽ–• ƒ”‡†‹••‘Ž˜‡†•–‡”‹Ž‹œ‡„›‹ŽŽ‹’‘”‡ϐ‹Ž–”ƒ–‹‘ȋϐ‹Ž–‡”•ƒ›–Š‹‰„‡–™‡‡ͲǤͶͷρ–‘ͲǤʹρ’‘”‡•‹œ‡ȌǤ

b)

Autoclave distilled water

…Ȍ

ȋ—’‡”‘’–‹ƒŽ„”‘–ŠȌǦ‡†‹ƒ

†Ȍ

Ǧ‰ƒ”Ǥ

‡Ȍ

ʹ Ž—…‘•‡ȋ•–‡”‹Ž‹œ‡„›‹ŽŽ‹’‘”‡ϐ‹Ž–”ƒ–‹‘Ȍ

ˆȌ

ʹ‰Ž2Ǥ͸2ȋʹ‰Ž2Ǥ͸2ǣŠ‹••‘Ž—–‹‘‹•ƒ†‡„›†‹••‘Ž˜‹‰ ͳͻ ‰ ‘ˆ ‰Ž2 ‹ ͻͲ Ž ‘ˆ †‡‹‘‹œ‡† 2Ǥ †Œ—•– –Š‡ ˜‘Ž—‡ ‘ˆ –Š‡ •‘Ž—–‹‘–‘ͳͲͲŽ™‹–Š†‡‹‘‹œ‡†2ƒ†•–‡”‹Ž‹œ‡„›‹ŽŽ‹’‘”‡ϐ‹Žtration). –5–

A Practical Textbook of Genetic Engineering in Bacteria

‰Ȍ

ͳͲͲ‰ȀŽ’‹…‹ŽŽ‹†‹••‘Ž˜‡†‹ƒ—–‘…Žƒ˜‡††‹•–‹ŽŽ‡†™ƒ–‡”

ŠȌ

ͳ  •‘Ž—„‹Ž‹œ‡†‹ƒ—–‘…Žƒ˜‡††‹•–‹ŽŽ‡†™ƒ–‡”Ǥ

‹Ȍ

ͷͲ‰ȀŽǦ ƒŽ†‹••‘Ž˜‡†‹ǯ†‹‡–Š›Žˆ‘”ƒ‹†‡Ǥ

SOB Medium 

‘ͻͷͲŽ‘ˆ†‡‹‘‹œ‡†2ǡƒ††ǣ ƒ…–‘–”›’–‘‡ 



ʹͲ‰



ƒ…–‘›‡ƒ•–‡š–”ƒ…–



ͷ‰



ƒŽ



ͲǤͷ‰





Šƒ‡—–‹Ž–Š‡•‘Ž—–‡•Šƒ˜‡†‹••‘Ž˜‡†Ǥ††ͳͲŽ‘ˆƒʹͷͲ•‘Ž—–‹‘ ‘ˆŽǤȋŠ‹••‘Ž—–‹‘‹•ƒ†‡„›†‹••‘Ž˜‹‰ͳǤͺ͸‰‘ˆŽ‹ͳͲͲŽ‘ˆ†‡‹‘‹œ‡†™ƒ–‡”ȌǤ†Œ—•––Š‡’–‘͹ǤͲ™‹–ŠͷƒȋͲǤʹŽȌǤ†Œ—•––Š‡˜‘Ž—‡ ‘ˆ–Š‡•‘Ž—–‹‘–‘ͳŽ‹–”‡™‹–Š†‡‹‘‹œ‡†2Ǥ–‡”‹Ž‹œ‡„›ƒ—–‘…Žƒ˜‹‰ˆ‘”ʹͲ ‹—–‡•ƒ–ͳͷŽ„Ȁ•“‹‘Ž‹“—‹†…›…Ž‡Ǥ SOC (Super Optimal broth with Catabolite repression) Medium: ‡†‹—‹•‹†‡–‹…ƒŽ–‘‡†‹—ǡ‡š…‡’––Šƒ–‹–…‘–ƒ‹•ʹͲ‰Ž—…‘•‡ƒ†ʹͲ‘ˆ‰Ž2Ǥ ‘…—Žƒ–‡–Š‡‡†‹ƒ™‹–ŠͲǤͷŽ‘ˆ‹†ǦŽ‘‰’Šƒ•‡ „ƒ…–‡”‹ƒŽ…—Ž–—”‡ƒ†ƒŽŽ‘™‹––‘‰”‘™—–‹ŽͷͶͲ‹•„‡–™‡‡ͲǤͷȂͲǤ͸Ǥ Luria - Bertani (LB) Medium



ƒ…–‘–”›’–‘‡ 

ͳͲ‰

Bacto yeast extract 

ͷ‰

ƒŽ





ͷǦͳͲ‰

dH2





ͳŽ‹–”‡

†Œ—•––Š‡’–‘͹ǤͲ™‹–ŠͳͲƒȋͲǤͶŽȀŽ‹–”‡ȌǤ LB agar plates: ͳͷ‰‘ˆ ƒ…–‘ƒ‰ƒ”ȀŽ‹–”‡‡†‹ƒǤ 10% Glycerol: ͳͲŽ ‘ˆ ƒ—–‘…Žƒ˜‡† ‰Ž›…‡”‘Ž †‹••‘Ž˜‡† ‹ ͹ͲŽ ‘ˆ ƒ—toclaved distilled water and after dissolution the volume is made up to ͳͲͲŽ™‹–Šƒ—–‘…Žƒ˜‡††‹•–‹ŽŽ‡†™ƒ–‡”Ǥ

–6–

Cloning of DNA Fragments into Plasmid Vectors

Method I. (CaCl2. 2H2O mediated transformation) 1.

Streak E. coliͷȽ…‡ŽŽ•‘Ǧƒ‰ƒ”’Žƒ–‡ƒ†‹…—„ƒ–‡ƒ–͵͹ ͲC for ‘˜‡”‹‰Š–Ǥ‹…ƒ•‹‰Ž‡…‘Ž‘›ˆ”‘–Š‡’Žƒ–‡ƒ†‰”‘™—’–‘ͷͶͲ = ͲǤ͸ȋͳͲŠ”•‰”‘™–ŠȌǤ

ʹǤ

‘…—Žƒ–‡ͷͲŽ‘ˆ–Š‹•…—Ž–—”‡–‘ͳͲͲŽ ‘ˆ‡†‹ƒ…‘–ƒ‹‹‰ ʹͲ‘ˆ‰Ž—…‘•‡ƒ†ʹͲ‘ˆ‰Ž2Ǥ͸2ƒ†‰”‘™ƒ–͵͹ Ͳˆ‘”ͷ Š”•™‹–Š…‘•–ƒ–•Šƒ‹‰ƒ–ʹͷͲ”’ȋ•—…Š–Šƒ–ǤͷͶͲαͲǤͷȌǤ

͵Ǥ

‡ƒ˜‡–Š‡‰”‘™E. coliͷD…—Ž–—”‡‘‹…‡ˆ‘”͵Ͳ‹—–‡•Ǥ‡ŽŽ‡––Š‡ …‡ŽŽ•„›…‡–”‹ˆ—‰‹‰ƒ–͹ǡͲͲͲ”’ˆ‘”ͳͲ‹—–‡•ƒ–ͶͲC.

ͶǤ

‹Ž—–‡–Š‡•–‘…ͳ…ƒŽ…‹—…ŠŽ‘”‹†‡–‘ͲǤͳƒŽ2. 2H2ƒ†…Š‹ŽŽ‹– „›‡‡’‹‰‹–‹‹…‡ƒ†•—•’‡†–Š‡’‡ŽŽ‡–‹ͳͲŽ‘ˆ…Š‹ŽŽ‡†ͲǤͳ CaCl2. 2H2ǡ‘”‹Ǥ‡ƒ˜‡‹–‹‹…‡ˆ‘”͵Ͳ‹—–‡•Ǥ[Note: This is 1/10 volume of the bacterial culture volume].

ͷǤ

‡–”‹ˆ—‰‡–Š‡…‡ŽŽ•ƒ–͹ǡͲͲͲ”’ˆ‘”ͳͲ‹—–‡•ƒ–ͶͲC. Discard the supernatant very carefully as the pellet would be loose and suspend –Š‡ ’‡ŽŽ‡– ‹ ͳ Ž ‘ˆ ‹…‡ …‘Ž† ͲǤͳ ƒŽ2. 2H2 ‘” Ǥ – –Š‹• •–ƒ‰‡ these cells are called as competent cells and they can be stored in ʹͷΨ ˜Ȁ˜ ‰Ž›…‡”‘Ž ȋ͹ͷͲρŽ …‘’‡–‡– …‡ŽŽ• ƒ† ʹͷͲρŽ ͳͲͲΨ •–‡”‹Ž‡ ‰Ž›…‡”‘ŽȌƒ†‡’–ƒ–ǦʹͲͼǤ

͸Ǥ

ƒ‡ ʹͲͲ Ž ‘ˆ –Š‡ …‘’‡–‡– …‡ŽŽ• ‹ ƒ •–‡”‹Ž‡ ͳǤͷŽ ‹…”‘…‡–”‹ˆ—‰‡–—„‡ƒ†ƒ††ͳͲŽ‘ˆ–Š‡’Žƒ•‹†ȋ…‘–ƒ‹‹‰ͳ–‘ͳͲ‰‘ˆ–Š‡ Ȍƒ†Ž‡ƒ˜‡‹–‘‹…‡ˆ‘”͵Ͳ‹—–‡•Ǥȋ …ƒ•‡‘ˆ…Ž‘‹‰‘ˆƒ fragment or PCR product or cDNA or genomic library preparation ’”‡’ƒ”‡ͷ–—„‡•…‘–ƒ‹‹‰ʹͲͲŽ‘ˆ–Š‡…‘’‡–‡–…‡ŽŽ•ƒ†–‘–Šƒ– add one DNA control mentioned in Table 1 such that you have four controls and one tube contains the ligation mixture).

͹Ǥ

‡‡’–Š‡ƒ„‘˜‡–—„‡•ƒ–Ͷʹ Ͳˆ‘”ͻͲ•‡…‘†•ƒ†–Š‡‹‡†‹ƒ–‡Ž› leave them on ice for 2 to 3 minutes.

ͺǤ

†† ͺͲͲ Ž ‘ˆ  ‡†‹ƒ ƒ† ‰”‘™ ƒ– ͵͹ ͲC for 1hr with constant •Šƒ‹‰ƒ–ʹͷͲ”’Ǥ

ͻǤ

Žƒ–‡Ǧ‰ƒ”’Žƒ–‡•™‹–ŠͳͲͲ‰ȀŽƒ’‹…‹ŽŽ‹ǡƒ†‹…—„ƒ–‡ƒ–͵͹ Ͳ ˆ‘”‘˜‡”‹‰Š–ǡˆ‘ŽŽ‘™‹‰†ƒ›•…”‡‡–Š‡’Žƒ–‡•–‘‡•–ƒ„Ž‹•Š–Š‡•—…cessful transformation taking into consideration of controls (Table ͳ ƒ† ‡“ǤͳȌǤ …‡ –Š‡ ”‡…‘„‹ƒ–• ƒ”‡ ƒ•…‡”–ƒ‹‡†ǡ ’”‡’ƒ”‡ ˆ‘” –7–

A Practical Textbook of Genetic Engineering in Bacteria About the Author Dr P.V.G.K. Sarma was born in Vijayawada, Andhra Pradesh, India and grew up in Shillong, India. He obtained his Ph.D in Biotechnology from Jawaharlal Nehru Technological University, Hyderabad in 2000. He worked as guest researcher at the National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA where he worked on cloning and expression of various genes from Mycobacterium phlei. One of his most exciting educational experiences was during his tenure at NIST, USA where his interactions with his mentor made him to learn intricacies in Molecular Biology directly from the scientific literature. He joined Sri Venkateswara Institute of Medical Sciences and University, Tirupati, India in 2004 and is working presently as Associate Professor and Head of the Department. His research was published in very well established journals across the globe which has made lot of impact in the field of biotechnology. His book A Practical Text Book of Molecular Biology was published by MJP Publishers.

ISBN 978-93-88694-41-4

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