Reading Lecture 3: 24-25, 45, Lecture 4: 66-71, Lecture 3. Vectors. Definition Properties Types. Transformation

Transcription

1 Lecture 3 Reading Lecture 3: 24-25, 45, Lecture 4: 66-71, Vectors Definition Properties Types Transformation 56

2 VECTORS- Definition Vectors are carriers of a DNA fragment of interest Insert Allow the fragment to be replicated in a host cell Means of producing many copies of a specific fragment Bacteria does all the work 57

3 Types of Vectors Plasmids DNA inserts less than 20 kb Phage Inserts in the 20 kb range Cosmids Inserts up to 45 kb BACs DNA fragments up to 300 kb YACs DNA fragments greater than 1 Mb 58

4 Requirements of a Vector Needs a replicon Needs to be able to replicate itself Origin of replication Needs a place to put DNA to be cloned Unique restriction enzyme cleavage site Cloning site Must be selectable 59 Need to be able to distinguish which cells carry the vector Selectable marker gene (antibiotic resistance)

5 Plasmids Extrachromosomal replicating DNA Double-stranded circular DNA Genetically homogenous Stably inherited Can be present in a bacterial cell in multiple copies Not essential for cell s existence Plasmids E. Coli chromosome 60

6 Plasmid Discovery Plasmids are found in bacteria with different phenotypes 3 Classes Virulence plasmids Encode toxin genes Drug-resistance plasmids Confer resistance to antibiotics Conjugation plasmids Encode genes required for bacterial conjugation 61

7 Laboratory Plasmid Characteristics: Origin of Replication Origin of replication Various mechanisms Figs. 4.4 and 4.5 in text F plasmid ori One copy per cell ColEI ori Relaxed replication ~25 copies per cell 62

8 Laboratory Plasmid Characteristics: MCS A polylinker or multiple cloning site (MCS) is an artificially synthesized DNA sequence that is inserted into a vector and carries a set of restriction enzyme cleavage sites unique in the plasmid. The polylinker is integrated into the lacz gene. 63

9 Laboratory Plasmid Characteristics: Selectable Marker- Antibiotic Resistance Ampicillin resistance (Amp r ) Ampicillin inhibits cell wall synthesis Stops bacterial growth Amp r gene product hydrolyzes ampicillin Eliminates ampicillin, cells with plasmid grow 64 Tetracycline resistance (Tet r ) Tetracycline inhibits translation Cell death Tet r gene product prevents tetracycline accumulation Eliminates tetracycline, cells with plasmid grow

10 Transformation Bacterial take-up of DNA Way to introduce your plasmid into an E. coli host Bacteria take up DNA and become transformed Bacteria grow and form colonies Plasmid can replicate and express its genes in the bacterial host Growth of bacteria of selective media will depend on the presence or absence of plasmid 65

11 Selective Media Transformed Bacteria 66 LB LB + Amp

12 Evolution of Plasmid Cloning pbr322 First plasmid cloning vector Ori Few unique restriction enzyme sites Two selectable markers Amp r Tet r Vectors 67

13 More Advanced Plasmid Vectors: puc Developed at UCD by J. Messing puc plasmids carry an ori mutation Increased copy number MCS Polylinker with multiple unique RE sites Amp R and Blue/white selection LacZ gene 68

14 Blue/White Selection for Insert a-complementation system using the E. coli lac operon The lac operon In bacteria, an operon is a cluster of genes Encodes 3 genes LacZ - b-galactosidase LacY- Permease Lac A - Transacetylase Regulated by the lac promoter and repressor The Lac Operon Lacp lacz lacy laca 69

15 The Lac Operon Repressor protein binds to the operator Blocks transcription IPTG can induce expression of the lac operon Lessens repressors affinity for operator 70

16 Product of the lacz gene Activity can be measured in live cells Can cleave the chromogenic substrate X-gal Cleavage of X-gal produces a blue color Can exploit this by using a-complementation Vector expresses N-terminal (a-fragment) of b-gal Host cell expresses C- terminal (w-fragment) Two fragments combine to form functional b-gal 71 b-galactosidase

17 Blue/White Selection in Plasmids DNA for the lac promoter and N- terminus of the lacz gene flank the MCS Transform vector into cells producing C- terminus Plate on media containing IPTG and X- gal 72

18 73 Blue/White Color Selection Vector WITHOUT insert Get blue colonies LacZ gene product is made Cleaves X-gal Get blue precipitate Vector WITH insert Get white colonies Insert disrupts the coding of the lacz gene product No functional b-gal Can t cleave X-gal No blue precipitate

19 pbluescript Commonly used modern plasmid Sold by Stratagene 2 origins or replication ColEI f1 origin Can do ss DNA synthesis T7 and T3 RNA polymerase promoters ss RNA for probes Universal primers 74

20 75 MCS of pbluescript

21 Methods of Transformation Competent cells Chemically treated with CaCl 2 Makes membrane leaky Heat shock to stimulate uptake 76

22 Methods of Transformation Electroporation Uses a short pulse of electrical charge Causes transient opening in cell membrane DNA can get in More efficient than competent cells Used frequently for mammalian cells 77