Cloning vectors. E. coli Yeast Plants Insects

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1 Cloning vectors E. coli Yeast Plants Insects

2 Cloning vectors for E. coli The simplest cloning vectors are based on small bacterial plasmids Desirable properties: -Easy purification -High transformation efficiency -Selectable markers -Ability to clone reasonably large (up to about 8 kb) pieces of DNA

3 The nomenclature of plasmid cloning vectors The name pbr322: - p for plasmid - BR for Bolivar and Rodriguez, the two researcher who developed it distinguishes this plasmid from others constructed in the same laboratory

4 One of the first vectors to be developed was pbr322 -Small size (4363 bp) -Two selectable markers (antibiotic resistance genes, ampicillin and tetracycline) -Unique restriction sites for cloning -Reasonably high copy number (15 copies/cell) - Conjugative plasmid

5

6

7 Selecting cells that contain pbr322 plasmids

8 Incubation at 37ºC for 1h before plating out improves the survival of the transformants on selective medium

9 Identification of recombinants by insertional inactivation How to distinguish between cells that have recombinant DNA molecules from the ones that contain self-ligated vector?

10

11 Screening for pbr322 recombinants by insertional inactivation of the tetracycline resistance gene

12 The puc plasmids -high copy number ( per cell) -identification of recombinant cells can be achieved in one step -a multicloning site clustered and allowance to clone fragments with two different sticky ends

13 β-galactosidase splits lactose into glucose and galactose, but the lactose analogue X-gal (5-bromo-4-chloro-3- indolyl-β-dgalactopyranoside) which is broken down by β-galactyosidase produces a coloured blue compound -cells producing β- galactosidase turn blue

14

15 Cloning vectors based on M13 bacteriophage Vectors based on M13 offer the possibility of obtaining singlestranded versions of cloned DNA Important for sequence determination and site-directed mutagenesis All the phage genes are important and is very difficult to do a lot of changes

16 Construction of M13mp7

17

18 Phagemid a hybrid plasmid- M13 vector

19 Cloning vectors based on λ bacteriophage Two problems have to be solved before λ-based cloning vectors could be developed: 1) Only 3kb can be added to be packed into λ head 2) Many recognition sequences for many restriction endonucleases

20 The λ genetic map showing the position of the nonessential region that can be deleted without affecting the ability of the phage to follow the lytic cycle About 15 kb were deleted and as much as 18 kb of new DNA can be inserted

21 Natural selection can be used to isolate modified λ that lack certain restriction sites

22

23 Long DNA fragments can be cloned using a cosmid Cosmids are hybrids between a phage DNA molecule and a bacterial plasmid, and their design centres on the fact that the enzymes that package the λ DNA molecule into the phage protein coat need only the cos sites in order to function The in vitro packaging reaction works with any molecule that carries cos sites separated by kb of DNA

24 A typical cloning with a cosmid

25 λ and other high capacity vectors enable genomic libraries to be constructed A genomic library is a set of recombinant clones that contain all of the DNA present in an individual organism Genomic libraries can be retained for many years and copies can be sent from one research group to another How many clones are need for a genomic library? N= ln (1-P) ln (1-a/b) N- number of clones required P- probability that any given gene will be present a- the average size of the DNA fragments inserted into the vector b- the total size of the genome

26

27 Insert capacities for common vector systems

28 Cloning vectors for yeast The yeast Saccharomyces cerevisiae is very important in Biotechnology: brewing, breadmaking and a host organism for the production of many pharmaceuticals The development of cloning vectors was accelerated by the discovery of the 2 µm plasmid Small plasmid copies/cell Plasmid origin of replication

29 Selectable markers for the 2 µm plasmid

30 Yeast episomal plasmids (YEps) YEps are called shuttle vectors because they can replicate both in E. coli and S. cerevisiae -2 µm origin of replication -Selectable LEU2 gene -Entire pbr322 sequence -copy number between YEp13

31

32 A YEp may insert into yeast chromosomal DNA (homologous recombination)

33 Yeast integrative plasmids (YIps) are bacterial plasmids carrying a yeast gene URA3 codes for an enzyme involved in the biosynthesis of pyrimidine nucleotides (T, C) Its survival depends on integration into the yeast chromosomal DNA Yeast replicative plasmids (YRps) carry a chromosomal DNA sequence that includes a yeast origin of replication TRP1 codes for an enzyme involved in the biosynthesis of tryptophan Copy number between 5-100; unstable

34 Yeast artificial chromosomes (YACs) can be used to clone long pieces of DNA Key components of a chromosome: -The centromere, required for the correct partition of the chromosome to the daughter cells -Two telemores, needed for the ends of the chromosome to be replicated and to protect from exonucleases -The origins of replication, positioned along the chromosome at which DNA replication occurs

35

36 Cloning vectors for higher plants Vectors based on naturally occuring plasmids of Agrobacterium Ti (tumor inducing)

37

38 The binary vector strategy The Ti plasmid has a large size and the genetic manipulation is very difficult The T-DNA does not need to be physically attached to the rest of the Ti plasmid

39 The cointegration strategy

40 The only parts of the T-DNA that are involved in the infection process are two 25 bp repeats found at the left and right borders of the T-DNA pbin19 binary vector (disarmed Ti) Limitation of cloning with Agrobacterium plasmids: -only for dicotyledonous plants (tomato, tobacco, potato, peas, beans)

41 Ri plasmid from Agrobacterium rhizogenes cause hairy root disease Biotechnological potential to obtain large amounts of recombinant proteins from fast growing roots Valuable source of root derived phytochemicals that are useful as pharmaceuticals, cosmetics, and food additives. Arabidopsis thalina with hairy root

42 Transfer of genes into the chloroplast genome As each cell has many chloroplasts, a gene inserted into the chloroplast genome expresses at higher levels

43 Cloning vectors for insects Cloning in Drosophila makes use of a transposon called the P element A transposon is a short piece of DNA that can move from one position to another in the chromosome of a cell

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