3 Transformation - Lab Description PDF

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Lab Description...

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Bacterial Transformation Major objective of the lab The purpose of this lab is to make competent E. coli cells and to introduce isolated plasmid DNA from the DNA purification lab into these cells. Techniques • Make cells competent by calcium chloride exposure • Design and perform serial dilutions • Perform a transformation reaction • Bacterial plating using beads Theoretical concepts • Understand transformation as a means of introducing genetic material • Using plasmid genes as a method of selection and identification membrane in a way that favors the uptake of exogenous plasmid DNA. You will transform each of the two plasmids separately as well as mixed in order to determine the transformation frequency of plasmids. To determine the total number of bacteria, you will plate a serial dilution of untransformed bacteria on nutrient agar plates.

INTRODUCTION! Transformation is the genetic alteration of a cell resulting from the direct uptake and expression of exogenous genetic material from its surroundings. Bacteria that are capable of being transformed, whether naturally or artificially, are called competent. Transformation is one of three processes by which genetic material may be introduced into a bacterial cell, the other being conjugation (transfer of DNA between two cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage).

Recall that plasmids contain unique genes (reporter genes) that can help distinguish them. Following transformation, you will screen the bacteria for ampicillin resistance, betagalactosidase activity (results in blue colonies in the presence of X-Gal), and Green Fluorescent Protein (GFP) (a gene that causes a colony to glow green when exposed to UV light). Some plasmids may also have reporter genes under the control of specific promotors. Genes under the regulatory control of the arabinose promotor are only expressed in the presence of arabinose. Based on these features (plasmid size, restriction site positions, reporter genes and presence or absence of the arabinose promotor), you will identify the plasmids from several given plasmid maps.!

Under normal conditions, transformation occurs very infrequently – less than one in a million bacteria will take up naked DNA. The frequency of transformation can be improved by:

•Exposing

the bacteria to high concentration of calcium chloride

•Rapidly

changing the temperature (heat shock) Today you will be using the products of the two plasmid preps from the earlier DNA Purification Lab to transform E. coli cells. You will make the cells competent by a calcium chloride treatment. Cold calcium chloride (CaCl2) alters the cell -1  -

Bacterial Transformation

Transformation (Keep cells on ice at ALL times unless otherwise directed)

PROTOCOL - Week 1 Making Cells Competent

o Label 3 sterile 1.5 ml microcentrifuge: A, B and A+B.

o Obtain two microcentrifuge tubes. Pipet 1.5 ml of log phase E. coli culture into each.

o Using a sterile tip, add 50 µl of the competent cells to each of the transformation tubes.

o Spin the tubes for 1 minute at 8,000 RPM in the centrifuge. o The cells will form a pellet at the bottom of the tube. Discard supernatant and refill microcentrifuge tube with an additional 1.5 ml of E. coli culture. o Spin the tubes for 1 minute at 8,000 RPM in the centrifuge. o Discard supernatant

o Use a separate sterile pipette tip for each, transfer 2 µl (and no more) of the appropriate plasmid DNA to the new transformation tube of the same name. For the tubes labeled ‘A+B’, add 2 µl l of each plasmid. Pipette slowly up and down to mix. Do this on ice. o Incubate the tubes on ice for 30 minutes. This will allow time for the plasmids to adhere to the cell surface so they can enter the cell. o Transfer the tubes from the ice to a 45°C heat block for exactly 40 sec. Place immediately back on ice for two minutes. The heat shock will make the membrane more fluid as it heats up, allowing the plasmid to enter the cell. Because the cells are fragile, it is important to hold to the 40 sec. heat shock. o Using a sterile pipette tip, transfer the whole volume from each tube, to respectively labeled test tubes containing 0.4 ml SOC broth. Incubate for 45 minutes on a shaker in a 37°C water bath. This is the recuperation period, designed to allow the cells to recover from the calcium chloride treatment and the heat shock. Furthermore, the incubation period will allow the replication of the plasmid DNA that entered the cells allowing the bacteria to begin expressing the ampicillin resistance gene.

o Add 0.75 ml of ice-cold 50mM CaCl2 to each tube and use a p1000 pipette to gently resuspend the pellet until all the cells are uniformly suspended. Add an additional 0.75 ml of ice-cold CaCl2 to each tube. o Incubate the tubes on ice for 20 minutes. o Spin the tubes for 1 minute at 8,000 RPM in the centrifuge. o Discard supernatant. o Add 0.133 ml of ice-cold CaCl2 to each tube and resuspend with a pipette until all the cells are uniformly suspended. o Transfer the contents of both tubes into one microcentrifuge tube. Label the top “CC” for competent cells. o Place the competent cells back on ice and keep them there until needed.

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Bacterial Transformation

Plating Cells o Label 3 plates for transformed bacteria and contain Ampicillin and XGal.

o Use the glass bead method to spread 100µl of each culture on the corresponding plate. Spread for 15-20 seconds. Your TA will demonstrate this method. o Leave the plates at room temperature for 15 min to allow the liquid to be absorbed.

Transformation Plates - w/ Ampicillin & X-Gal

A

B

o Invert the plates and store in the labeled rack. They will be incubated at 37°C overnight, and transferred to the cold room until the next lab period.

A+B

o Add 50 µl of competent cells to 100 µl of LB broth and mix. (this will be your ‘undiluted cells’)

o ❑ The other 4 plates are for different dilutions of untransformed bacteria and do not contain ampicillin. These plates will be used for determining the total concentration of bacteria in your undiluted competent cells

o Make a 1:100, 1:10,000 and 1:1,000,000 serial dilution of this solution. Before you begin, diagram the serial dilutions (include how volume of each tube as well as how LB Plates (no ampicillin) - for determining bacterial titer much is transferred) and check with your TA before you proceed. o Use the glass bead method to spread 100µl of each dilution onto LB/nonAmp plates. undiluted 1:100 1:10,000 1:1,000,000 o Leave the plates at room temperature for 15 min to allow the liquid to be absorbed. o Invert the plates and store in the ! labeled rack. They will be incubated at 37°C overnight, and transferred to the cold room until the next lab period.

note - plates should always be labelled on the edge of the bottom half (TA will demonstrate), and include information on contents, dilution (if necessary), date, initials of both partners & section number! -3  -

Bacterial Transformation

PROTOCOL - Week 2

PROTOCOL - Week 3

o Create a data table to accommodate the counts from each plate. o Examine your plates. Identify and count freestanding colonies on each transformation plate. Observe your plates under UV light and determine which fluoresce (contain GFP). o Count colonies on one of each of the bacterial titer plates. You should count the plate with the most colonies but not so much that most of colonies are touching. Calculate original titer.

o Create a data table to accommodate the counts from each plate. o Examine the phenotype of each colony with and without UV light.

o Calculate the original concentration of each of the two competent cell types. o For each of the three plasmids, calculate the proportion of cells that were transformed. Colony Transfer You will now use colonies from each of your plates to determine if the plasmids contain a reporter gene under the control of the arabinose promotor (a gene that is only expressed in the presence of the sugar arabinose) o Obtain 3 plates containing arabinose. o Draw grids on each plate to accommodate 20 colonies. o Following the technique demonstrated by your TA, transfer colonies from each of your three transformation plates to the three arabinose plates

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Bacterial Transformation

TRANSFORMATION POST LAB Name _______________________ Lab Section __________________

1. Record the results of transformation (include #s of colonies) (2 pts) Unknown& Plasmid

Amp& Resistance

Beta-Galactosidase& Activity

Arabinose& promotor

GFP

A B

2. Based on your results (including restriction digest from the previous lab), identify plasmids A & B from the list of plasmid maps provided by your TA. If your data is not perfectly consistent with plasmids on the list, suggest reasons for the discrepancy. (4 pts)

3. Give the purpose for each of the following steps in transformation procedure. How might transformation efficiency differ if the time for each step is increased? decreased? (2 pts) a. Calcium Chloride treatment:

b. 45 min shacking at 37°C:

c. Heat shock at 42°C:

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Bacterial Transformation

4. What is the original concentration of your “undiluted” competent cells? (Show your work) (4 pts)

5. How efficiently has each of the plasmids transformed (what proportion of cells were transformed? For both plasmids mixed together? (show your work) (4pts)

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Bacterial Transformation

6. Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 µg of plasmid into a given volume of competent cells. The term is somewhat misleading in that 1 µg of plasmid is rarely actually transformed. Instead efficiency is routinely calculated by transforming 100 pg-1 ng of highly purified supercoiled plasmid under ideal conditions. (source: New England Biolabs) Transformation efficiency (TE) equation: TE = Colonies/µg/Dilution Colonies = the number of colonies counted on the plate µg = the amount of DNA transformed expressed in µg Dilution = the total dilution of the DNA before plating TE calculation example: Transform 2 µl (100 pg) of control pUC19 DNA into 50 µl of cells, outgrow by adding 250µl of NEB 10-beta/Stable Outgrowth Medium and dilute 10 µl up to 1 ml in NEB 10-beta/Stable Outgrowth Medium prior to plating 30 µl. If you count 150 colonies on the plate, the TE is: Colonies = 150 µg DNA = 0.0001 Dilution = 10/300 x 30/1000 = 0.001 TE = 150/0.0001/0.001 = 1.5 x 109 cfu/µg

What is the transformation efficiency for each plasmid? what about when combined? (4 pts)

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