Bacterial Transformation and Plasmid Purification. Chapter 5: Background

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1 Bacterial Transformation and Plasmid Purification Chapter 5: Background

2 History of Transformation and Plasmids Bacterial methods of DNA transfer Transformation: when bacteria take up DNA from their environment Conjugation: process of transferring DNA by a pilus (bridge) from one bacteria to another Transduction: when bacterial DNA is transferred from one bacteria to another by viruses 2

3 Origin of Plasmids Joshua Lederberg and William Hayes independently discovered plasmids while studying conjugation 1952 Lederberg proposed the name plasmid 1961 Tsutomu Watanabe and Toshio Fukasawa found that some plasmids carried antibiotic resistance genes 1962 Allan Campbell determined that plasmids were circular 1973 Peter Lobban proposed using restriction enzymes to help recombine DNA 1973 Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling published a paper describing how to construct a functional plasmid 1976 Herbert Boyer and Robert Swanson founded Genentech using plasmids to manufacture insulin 2009 Genentech was sold to Roche for $46 billion 3

4 Plasmids: Structure and Function Most are extrachromosomal loops of DNA that can self-replicate in the cytosol of bacteria They have an origin of replication (ori on the map) Are designated with a p in the name Have genes that code for proteins. They are symbolized by an arrow in the direction of transcription Genes are preceded by a promoter The location for RNA polymerase to bind They are followed by a terminator The location that causes the polymerase to stop transcribing Number of plasmids ranges from 5 to 1,000 per bacterial cell Low copy number plasmids High copy number plasmids 4

5 Plasmid Uses Two main uses To express recombinant proteins To house genes that have been cloned These can then be placed into other organisms (e.g. corn) 5

6 Modern Plasmids Plasmids are constructed to make cloning easy. They have an area called a multiple cloning site (MCS) that has a series of unique restriction enzyme recognition sites This MCS is used to open up the plasmid to receive the gene of interest Plasmid with a gene (red) inserted into the MCS (green) 6

7 Recombinant DNA Using Plasmids Steps Extract and purify plasmid and DNA of interest Digest plasmid and DNA of interest with restriction enzymes PCR can be used to amplify gene of interest Mix the two different DNA fragments together and add DNA ligase Transform plasmid into host cell Grow and select for cells that have insert 7

8 Transcriptional Regulation of Plasmids How operons work Jacob and Monod in 1961 discovered how the lac operon work in bacteria 8

9 Transcriptional Regulation of Plasmids How pbad operon works An operon in which arabinose is the inducer instead of lactose Different operons have different inducers 9

10 Transcriptional Regulation of Plasmids If the three genes BAD are cut out by restriction enzymes and GFP is ligated in their place, a recombinant operon is produced that expresses GFP 10

11 Other Types of Plasmids Shuttle plasmids Plasmids that can be inserted into bacteria initially to be cloned, then transformed into eukaryotic cells once duplicated and isolated Ti plasmid For example, to grow in E. coli, a plasmid needs a prokaryotic origin of replication and an antibiotic-resistant gene To grow in a eukaryote, it would need a eukaryotic origin of replication, a sequence for a poly A tail, a promoter, and a terminator sequence that would function in a eukaryotic cell Found naturally in Agrobacterium tumefaciens Causes crown gall disease in plants Can be modified to carry genes of interest into plants 11

12 Transforming Cells Two major methods of transformation Calcium chloride Electroporation 12

13 Calcium Chloride Transformation Steps Suspend bacterial colonies in 50 mm (0.05 M) calcium chloride Add plasmid DNA Place tubes on ice Heat shock at 42ºC and place on ice Incubate with nutrient broth Streak plates 13

14 Transformation of Bacteria Play video: Bacterial Transformation 14

15 How the Calcium Chloride Method Works In the presence of calcium chloride, plasmids are mixed with bacteria and heat shocked Plasmids move into the bacteria GFP Beta-lactamase Ampicillin Resistance 15

16 Why Calcium Chloride? Helps to neutralize the charge on DNA molecule, increasing probability of that molecule moving into the cell Ca ++ Ca ++ Ca ++ O O P O O CH 2 O Base Sugar O O P O Base O CH 2 O Sugar OH 16

17 What Happens in Each Step? Incubate on ice Slows fluid cell membrane Heat shock Increases permeability of membranes Nutrient broth incubation Allows beta-lactamase expression 17

18 Electroporation Electroporation works by Using electricity to disrupt the bacterial wall and membranes Plasmids move in during disruption 18

19 Other Methods of Moving DNA into Cells Biolistics Using microparticles to shoot or blast small particles coated with DNA into cells Plants have a cell wall that is difficult to disrupt to move DNA into cells Transfection Plasmids are placed into lipid vesicles The vesicles merge with cell membranes and deliver DNA into the cells 19

20 Methods to Select Transformed Cells Antibiotic selection When bacteria are plated onto agar that contains antibiotic Bacteria that successfully incorporate a plasmid can grow in the presence of antibiotics due to the new enzyme on the plasmid 20

21 Selection of Transformed Cells Blue-white screening The β-galactosidase enzyme cleaves X-gal converting the X-gal into a blue color If a gene is successfully inserted into the MCS (shown in green), then it disrupts the cleavage of X- gal and will be white in color Antibiotic selection is also used to ensure that the bacteria were successfully transformed initially 21

22 Selection of Transformed Cells with an Insert pjet1.2 plasmid The plasmid contains the Eco47IR gene, which codes for a restriction enzyme that is toxic to E. coli If an insert is successfully inserted, then the Eco47IR gene is disrupted and the bacteria survive Antibiotic selection is still part of the plasmid 22

23 Transformation Efficiency Measurement of the number of transformed cells per microgram of plasmid DNA utilized Electroporation is the most efficient method Transformation with plasmid DNA is more efficient than with plasmid that has been ligated Transformation with ligated DNA requires cells with very high transformation efficiency (>10 6 CFU/µg of DNA) 23

24 Calculating Transformation Efficiency Example: 50 ng of plasmid DNA is transformed into a final transformation volume of 500 μl, and 10 μl of this volume is spread on an agar plate. Assume that 60 CFU are observed on the agar plate Note: 1 μg is 1,000 ng, so 50 ng = 0.05 μg of DNA 24

25 Calculating Transformation Efficiency Steps: First, count the number of colonies growing on the LB/ampicillin (LB/amp) agar plate. In this case, the CFU is 60 Next, determine the amount of plasmid DNA (in μg) spread on the LB/amp agar plate. In this example, only 10 μl of a 500 μl transformation was spread on the plate 25

26 Calculating Transformation Efficiency Steps: Next, calculate transformation efficiency by dividing the CFU by the amount of DNA spread on plate 26

27 Maximizing Transformation Efficiency E. coli divides once every 17 minutes Cells for purification of plasmids are typically harvested late in growth phase E. coli is optimally grown for hours at 37ºC with shaking 27

28 Purification of Plasmids Alkaline lysis method Uses detergent to lyse cells, releasing the DNA into solution Alkaline environment makes DNA single-stranded (plasmid and genomic) Acid allows the smaller plasmids to re-anneal; the longer genomic DNA strands only partially re-anneal Centrifuging pulls cell debris and genomic DNA to the bottom of the cell Plasmids are in the supernatant (liquid on top) 28

29 Purification of Plasmids 29

30 Purifying Plasmid Play video: Alkaline Lysis Miniprep 30

31 DNA Quantitation Gel quantitation Matching the intensity of bands on a gel with a band on the same gel that has a known quantity Unknown DNA band to quantify Known bands to compare 31

32 DNA Quantitation Spectrophotometric quantitation DNA absorbs UV light at 260 nm An absorbance of 1 at 260 (A 260 ) is equivalent to 50 µg/ml of doublestranded DNA So an absorbance of 0.5 would be equivalent to 25 µg/ml Single-stranded DNA with an absorbance of 1 is 33 µg/ml Single-stranded RNA with an absorbance of 1 is 40 µg/ml Often DNA is diluted before it is quantified, because it is very precious and one would not want to use it up to quantify it. It is often diluted from tenfold to 100-fold If the DNA is diluted, the dilution must be accounted for in the final concentration 32

33 DNA Quantitation 33

34 Determining the Concentration of DNA Play video: DNA Quantitation Using a Spectrophotometer 34

35 DNA Purity Spectrophotometer can be used to test DNA purity Often DNA is contaminated with protein. Proteins absorb UV at 280 nm This is tested by taking the absorbance at 260 nm and 280 nm A 260 :A 280 Pure DNA is >1.8 Pure RNA is >2.0 35

36 DNA Quantitation Fluorometer DNA is bound to a dye that fluoresces at a particular wavelength The fluorometer excites the sample at a particular wavelength and then measures emitted wavelengths Can measure samples at a much lower concentration than a spectrophotometer >1µg for a spectrophotometer nanograms for a fluorometer 36

37 Chapter 5 Summary Background History of Plasmids Plasmid Structure and Function Uses of Plasmids Recombinant Plasmids Transcriptional Regulation Transformation Transformation Selection Efficiency Purification of Plasmids DNA Quantitation Spectrophotometer Purity Fluorometer 37