Molecular biology

Lab 4:

Transformation of E.coli with pGAL.

Introduction: Bacterial transformation is of central importance in molecular biology. It allows for the introduction of genetically engineered or naturally occurring plasmids in bacterial cells. This makes possible the propagation, genetic expression and isolation of DNA plasmids. The transformation process involves the uptake of exogenous DNA by cells which results in a newly acquired genetic trait that is stable and heritable. Bacterial cells must be in a particular physiological state before they can be transformed. This state is referred to as competency. Most of the current transformation experiments involve E. coli. This organism does not enter a stage of competency unless artificially induced. Treatment to achieve competency involves the use of chloride salts, such as calcium chloride, and sudden hot and cold temperature changes. The metal ions and temperature changes affect the structure and permeability of the cell wall and membrane so that DNA molecules can be absorbed by the bacteria. Many different plasmids serve as useful tools in molecular biology. One example is the pGAL plasmid, present in multiple copies in specified host E. coli host cells. It contains 6751 base pairs and has been cleverly modified by genetic engineering. In the cell, it does not integrate into the bacterial chromosome, but replicates autonomously. The pGAL plasmid contains the E. coli gene which codes for -galactosidase. In the presence of artificial galactosides such as 5Bromo-4 Chloro 3-indolyl--D-galactoside (X-Gal), pGAL colonies appear blue when X-Gal is cleaved by -galactosidase and forms a colored product. This experiment has been designed to utilize EDVOTEK Transformation LyphoCells. It also contains the proprietary plasmid, pGAL (Blue Colony), which was engineered by EDVOTEK. Plasmid pGAL carries the complete gene for galactosidase. Since the host E. coli does not contain a -galactosidase gene, only cells transformed by the pGAL plasmid will produce the functional galactosidase enzyme. Cells that express -galactosidase will cleave XGal and the pGAL transformed colonies will be blue. In addition to the expression and cleavage of X-Gal by -galactosidase, transformation by pGAL is also demonstrated by resistance to ampicillin. E. coli host cells used in this experiment are not naturally resistant to ampicillin. The plasmid pGAL contains the gene which encodes for -lactamase that inactivates ampicillin. E. coli cells transformed by pGAL will express the resistance gene product -lactamase as an extracellular enzyme excreted from E. coli cells. Once outside the cell, the enzyme diffuses into the surrounding medium and inactivates ampicillin. Purpose: Preparation of E.colicompetent cells for transformation Develop and understanding of the biological process of bacterial transformation by plasmid DNA.

Opportunity to observe an acquired phenotypic trait of the transformed bacterial cells. Determination of transformation efficiency.

Materials and methods: Materials needed for this experiments: Transformation lyphocells, supercoiled pgal, control buffer (no dna), ampicillin, x-gal in solvent ,cell reconstitution medium, solvent for induction of competency, bottle ready pour luria broth agar, bottle luria broth medium for recovery, petri plates, plastic microtipped transfer pipets , inoculating loops (sterile) , microtest tubes with attached lids, automatic micropipet (5-50 l) and tips, two water baths (37c and 42c), thermometer, incubation oven (34c and 37c), hot plate or microwave oven,pipet pumps or bulbs, ice, marking pens, bunsen burner. DAY 1 Objectives: -Prepare Lypho Cells (overnight incubation). -Prepare agar plates Part I: Preparation of Lypho cells A 10 ml sterile pipet was used to add 6 ml sterile cell reconstitution medium (Component F) to the vial of LyphoCells. The rubber stopper was replaced and capped. It was then mixed by inverting until the freeze dried plug was dissolved. The cell suspension was shaked vigorously and the vial was incubated at 34-37C for 16 - 24 hours (overnight) in an incubation oven. For optimal results, LyphoCells were incubated for 19 hours. Part II: Preparation of agar plates The ReadyPour LB Medium was heated, the water bath was Equilibrate at 60C. The cap on the ReadyPour medium bottle was loosened but not removed to allow for the venting of steam during heating. The plastic bottle was squeezed and vigorously shaked to break up the solid agar into chunks. The bottle of ReadyPour medium was heated by Hot plate or burner method outlined below. When completely melted, the amber-colored solution should appear free of small particles.

Hot plate or burner method: The bottle was placed in a beaker partially filled with water. The beaker was than heated to boiling over a hot plate or burner. Using a hot glove, the beaker was occasionally swirled to expedite melting. The melted ReadyPour medium was allowed to cool. The bottle was placed in a 60C water bath which allowed the agar to cool, while preventing it from prematurely solidifying. When the ReadyPour medium reaches approximately 60C, the bottle will be warm to the touch but not burning hot. A lab marker was used to "stripe" the sides of twenty (20) 60 x15 mm petri dishes. This will provide an easy method of differentiating between plates with ampicillin and plates without ampicillin. The plates with ampicillin was striped. The Plates were poured after the medium was cooled. Thaw and all of the X-Gal solution (Component E) was added to the molten and cooled ReadyPour medium. The bottle was recapped and swirled to mix. A fresh 10 ml pipet was used to pipet pump to pour 10 unstriped plates, 5 ml each. The ampicillin powder (entire contents of tube D) was then added to the remaining molten ReadyPour medium. The bottle was recapped and swirled to completely dissolve the ampicillin powder. The pipet from step 8 was used to pour 20 striped plates, 5 ml each. Pour extra plates with any remaining medium. The agar was allowed to cool and resolidify. DAY 2 Objective -Preparation of competent cells .Transformation of competent E.coli cells with pGAL . The competency induction solvent (G) was placed on ice, the vial of incubated cells was mixed and resuspended by inverting and gently shaking the vial was placed on ice for 10 minutes. 10 ml sterile pipet was used to add 3 ml of ice cold competency induction solvent (G) to the vial of cells.The the cells was mixed and induction solvent was mixed thoroughly by inverting the vial several times, the cells was kept on ice for a minimum of 30 minutes. Dispensing the Cells just prior to the Experiment The cells was mixed by inversion to obtain an even suspension. a sterile 1 ml pipet was used to aliquot 0.7 ml of cells to 10 ice cold tubes labeled "Cells the tubes were caped and placed on ice. Preparation of DNA and Control Buffer The tubes of supercoiled pGAL DNA (Component B) and control buffer (Component C) were placed on ice, . Before dispensing DNA and Control buffer the tubes were taped until all the sample is at the tapered bottom of the tube. Using an automatic micropipette 25 l of the supercoiled pGAL DNA was dispensed to each of the 10 microtest tubes labeled "pGAL DNA", the

tubes were caped and placed on ice. Using a FRESH micropipette 25 l of control buffer was dispensed to each of 10 microtest tubes labeled "Control Buffer" ,the tubes were caped and placed on ice. SETTING UP THE TRANSFORMATION AND CONTROL EXPERIMENT Initials or group number was written on the tubes labeled pGAL DNA (contains 25 l of plasmid DNA) and Control Buffer (contains 25 l of buffer) . Place them back on ice. Set up the Control: Using a sterile transfer pipet, 0.25 ml (250 l) of cell suspension was transferred from the tube Cells to the tube Control Buffer.The tube was caped and mixed by tapping. Place them back on ice Set up the transformation: Using a sterile transfer pipet transfer 0.25 ml (250 l) of cell suspension was transfered from the tube Cells to the tube pGAL DNA.the tube was caped and mixed by tapping. Place them back on ice. The cells prepared was incubated on ice for 10 minutes, both transformation tubes was kept at 42Cfor 90 seconds. (This heat shock step facilitates the entry of DNA in bacterial cells.) both tubes was returned to the ice bucket and incubated for for 1 minute , 0.75ml of the Recovery Broth was added to the tube Control Buffer. 0.75ml of the Recovery Broth was added to the tube pGAL DNA. the closed tubes was incubated in a 37C water bath for 30 minutes for a recovery period. While the tubeswere incubating, 3 agar plates were labeled as indicated below. one unstriped plate: X-GAL/Control 1 one striped plate: AMP/X-GAL/Control 2 one striped plate: AMP/X-GAL/pGAL After the recovery period, the tubes were removed from the water bath and placed on the lab bench. Plating cells from the tube labeled "Control": A sterile 1 ml pipet was used to transfer recovered cells from the tube "Control Buffer" to the middle of the following plates: 0.25 ml to the plate labeled X-GAL/Control 1 0.25 ml to the plate labeled AMP/XGAL/Control 2 the cells were spread over the entire plate with a sterile inoculating loop, both control plates were covered to allow the liquid to be absorbed. Plating cells from the tube labeled "pGAL DNA"

A sterile 1 ml pipet was used to transfer recovered cells from the tube "pGAL DNA" to the middle of the following plate: 0.25 ml to the plate labeled AMP/X-GAL/pGAL the cells were spread over the entire plate with a sterile inoculating loop, both control plates were covered to allow the liquid to be absorbed (approximately 15-20 minutes). Preparing Plates for Incubation The plates were stacked on top of one another and taped and was left in the upright position to allow the cell suspension to be absorbed by the agar.

Results & discussion: The main aim of the following experiment was to make bacteria take up a plasmid and by placing the bacterial cells mixture with plasmid on different growth conditions to check for the transformation efficiency. As performed, 3 spread plates of LB media each contain different constituents: 1. Plate 1 X-GAL (Control 1) 2. Plate 2- X-GAL/AMP (Control 2) 3. Plate 3 X-GAL/AMP/pGAL

Figure1: Illustrate the plate 1 with X-GAL/ Control 1(left) and plate 3 with AMP + X-GAL + PGAL (right)

Figure 2 Plate 2- X-GAL/AMP (Control 2)

Plate 1 Here white colonies appear. Bacteria grown on this plate have no plasmid as transformation didnt take place however there was growth because the agar constituents were X-GAL only and thus the growth of bacterial colonies was demonstrated, as shown in figure 1. Plate 2 Here there was no growth of bacterial cells. The presence of AMP constituent in the media prevented bacterial growth, as shown in figure 2. Plate 3 Blue colonies of bacteria here indicate that transformation of pGAL plasmid into the bacteria occurred successfully. Presence of beta-galactosidase gene on the pGAL plasmid broke down XGAL in the LB media and oxidized it forming blue color colonies, as shown in figure 1.

In gene cloning, X-gal is used as a visual indication of whether a cell expresses a functional -galactosidase enzyme in a technique calledblue/white screening. This method of screening is a convenient way of distinguishing a successful cloning product from other unsuccessful ones.The blue/white screening method relies on the principle of -complementation of the -galactosidase gene, where a fragment of the lacZ gene (lacZ) in the plasmid can complement another mutant lacZ gene (lacZM15) in the cell.

EFFICIENCY: determine transformation efficiency

References: http://www.edvotek.com/221 http://sargentwelch.com/transformation-of-e-coli-with-pgal-dna/p/IG0040536/