Bacterial Transformation and Plasmid Purification Chapter 5: Background

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.

Origin of Plasmids Joshua Lederberg and William Hayes independently discovered plasmids while studying conjugation. 1952 Lederbergy proposed the name plasmid. In 1961 Tsutomu Watanabe and Toshio Fukasawa found that some plasmids carried antibiotic resistance genes. 1962 Allan Campbell determined that plasmids where 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.

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 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. Range from 5-1000 in a bacteria. Low copy number plasmids High copy number plasmids

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

Modern Plasmids Plasmids are constructed to make cloning easy. They have a 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 be ready to receive the gene of interest. Plasmid with a gene(red) inserted into the MCS (green)

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.

Transcriptional Regulation of Plasmids How operons work… Jacob and Monod in 1961 discovered how the Lac Operon worked in bacteria.

Transcriptional Regulation of Plasmids How pBAD operon works… A different operon where arabinose is the inducer not lactose. Different operons have different inducers.

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 the gene of interest.

Other types of plasmids Shuttle Plasmids Plasmids that can be inserted into bacteria initially to be cloned, then transformed into eukaryote cells once duplicated and isolated. 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, and a promoter and terminator sequence that would function in a eukaryote cell. Ti plasmid Found naturally in Agrobacterium tumefaciens. Cause crown gall disease in plants. Can be modified to carry genes of interest into plants.

Transforming cells in biotechnology labs Two major methods of transformation Calcium Chloride Electroporation.

Calcium chloride transformation protocol 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

How to transform bacteria Play video: Bacterial Transformation

How 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

Why calcium chloride? Ca++ O Helps to neutralize the charge on DNA molecule, increasing probability of moving into the cell. Ca++ O P O Base O O CH2 Sugar O Ca++ O P O Base O CH2 O Sugar OH

Why incubate on ice, heat shock and incubate with broth? slows fluid cell membrane Heat-shock Increases permeability of membranes Nutrient broth incubation Allows beta-lactamase expression

Electroporation Electroporation works by… Uses electricity to disrupt the bacterial wall and membranes. Plasmids move in while briefly disrupted.

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

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.

Methods to select cells that were not only transformed but have an insert. Blue/White Screening The β-Galactosidase enzyme breaks a X-gal down converting the X-gal into a blue color If a gene is successfully inserted into the MCS (shown in green), then it will be white in color. Antibiotic selection is also used to ensure that the bacteria were successfully transformed to begin with.

Methods to select cells that were not only transformed but have an insert. pJET1.2 plasmid The plasmid containes 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.

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 plasmid that has been ligated. Transformation with ligated DNA requires cells with very high transformation efficiency. >106 CFU/µg of DNA

Sample Transformation Efficiency 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. Let’s 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.

Sample 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.

Sample Transformation Efficiency Steps Next, calculate transformation efficiency by the CFU by the amount of DNA spread on plate.

E. coli divides once every 17 minutes. When streaking bacteria they need to be actively growing get maximum transformation efficiency. E. coli divides once every 17 minutes. Cells are typically harvested late in growth phase for purification of plasmids. E. coli is optimally grown for 16-24 hours at 37ºC with shaking.

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)

Purification of plasmids

How to purify plasmid Play video: Alkaline Lysis Miniprep

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

DNA Quantitation Spectrophotometric Quantitation DNA absorbs UV light at 260 nm An absorbance of 1 at 260 (A260 ) is equivalent of 50 µg/ml of double stranded DNA. So an absorbance of 0.5 would be equivalent to 25 µg/ml Single stranded DNA with the absorbance of 1 is 33 µg/ml Single stranded RNA with the 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 10 to 100 fold. If the DNA is diluted, the dilution must be accounted for in the final concentration.

DNA Quantitation

Determining the concentration of DNA Play video: DNA Quantitation Using a Spectrophotometer

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 260nm and 280nm. A260:A280 Pure DNA is >1.8 Pure RNA is >2.0

DNA Quantitation Flurorometer DNA is bound to a dye that fluoresces at a particular wavelength. The flurorometer 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.

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