bacteriophage vectors used for genomic or cDNA library creation in cloning

1. Lambda Bacteriophage vectors
Dhanya Mol T S
Assistant professor(Contract)
Applied Biotechnology
St Mary’s college
Thrissur

2. Lambda phage
49 kb
30 kb-lytic cycle

3. Types
1. Insertion vector
• Contain a unique cleavage site whereby foreign DNA
with size of 5–11 kb may be inserted.
• Contain single site for cloning DNA inserts
• Designed to be packaged with/without inserted
DNA
• Minimum size for packaging – 38 kb so can only
clone in 10 kb (or less) inserts
• Used in cloning cDNA in expression vectors
2. Replacement vector
• The cleavage sites flank a region containing genes
not essential for the lytic cycle, and this region
may be deleted and replaced by the DNA
insert in the cloning process, and a larger sized DNA
of 8–24 kb may be inserted.

4. Insertion v/s replacement vectors

5. Insertion vector-lambda gt10
• allows screening using insertion inactivation of cI gene
• cI gene in vector has unique EcoRI site
• cloned insert prevents lysogeny
• only lytic growth – clear plaques
• without insert lysogens survive in plaques( turbid plaques)
• screen using hfl mutant host
• no Hfl protease, no cleavage of cII gene product
• lambda + and lambda gt10 without insert always lysogenize
• if cI insertion inactivated – no lysogeny – plaques

7. lambda gt11
Has complete lacZ gene cloned with unique EcoRI site
• Is an expression vector where DNA is expressed as β-galactosidase fusion protein
Recombinant phage will form white colonies and non-recombinant blue
colonies(with X-gal, IPTG)
• Recombinants can be screened using antibody probes
Potential problems
• insert must be in frame
• antisera must recognise protein produced in E. coli
• proper folding, glycosylation may be problem
• fusion protein must not be lethal
• kills cells before progeny phage produced

8. Advantages
Applications: For construction of genomic and cDNA libraries

9. Construction of cDNA Libraries
in lambda gtl0 or lambda gtll
Steps
• Preparation of Plating Cells f or Infection with lambda gtl0 or lambda gtll
• Titering and Plaque purification of lambda gt10 Stocks
• Preparing gt10/11 DNA
• Eco R1 digestion of lambda
• Phosphatasing lambda
• Purification of cDNA Linkers on Ultra gel
• Ligation of cDNA and Vector
• In Vitro Packaging
• Plating Out gt 10/11 Libraries for Screening

10. References
• https://onlinecourses.swayam2.ac.in/cec24_bt11/course
• https://www.youtube.com/watch?v=QpstvqDy7pk
• https://www.youtube.com/watch?v=vMg4pF29BG8&t=335s
• Burrell, M. M. Construction of cDNA Libraries in gtl0 or gt ll.