pUC Vectors - pUC 8,18,19 Microbial biotechnology

pUC vectors- microbial biotechnology

1. VIVEKANANDHA ARTS AND SCIENCE COLLEGE FOR WOMEN, VEERACHIPALAYAM, SANKAGIRI, 637303 AFFILIATED WITH PERIYAR UNIVERSITY, SALEM DT., RECOGNISED UNDER SECTION 2(f) & 12(b)UGC ACT 1956. SUBJECT: MICROBIAL BIOTECHNOLOGY TOPIC: PUC SUBJECT INCHARGE, R. PANDIAN, ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY VIAAS, SANKAGIRI. SUBMITTED BY, Nandhini Vengadachalam, III BSc Microbiology, Department of Microbiology, VIAAS, sankagiri.

2. PUC VECTORS

3. INTRODUCTION Puc are obtained by modifying the PBR322 vector. Puc vectors are smaller than PBR322 of being only 2.7kb.But comparatively they have a high copy number. A mutation within the origin of replication produce 500 to 600 copies of the plasmid per cell without amplification. “P” indicates the plasmid “UC” Stands for university of california where it was first developed by J. Mess ing et al. We also see many numbers after this like pUC8, pUC18, pUC19 and so on. They are just to separate from each other.

4. The construction of pUC vectors Origin of Replication: It is derived from the origin of replication of pBR322. The COIEI origin of replication of pBR322 has been modified by carrying out a chance mutation so that each transformed E . C oli cell has 500 to 600 copies of the plasmid. Selectable marker: It has an amppicilin resistant gene. The transformed host cells can grow on media having ampicillin where non transformed cells die. Lac‘z gene having multiple cloning sites(MCS). Cloning with pUC8 involves insertional inactivation of the lac z gene with recombinants identified because of their inability to synthesize beta- galactosidase.

5. Uses of pUC vectors pUC vectors can be used both as cloning vector and expression vector when used an expression vector it’s sequence are slightly modified to meet neccesary requirements.

6. PUC vector types There are three types of pUC vector. Then, PUC 8 PUC 18 PUC 19

7. pUC 8 One of the plasmids to be used in recombinant genetics was called pBR322. It is approximately 4300 bp length and has two antibiotic resistance genes. Ap (Ampicillin) and Tc (Tetracycline). Bacteria cells that are successfully transformed with this plasmid are able to grow in the presence of both ampicillin and tetracycline antibiotics.

8. pUC 8 plasmids

9. Improving the use of pUC8 While it was possible to isolate the bacteria transformed with pUC8 on lactose and ampicillian containg media, a problem existed.

10. The solution to the problem of separating the JM101 cells transformed with recombinants plasmid from the JM101 cells transformed with non recombinants plasmid was solved using the chemicals called q X-Gal and IPTG. The technique could be successfully employed with any type of bacteria cells that was used.

11. PUC 18 pUC18 is a plasmid cloning vector used in E. Coli that’s known for its small size and ability to clone large DNA fragments:
Size: pUC18 is a double-stranded circular DNA molecule that’s 2,686 base pairs Multiple cloning site: pUC18 has a 54 base-pair multiple cloning site polylinker that contains unique sites for 13 different restriction endonucleases.
Beta-galactosidase: pUC18 complements defects in beta-galactosidase in appropriate host strains.
Origin of replication: pUC18 has the pMB1 origin of replication.
Storage: pUC18 should be stored at -20°C.
Cloning: pUC18’s small size allows for the successful cloning of large DNA fragments.
Ampicillin resistance: pUC18 confers ampicillin resistance to appropriate host strains.

12. Cloning pUC 18

13. Summary First generation plasmid cloning vectors include pBR322 and pUC plasmids. pUC has – 1 antibiotic resistance gene ampicillin resistance gene. Variety of unique restrictions sites in lacZ gene for inserting foreign DNA. Most of there sites interrupted lacZ gene making screening straight forward. Thus MCS in lacZ facillitates disestional cloning.

14. PUC 19 P = plasmid
UC = University of California
19 = numerical designation pUC plasmids were first developed by Joachim Messing and his co-workers. It is a commonly used cloning vector in the bacteria E. Coli.
pUC19 is 2686 bp in length. The molecular weight of the pUC19 vector is 1.75×106 Da. It is a small plasmid with a high copy number. It contains the lacZ gene and has multiple cloning sites.
Hence, it is widely used as a cloning vector. pUC19 plasmid is similar to pBR322 plasmid in structure.

15. PUC 19 diagram

16. Structure of PUC 19 It is a 2686 bp long plasmid.
Origin of replication – The origin of replication of the pUC19 plasmid is derived from pMB1.
Multiple Cloning Sites – There is a short sequence of 2.8 kb which contains sites for various restriction enzymes. This increases the number of potential restriction sites available, enabling the production of the desired fragment for cloning.
Selectable markers – The pUC19 plasmid contains an Ampicillin resistance gene which can be used to screen the recombinants. The plasmid also contains the E. Coli gene lacZ, which encodes for β-galactosidase (β-galactosidase hydrolyses lactose).
Restriction sites – The pUC19 vector carries a 54 bp long multiple cloning site poly-linker containing 13 different hexanucleotide-specific restriction endonucleases sites.

17. PUC 19 Vectors

18. The Blue-White screening method is used for pUC19 vectors. The process of screening is as follows:
This method of screening is based on the fact that the blue pigment is formed when β-galactosidase catalyses the hydrolysis of a synthetic substance known as X-gal in the medium.
When X-gal is hydrolysed, it forms galactose and 5-bromo-4-chloro-3-hydroxy indole.
The later product undergoes dimerization (spontaneous) and oxidation.
As a result of dimerization and oxidation, a blue pigment is formed.
The cells which contain the β-galactosidase activity form blue colonies, whereas the cells which do not show β-galactosidase activity form white colonies on the agar medium containing X-gal.
The recombinant cells, which contain newly inserted DNA fragments, lack the β-galactosidase activity and hence appear white on the agar plates.
This method of screening the recombinant cells is the easiest and fastest method.

19. Application Cloning : The pUC 19 vector can be used to clone and express genesof interested in E. Coli. Blue – white selection: The pUC19 vectors multiple cloning site (MCS) is inserted into the lacZ gene which allows for blue white selection of plasmid DNA that contains inserts. Amplifying cDNA sequences: The pUC 19 vector can be used to amplify ORF cDNA sequences by PCR.

20. Advantages High copy number of 500-600 copies per cell. Easy and single step selection The unique restrictions sites used for cloning are clustered within the MCS. This allows cloning of a DNA fragment having two different sticky ends.

21. Disadvantages It cannot accommodates a genes of interest larger then 15kb.