DAIRY BIOTECHNOLOGY

Module 3. Genetic engineering technology / recombinant DNA technology

Lesson 11
VECTORS

11.1 What is a Vector?

A vector (a carrier, a Latin word meaning bearer) is a DNA molecule having its own origin of replication that carries foreign DNA into a host cell and produces many copies of itself and that of foreign DNA. They are categorized into following types.

11.1.1 Plasmids

Plasmid vectors can be used for cloning of DNA fragments of size generally ranging from 0.1 to 10 kb. The most common examples of plasmid cloning vectors are pUC18/19, pBluescript, pGEX, pET series etc.

11.1.2 Lambda phage

Lambda phage can also be used as vector for cloning gene of interest of size ranging from 8-25 kb fragments. This is based on lambda phage genome which is 48,502 bp with a central 33% (stuffer region) which is not critical for lytic growth and can be replaced with 8-25 kb gene inserts e.g. gt11, charon phages, lambda ZAP vectors. Lambda vectors can be introduced into cells at a very high efficiency.

11.1.3 Cosmids

Cosmids vectors (5-7 kb) are hybrid between plasmids and phages. They contain antibiotic genes and replication origin from plasmids and ‘cos’ sites from phages which are necessary for packaging of DNA. These vectors can clone larger fragments of 35 size – 50 kb.

11.1.4 BAC vectors (Bacterial artificial chromosomes)

BAC vectors contain sequences from the E. coli F plasmid and they have the ability to clone up to 75 - 200 kb fragments.

11.1.5 YAC vectors (Yeast artificial chromosomes)

YAC vectors contain sequences required to replicate and maintain chromosome in budding yeast (like l, end up as a linear molecule) and contain a yeast origin of replication, a centromere, and a telomere at each end. These vectors are able to clone very large fragments of >2,000 kb inserts (2 Mb).

Out of the above mentioned vectors, we shall study plasmid cloning vectors in detail as the plasmids are the most commonly used vectors in majority of the laboratories.

11.2 Definition of Plasmids

Plasmids are small extra-chromosomal circular DNA molecules (replicons) that can replicate independently in bacteria (i.e. they have their own origin of replication) and are present in one or multiple copies in a bacterial cell (Fig. 11.1). Plasmids naturally occur in different types of bacterial cells. However, most of the plasmids are cryptic in nature i.e. their functions remain by and large unknown.

11.3 Classification of Plasmids

Plasmids can be classified differently based on their conformation, copy number and ability to transfer.

11.3.1 Conformation/forms of plasmids

Plasmids can exist in four forms as shown in Fig. 11.2.

i) Relaxed form – In this state, plasmid does not possess super coils and both the strands remain intact without any stress / torsion

ii) Super coiled form or covelantly closed circular form (CCC form) – In this form, plasmid is twisted enzymatically and is highly compact

iii) Nicked open circular form – the form of plasmid in which one of the two strands is nicked or cut as a result of which it loses super coiled configuration and remain in circular form

iv) Linear form – As both the strands of DNA are cut at the same site, the plasmid DNA becomes linear.

Out of the aforesaid forms, super coiled or CCC form is the most prevalent form that occurs in the bacterial host under natural conditions.

11.3.2 Based on copy number

Based on the copy number, plasmids are classified as

11.3.2.1 Stringent plasmids

Stringent plasmids are low copy number plasmids of relatively larger size and exist in 1-3 per cell. These plasmids replicate along with the main bacterial chromosome.

11.3.2.2 Relaxed plasmids

These are high copy number plasmids of relatively small size that can exist in > 25 copies per cell. These are the preferred type of vectors used extensively in molecular cloning and recombinant DNA experiments where high level of expression of the recombinant protein is required for commercial application.

11.3.3 Based on ability to transfer

11.3.3.1 Conjugative plasmids

The conjugative plasmids contain ‘tra’ genes (transfer genes) as well as ‘mob’ genes (mobility genes) which enable them to be transferred to other bacteria through conjugation.

11.3.3.2 Non-conjugative plasmids

The non-conjugative plasmids lack the ability to be transferred to other bacteria as they do not possess ‘tra’ genes. However, they can be transferred with the help of other conjugative plasmids if they harbor ‘mob’ gene.

11.4 Properties of Plasmid Cloning Vectors

Artificially constructed plasmids used for cloning of DNA / genes are called Plasmid Cloning Vectors e.g. pBR322 and its derivatives which were the first plasmids to be constructed and used in Molecular Biology. With the advancements in molecular biology, many more efficient plasmid vectors are now available commercially and used world wide for cloning and expression of heterologous proteins in prokaryotic and eukaryotic host systems. Some of the examples are pBR322, pUC18/19, pBluescript KS+ / SK+, pGEM series etc. (Fig. 11.3)

Fig. 11.3 Genetic map of pBR322, pUC18/19 and pBluescript

The plasmid cloning vectors possess the following properties as shown in the Fig. 11.4.

11.4.1 Origin of replication (Ori site)

Plasmid vectors contain sequences that permit propagation of vectors in bacteria or yeast i.e origin of replication. Origin of replication helps in replication of DNA and is recognized by DNA polymerase for plasmid replication.

11.4.2 Multiple cloning or Poly cloning site (MCS or PCS)

Plasmid vectors must possess cloning site to insert foreign DNA. Most of the commercially available vectors are designed in such a way that they have a multiple cloning site or poly cloning site by introducing a large number of restriction sites for different restriction endonucleases to widen their cloning prospects. However, restriction enzyme sites present in MCS /PCS should not be present anywhere else in the rest of the plasmid sequence. Genes of interest to be cloned or expressed are introduced into the cloning vector at restriction enzyme sites by using MCS /PCS.

11.4.3 Selection marker

Plasmid vectors possess selection markers e.g. antibiotic resistance markers (ampr, tetr, chlr) or histochemical markers e.g. lacZ – such that selection of recombinants is based on insertional inactivation (selection marker i.e. antibiotic resistance gets inactivated when a foreign gene is inserted) or alpha complementation such as using lacZ gene for selection of recombinant clones based on blue white selection.

11.4.3.1 Inactivation of Antibiotic resistance

The incorporation of foreign DNA fragment into the antibiotic resistance gene resulting into inactivation of that gene is called insertional inactivation. Insertional inactivation strategy is based on using two antibiotic resistance marker genes in the vector (e.g. ampicillin and tetracycline), one of which is inactivated due to insertion of foreign gene and the other remains intact and thus helps in the selection of recombinants as has been shown in Fig. 11.5.

11.4.3.2 Alpha complementation

Alpha complementation is a commonly used process wherein alpha portion of ‘lacZ’ gene (that encodes β–galactosidase) is incorporated into the vector and E. coli host contains ω portion of ‘lacZ’ gene. Neither of them alone is able to express functional β –galactosidase. However, when both the fragments are present simultaneously in the host, they complement each other and produce a functional β -galactosidase. The insertion of foreign gene at the multiple cloning site located in ‘lacZ’ region of the vector results into inactivation of alpha-complementation by disrupting the open reading frame of ‘lacZ’ gene. This criteria forms the basis of blue white screening for selection of recombinant clones by adding substrate X-gal (5 bromo-4 chloro-3-indolyl- β –D-galactoside) into the medium. If ‘lacZ’ gene is not inactivated, the colonies give blue colour otherwise white colour due to insertion of the foreign DNA (insert). Ths alpha complementation phenomenon has been illustrated in Fig. 11.6. IPTG (Iso- propyl- thio - β -galactoside) is also used as gratuitous inducer if the promoter is inducible.

Selection markers are also required for maintenance of plasmids in the bacterial cells e.g. plasmid containing ampicillin or tetracycline markers will be lost if the medium will be devoid of these antibiotics which are used as positive selective pressure for their maintenance.

11.5 Plasmid as Cloning and Expression Vectors

Plasmids can be explored both as cloning and expression vectors as described below.

11.5.1 Cloning vectors

Vector that is used for reproducing the insert DNA For example pBR322 and pUC18/19. PCR products can be directly cloned into PCR cloning vectors known as TA cloning vectors e.g. pGEM-T, pGEM-TEasy, pDrive (Fig. 11.7). These vectors use the terminal transferase activity of some DNA polymerases. Taq polymerase adds additional A at 3’ end to each end of the PCR product which is exploited for cloning of PCR products in pGEMT vector since T is incorporated at the 3’ end of the vector. TA Cloning makes it possible to clone the PCR product into a cloning vector with 3'-T overhangs.

Fig. 11.7 Genetic map of pGEMT-easy and pdrive vectors

11.5.2 Expression vectors

Vector that allow the exogenous DNA to be inserted, stored, and expressed in an appropriate host are called expression vectors. Some of the expression vectors available commercially include pUC, pBluescript, pET series, pGEX series (Fig. 11.8) etc. These vectors also contain Ribosome binding sites (RBS), strong promoters (T3, T7, SP6) and transcription termination sequences along with fusion tags for protein purification as well as other components of cloning vectors. Most of the extensively used plasmid expression vectors use T7 promoter which is considered as a strong promoter for enhancing the expression of heterologous proteins for large scale production. Expression vectors are basic tools for the production of heterologous proteins of food, feed and pharmaceutical interest e.g. Insulin, Hepatitis B vaccine, Chymosin, Human lactoferrin and Phytase etc.

Fig. 11.8 Genetic map of expression vector pET

Books

Molecular Biotechnology - Second Edition, S. B. Primrose, Blackwell Science Inc., ASIN: 0632030534

Introduction to Biotechnology, Brown, C.M., Campbell, I and Priest, F.G. Panima Publishing Corporation, 2005. ISBN : 81-86535-42-X

DNA and Biotechnology, Fitzgerald-Hayes, M. And Reichsman, F. 2nd Amsterdam : Elsevier, 2010. ISBN : 0-12-048930-5

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Glick, B.R., Pasternack, Jack, J and Patten, Cheryl, L (Eds)., 4th Washington., ASM Press, 2010. ISBN : 1-55581-498-4

Molecular Biology and Biotechnology : a guide for students, Krauzer, H. And Massey, A.(Eds) 3rd Washington DC : ASM Press, 2008, ISBN : 978-155581-4724

Recombinant DNA and Biotechnology : a guide for teachers, Kreuzer, H and Massey, A. 2nd Washington : ASM Press, 2001, ISBN : 155581-175-2

Molecular Biotechnology, Primrose, S.B. 2nd New Delhi : Panima, 2001. ISBN : 81-86535-21-7

Molecular Biology and Biotechnology, Smith, C.A. and Wood, E.J. London : Chapman and Hall, 1991.ISBN : 0-412-40750-7

Introduction to Biotechnology, Thieman, W.J and Pallidino, M.A. 2nd New York : Pearson, 2009, ISBN : 978-0-321-58903-3

Molecular Biology and Biotechnology, Walker, J.M and Rapley, R. 4th – New Delhi : Panima Publishing Corporation, 2003, ISBN : 81-86535-40-3

Gene Biotechnology, Wu William, Welsh, M.J., Kaufman, P.B and Zhang, H.H.
2nd Boca Raton : CRC press, 2004. ISBN : 0-8493-1288-4

From Genes to Genomes: Concepts and Applications of DNA Technology, 3rd Edition, Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant (Eds), Wiley-Blackwell, 2011, ISBN: 978-0-470-68386-6

Microbial Genetics, 2nd Edition, Stanly R Maloy, John Cronan, David Freifelder, Narosa, ISBN: 8173196974

Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1

Internet Resources

http://en.wikipedia.org/wiki/Plasmid

http://www.ncbi.nlm.nih.gov/books/NBK21498/

www.youtube.com/watch?v=KQNyxwzBnjw

http://nhjy.hzau.edu.cn/kech/zwswjs/kc/ziliao/cloning.pdf

http://molecularhub.com/vectors-the-cloning-vehicles/

http://www.mfa.od.ua/page152.htm

http://en.wikipedia.org/wiki/Expression_vector

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Causey/pET.html