CLONING VECTORS FOR E.COLI BASED ON E.COLI PLASMIDS

Transcription

1 CLONING VECTORS FOR E.COLI BASED ON E.COLI PLASMIDS Different types Principle Nguyen Thu Giang-IPMB, VUB

2 General

3 What is a plasmid? Plasmids are small, extrachromosomal pieces of bacterial DNA that are often antibiotic resistant They are shuttle vectors to create, produce, and maintain recombinant DNA An example of the first cloning vector for E.coli based on E.coli plasmids is pbr322 And puc18 now is the most commonly used

4 Characters A way to maintain in engineered DNA in the cell s progeny A way to identify (or select) the cells containing DNA A place to insert the gene or piece of DNA you want to work with

5 General properties of plasmid vectors: small, easily isolated, ease of transformation high copy number polylinkers or multiple cloning sites (MCS) selection for transformation (drug resistance) ease of screening (lacz)

6 Specific properties of plasmid vectors: they must to appropriate to the host, e.g. ori, selection they must be appropriate to your objectives - expression or non-expression - expression of RNA or protein - integration and non-integration - low or high copy number - ease of sequencing - facilitate library construction ( the size problem ) - work in different species (shuttle vectors)

7 What is a plasmid?

8

9 Different types

10 pbr322 Nomenclature p indicates that it is indeed a plasmid BR identified the laboratory in which the vector was originally constructed 322 distinguishes this plasmid from others developed in the same laboratory

11 pbr322 pbr322 is an example of an early cloning vector that replicates in E.coli. pbr322 has some significant features: It has a small size of 4363 bp It carries two sets of antibiotic resistance genes It has reasonably high copy number

12 pbr322 Ori allows independent replication Single cleavage sites for various restriction enzyme (BamHI ect) tet resistance gene amp resistance gene Mechanism: insertion of foreign DNA at BamHI site tet resistance gene inactivated transformants carrying foreign DNA are amp resistant but tetracycline sensitive

13 pbr322 The pedigree amp gene originally resided on the plasmid R1 tet gene is derived from R6-5 The replication origin of pbr322 is originally from pmb1

14 Evolution of Plasmid Vectors Phagmid λzap Cosmids puc series Protein Purification pgex series pgem series Regulated Expression pet, pbad, ptet

15 pbr327 pbr327, one of the first derived from pbr322, was produced by removing 1089 bp from pbr322: pbr327 has a higher copy number pbr327 is non-conjugative Both are now no longer widely used

16 puc series next generation, although only replication origin and amp genes remain used in BIO lab collects all restrictions sites into MCS or polylinker 3 advantages: fortuitous: the manipulations involved in construction of puc series were accompanied by a chance mutation Identification of recombinant cells can be achieved by a single step process and puc series introduces screening with x-gal The clustering of restriction sites allows DNA fragments with two sticky ends.

17

18 pgem3z next generation, 2750bp very similar to puc vectors but has two promoters introduces RNA expression for in vitro or in vivo expression.

19 Principle

20 CLONING PROCESS Gene of interest is cut out with RE Host plasmid is cut with same RE Gene is inserted into plasmid and ligated with ligase New plasmid inserted into bacterium (transform)

21 PLASMID CLONING STRATEGY Involves five steps: 1.Enzyme restriction digest of DNA sample. 2.Enzyme restriction digest of DNA plasmid vector. 3.Ligation of DNA sample products and plasmid vector. 4.Transformation with the ligation products. 5.Growth on agar plates with selection for antibiotic resistance.

22 STEP 1. RE DIGESTION OF DNA SAMPLE

23 STEP 2. RE DIGESTION OF PLASMID DNA

24 STEP 3. LIGATION OF DNA SAMPLE AND PLASMID DNA

25 STEP 4. TRANSFORMATION OF LIGATION PRODUCTS The process of transferring exogenous DNA into cells is call transformation There are basically two general methods for transforming bacteria. The first is a chemical method utilizing CaCl2 and heat shock to promote DNA entry into cells. A second method is called electroporation based on a short pulse of electric charge to facilitate DNA uptake.

26 CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE

27 TRANSFORMATION BY ELECTROPORATION

28 STEP 5. GROWTH ON AGAR PLATES

29 STEP 5 Blue colonies represent Ampicillin-resistant bacteria that contain pvector and express a functional alpha fragment from an intact LacZ alpha coding sequence. White colonies represent Ampicillin-resistant bacteria that contain pinsert and do not produce LacZ alpha fragment

30 Thanks for your attention!

31 Merry Christmas and Happy New Year