experiment 8: bacterial transformationbiol 2281, spring 2016 e8: bacterial transformation...

Biol 2281, Spring 2016 E8: Bacterial Transformation Procedure/Report

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Experiment 8: Bacterial Transformation

Objectives:

At the end of this exercise, you will be able to

1. Understand the concepts of plasmids and genetic transformation.

2. Perform a bacterial transformation with appropriate laboratory techniques.

3. Describe the function of a selectable marker in the isolation of transformants. 4. Analyze the results and calculate the transformation efficiency.

This topic was adapted from pGLOTM

Bacterial Transformation kit, Bio-Rad Laboratories, Inc. Duplication of

any part of the document is permitted for classroom use only.

One of the most important techniques of modern molecular biology is the ability to introduce a specific

gene into an organism and have that genetic information expressed by the organism. This process,

called genetic transformation, played a critical role in the history of molecular biology and the

discovery that DNA was the genetic material. Four scientists, Griffith in the 1920's and Avery,

McCarty, and MacLeod in the 1940's, showed that the apparent "transformation" of one bacterial type

into another required DNA and no other biological molecule.

Today, transformation is not limited to bacteria, and in fact, is used routinely by molecular biologists

to manipulate and study the behavior of genes. In agriculture, genes coding for traits such as frost, pest,

or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be

genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by

defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick

person’s cells with healthy copies of the defective gene that causes the disease.

You will use a procedure to transform bacteria (Escherichia coli K-12:HB101) with a gene that codes

for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish

Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark.

Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and

produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet

light. In this activity, you will learn about the process of moving genes from one organism to another

with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more

small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more

traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and

forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to

new environments. The recent occurrence of bacterial resistance to antibiotics is due to the

transmission of plasmids.

Bio-Rad’s unique pGLO plasmid (p=plasmid) encodes the gene for GFP and a gene (bla) for beta-

lactamase that provides resistance to the antibiotic ampicillin. pGLO also incorporates a special gene

regulation system, which can be used to control expression of the fluorescent protein in transformed

cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the

cells’ nutrient medium (see Appendix ). Selection for cells that have been transformed with pGLO

DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type


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phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the

nutrient agar medium. Arabinose (C5H10O5) is found in nature and can be metabolized by

microorganisms as a carbon source.

In the lab, E. coli cells are more likely to incorporate foreign DNA if their cell walls and cell

membrane are altered so that DNA can pass through more easily. Such cells are said to be

"competent". E. coli cells are made competent by a process that uses calcium chloride and heat shock.

In addition, cells from exponential phase of growth are typically used in preparation of competent cells

since they give better transformation efficiency.

The transformation procedure involves three main steps. These steps are intended to introduce the

plasmid DNA into the host cells and provide an environment for the cells to express their newly

acquired genes.

Incubation: the cells mixed with a transformation solution of CaCl2 (calcium chloride) and the

plasmid.

Heatshock: the cells and DNA mixture are then subjected to a treatment at 42ºC for 50

seconds; the temperature changes destabilize cell membranes and create thermal imbalance.

Recovery: a short incubation period to begin expressing their newly acquired genes

Appendix

MAP of pGLO


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The Arabinose Operon: The three genes (araB, araA and araD) that code for three digestive

enzymes involved in the breakdown of arabinose are clustered together in what is known as the

arabinose operon. These three proteins are dependent on initiation of transcription from a single

promoter, PBAD. Transcription of these three genes requires the simultaneous presence of the DNA

template (promoter and operon), RNA polymerase, a DNA binding protein called araC and arabinose.

araC binds to the DNA at the binding site for the RNA polymerase (the beginning of the

arabinose operon). When arabinose is present in the environment, bacteria take it up. Once inside, the

arabinose interacts directly with araC which is bound to the DNA. The interaction causes araC to

change its shape which in turn promotes (actually helps) the binding of RNA polymerase and the three

genes araB, A and D, are transcribed. Three enzymes are produced, they break down arabinose, and

eventually the arabinose runs out. In the absence of arabinose the araC returns to its original shape and

transcription is shut off.

The Expression of Green Fluorescent Protein: The DNA code of the pGLO plasmid has

been engineered to incorporate aspects of the arabinose operon. Both the promoter (PBAD) and the araC

gene are present. However, the genes which code for arabinose catabolism, araB, A and D, have been

replaced by the single gene which codes for GFP. Therefore, in the presence of arabinose, araC protein

promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliant green as they

produce more and more GFP. In the absence of arabinose, araC no longer facilitates the binding of

RNA polymerase and the GFP gene is not transcribed. When GFP is not made, bacteria colonies will

appear to have a wild-type (natural) phenotype—of white colonies with no fluorescence.

This is an excellent example of the central molecular framework of biology in action:

DNA→RNA→PROTEIN→TRAIT


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Procedure Day 1

1. (TA) Streak E. coli HB101 on LB plate and incubate overnight at 37° C. LB plates should

be streaked for single colonies 24–36 hours before the transformation activity is planned.

Under favorable conditions, one cell multiplies to become millions of genetically identical

cells in just 24 hours. There are millions of individual bacteria in a single 1 mm bacterial

colony.

Procedure Day 2 Your workstation: (two students per group)

E. coli starter plate (shared by four

groups)

LB nutrient broth

Inoculation loops

Glass spreader

Ethanol beaker with lid

Bunsen burner and striker

Sterilized tip box

Micropipets: P200

Foam container full of crushed ice

Marking pen

Two sterilized microtubes

Microfuge-tube rack

Masking tape

Timer

1 LB plate (one black line)

2 LB/amp, (One black line, one red line)

1 LB/amp/ara (one black line, two red line)

Common workstation.

Transformation solution (CaCl2, 50 mM )

pGLO plasmid (0.05 μg/μl)

42°C water bath with floating tube rack, Timer

37°C incubator

1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your

group’s name. Place them on ice.

2. Use P1000 micropipet to transfer 400 μl of transformation solution (CaCl2) into the tube

labeled with -pGLO. Place the tubes on ice.

3. Use a sterile loop (flamed by using Bunsen burner) to pick up a single colony of bacteria

from your starter plate.

4. Immerse the loop into the transformation solution in the -pGLO tube. Spin the loop between

your index finger and thumb until the entire colony is dispersed in the transformation

solution (with no floating chunks). Place the tube back in the tube rack in the ice.

Transfer 200 μl of cell suspension from the -pGLO tube to the +pGLO tube. Discard the

used yellow tips in a small biohazard trashcan.

5. In dark room, examine the pGLO DNA solution with the UV lamp. Note your observations.

Would you see fluorescence in DNA tube? Why or why not? (On TA bench) Transfer 5 μl

pGLO DNA into the cell suspension of the +pGLO tube. Close the tube and return it on ice.

Do not add plasmid DNA to the -pGLO tube.

6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down to

make contact with the ice.


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7. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom (not

the lid) as follows:

• Label one LB/amp plate: + pGLO

• Label the LB/amp/ara plate: + pGLO

• Label the other LB/amp plate: - pGLO

• Label the LB plate: - pGLO

• Label all plates with sign-in numbers, section and the host strain.

8. Heat shock. Transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42oC,

for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the

bottom of the tubes stick out and make contact with the warm water. When the 50 seconds

are done, place both tubes back on ice. For the best transformation results, the transfer from

the ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2

minutes.

9. Remove the tubes from the ice and place on the bench top. Open a tube, add 200 μl of LB

nutrient broth to one tube and reclose it. Repeat with a new tip for the other tube. Close the

tube of LB and return it to TA. Incubate the tubes for 10 minutes at room temperature.

10. Tap the closed tubes with your finger to mix. Using a new tip for each tube, pipet 100 μl of

the cell suspensions from (+) pGLO and (-) pGLO tubes onto the appropriate nutrient plates.

11. Spread the suspensions evenly around the surface of the LB nutrient agar using glass-rod

spreader. Sterilize the spreader by dipping in the alcohol and passing through flame

before and after each spreading. Dispose excess cell suspension, used yellow tips in the

biohazard container.

12. Stack up your plates and tape them together. Put your group name and class period on the

bottom of the stack and place the stack of plates upside down in the 37°C incubator until

the next day. TA will store your plates at 4o C till next week. You will analyze the results

during your lab next week.


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Procedure Day 3 A. Data Collection

1. Carefully observe and draw what you see on each of the four plates under normal room lighting

and the ultraviolet light, respectively.

2. What color are the bacteria?

3. How many bacterial colonies are on each plate (use marking pen to count the colonies on the

bottom of plates).

4.

Plasmid added

LB/Amp

Plasmid added

LB/AmpP/Ara

No plasmid

LB/Amp

No plasmid

LB

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Group 8

Group 9

Group 10

Group 11

Group 12 0 50 (no fluorescene) 0 TNTC (too

numerous to count)

B. Analysis of Results

The goal of data analysis for this investigation is to determine if genetic transformation has

occurred.

1. Which of the traits that you originally observed for E. coli did not seem to become altered?

Which seem now to be significantly different after performing the transformation procedure?

2. From the results that you obtained, how could you prove that the presence of colonies on

LB/Amp/Ara is due to the effect of pGlO plasmid?

3. What are the two possible sources of fluorescence within the colonies when exposed to UV

light?” Note: Recall what you observed when you shined the UV light onto a sample of original

pGLO plasmid DNA and describe your observations. Which of the two possible sources of the

fluorescence can now be eliminated?


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4. Calculate Transformation Efficiency

The formula used below is one of the ways to calculate transformation efficiency. It tells us the

total number of bacterial cells transformed by one microgram of DNA. The transformation

efficiency is calculated using the following formula:

Transformation efficiency = Total number of cells growing on the agar plate

Amount of DNA spread on the agar plate (in μg)

1) Determine the Total Number of transformed Cells

Count the colonies on your LB/amp plate.

2) Determine the Amount of pGLO DNA in the Bacterial Cells Spread on the LB/amp Plate

a. Determining the Total Amount of pGLO plasmid DNA

(DNA in μg) = (concentration of DNA in μg/μl) x (volume of DNA in μl)

In this experiment you used 5 μl of pGLO at concentration of 0.05 μg/μl. Calculate the

total amount of DNA used in this experiment.

Total amount of pGLO DNA (μg) used in this experiment = __________

b. Determining the fraction of pGLO plasmid DNA (in the bacteria) that actually got spread

onto the LB/amp/ara plate:

Fraction of DNA used = Volume spread on LB/amp plate (μl)

Total sample volume in microfuge tube (μl)

You spread 100 μl of cells containing DNA from a test tube containing a total volume of

405μl of solution. Fraction of DNA = __________________

pGLO DNA spread = Total amount of DNA used in μg x fraction of DNA used

pGLO DNA spread on the LB/amp plate (μg) = ________

Now use the data above to calculate the efficiency of the pGLO transformation. Report your

calculated transformation efficiency in scientific notation.

Transformation efficiency = Total number of cells growing on the agar plate

Amount of DNA spread on the agar plate

Transformation efficiency = _________________ transformants/μg

(Optional) If a particular experiment were known to have a transformation efficiency of 3 x 104

cells/μg of DNA, how many transformants would be expected to grow on the LB/amp plate?

You can assume that the concentration of DNA and fraction of cells spread on the LB/amp

plate are the same as that of these procedures.

Number of colonies on LB/amp plate =____________________


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Laboratory Report: (20 pts total)

Turn in 2-a) Data analysis (printed on page 10) to your TA for grading at least 30 minutes before the

end of the lab session. You don’t have to repeat this section (part a) in your typed-up report.

Part (b) should be included in your report.

1. Include a title page and present data in a table format (see below) and drawings (or

photographs) with appropriate labeling. (2 pts)

Plates

Characteristics

Plasmid added Colony number Colony

fluorescence

2. Data analysis: (7 pts total)

a). During the lab, calculate the transformation efficiency of the following experiment using

the information and the hypothetical results listed below. Show your work and units.

DNA plasmid concentration: 6 x 10 -3

μg/μl

Cells incubated with 560 μl CaCl2 transformation solution and split into two tubes

20 μl pGLO plasmid solution to the tube of +pGLO

200 μl LB broth

50 μl cells spread on agar

213 colonies of transformants on the LB/amp plate

Total amount of pGLO DNA used (1pts) = __________

Fraction of DNA used (1pts) =________________

Micrograms of DNA spread on the plates (1pt) =____________

Transformation efficiency (1pts) =________________

b) Calculate the transformation efficiency of your OWN experiment after you obtain the

results the following week.

Number of colonies on LB/amp plate =______________

Total amount of pGLO DNA used = __________





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3. Discussion and conclusions: (Include the questions in your report)

1) Based on the experimental design, on which plate(s) should the genetically transformed

bacterial cells be located? Which plates should be compared to determine if successful genetic

transformation has occurred? What should growth on each of these plates look like? (2 pts)

2) What allows the transformed cells to grow on LB/Amp? How? (1 pt)

3) Why do transformed cells display fluorescence in the presence of arabinose? Explain the

mechanism. (2 pts)

4) Based on the analysis of your results, describe the evidence that indicates your transformation

experiment was successful or not successful? Offer a possible explanation if your result was

unsuccessful? Explain the possible errors resulting in the data of group 12 on page 6 (2 pts).

5) Which plate in your experiment is the negative control plate? What might cause growth on the

negative control plate? (1 pt)

6) What purpose does spreading –pGLO on the LB plate serve? What might cause your cells to

fail to grow on the LB plate? (1 pt)

7) If you spread 100 μl of the +pGLO transformation solution onto an LB plate, what should you

see after incubating the plate at 37-degrees for a few days and why? If you used the velvet

stamping method demonstrated in lab 3 to replica-plate the growth from this LB plate onto an

LB/Amp plate, what would the LB/Amp plate look like after a few days of incubation at 37

degrees. (2 pts)


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Section________________________ Name________________________Points_________

Turn in Part-a) Data analysis below to your TA for grading 30 minutes before the end of the lab

session. You don’t have to repeat this section in your typed-up report, but you may want to copy

your work and answers on page 8 for future reference.

a). During the lab, calculate the transformation efficiency of the following experiment using

the information and the hypothetical results listed below. Show your work and units.

DNA plasmid concentration: 6 x 10 -3

μg/μl

Cells incubated with 560 μl CaCl2 transformation solution and split into two tubes

20 μl pGLO plasmid solution to the tube of +pGLO

200 μl LB broth

50 μl cells spread on agar

213 colonies of transformants on the LB/amp plate

Total amount of pGLO DNA used (1pts) = __________




Biol 2281, Expt. 8

Drs. Wenju Lin & Elizabeth Pickett,

Spring 2016 1

Bacterial Transformation BIOL 2281

The Central Dogma of Molecular Biology

DNA

RNA

Protein

Translation

Transcription

Genetic transformation

• A process that introduces a specific gene into an organism and have that genetic information expressed by the organism.

• 1928- Griffith

• 1940- Avery, McCarty, and MacLeod – "transformation" of one bacterial type into

another required DNA and no other biological molecule.

Transformation Griffith’s experiment

Genetic transformation (cont.)

http://www.accessexcellence.org/RC/VL/GG/ecb/DNA_genetic_material.php

Transformation (cont.)

• Most naturally transformable bacteria develop competence (being able to take up DNA) only under specific conditions (nutritional stress).

• Cells are passively made permeable to plasmid DNA using laboratory procedures and are called competent cells. – calcium chloride and heat shock.

– electroporation, protoplast formation, and microprojectiles

Biol 2281, Expt. 8


Spring 2016 2

Plasmids • Small DNA molecules that are separate from the

chromosomal DNA

• Often contains 1. Gene of interest

2. Genes that confer a selective advantage to the bacterium harboring them

• Antibiotic resistance

3. Origin of replication

• Shuttle vector

4. “Polylinker” area (multiple endonuclease cleavage sites)

http://www.accessexcellence.org/RC/VL/GG/plasmid.php

Recombinant DNA technology

1. Obtain Plasmid DNA and DNA containing the gene of interest.

2. Insert the gene of interest into plasmid. 3. Put recombinant plasmid into bacterial cells.

– Efficiency? – Selection mechanism?

4. Obtain more of recombinant plasmid by growing bacterial cells.

5. The resulting recombinant plasmid can be used to transform another organism cells (bacteria or yeast cells) to change their phenotype.

Bacterial Transformation

A successful transformation requires: • Host cells: Escherichia coli K-12:HB101

• A plasmid containing a selectable marker and a gene of interest. – the gene (bla) for beta-lactamase

– the gene for GFP

• Procedures of placing the plasmid DNA into the host – Incubation

– heat shock

– recovery

• A selection mechanism: growth on media containing antibiotics (ampicillin, a member of the β-lactam family)

The Host Cells

Escherichia coli K-12:HB101

– Is a single-celled organism

– Is an organism which reproduces quickly.

– Is not able to infect plants or animals.

– Is not naturally antibiotic resistant • Can’t grow on antibiotic plates (LB/Amp)

LB, HB101, -pGLO LB/Amp, HB101 -pGLO

The Plasmid We Will Use

pGLO

• Selectable marker: the ampicillin resistant gene (bla or Ampr) which codes for beta-lactamase protein

• Origin of replication (ori)

• A “polylinker” area

• The gene for GFP (Green Fluorescent Protein)

• A special gene regulation system for GFP

– Promoter (PBAD) and araC gene

Biol 2281, Expt. 8


Spring 2016 3

pGLO plasmid Green Fluorescent Protein (GFP)

Originally isolated from the bioluminescent jellyfish, Aequorea victoria.

Excitation occurs when protein is exposed to ultraviolet light and emission is in the green portion of the visible spectrum.

Mutation of Chromophore Alters Color Genetics Terms Refresher

• Gene expression

– Constitutive

– Inducible

• RNA Polymerase

• Promoter – A region of DNA that initiates transcription of a particular gene

Expression of GFP in pGLO

• Inducible system – Arabinose present in growth

media • The gene for GFP will be switched on

• Transformed cells will appear fluorescent green under UV light

– No arabinose present in growth media • The gene for GFP will remain switched off

• Transformed cells will appear white under UV light

How Does Our Inducible System Work?

• Uses components of the arabinose operon

Biol 2281, Expt. 8


Spring 2016 4

The Arabinose Operon

In E.coli • Three enzymes are needed to digest arabinose as a food source.

• The genes coding for these enzymes are not expressed in the absence of arabinose.

• The genes are expressed when arabinose is present.

• This cluster of genes controlled by a single promoter, PBAD, and is called The Arabinose Operon.

• Transcription of these three genes requires the DNA template, RNA polymerase, a DNA binding protein called araC and arabinose.

Inducing GFP Expression from pGLO

The pGLO plasmid

• Contains the promoter (PBAD) and the araC gene from the arabinose operon.

• The three genes which code for arabinose catabolism have been replaced by the gene which codes for GFP.

Inducing GFP Expression From pGLO

The araC protein binds the pGLO DNA near the PBAD and blocks binding of RNA Pol.

Arabinose interacts with araC, changing its shape to a formation that helps recruit RNA Pol to the PBAD

Once RNA Pol binds, transcription starts. Translation follows and GFP is produced.

Cells transformed with pGLO

• In the presence of the ampicillin

– Cells can grow on media containing ampicillin

• In the presence of arabinose

– Cells fluoresce brilliant green as they produce more and more GFP.

• In the absence of arabinose

– Bacteria colonies will appear to be white colonies with no fluorescence.

Procedures of transformation

• Steps to enhance the transformation efficiency

1. Incubation: cells mixed with CaCl2

2. Heat shock: quick temperature changes

3. Recovery: short incubation

– Cells growing at log (exponential) phase are often used to

increase the transformation efficiency

In the lab

• Two groups of plates in this controlled experiment

– Transformation plates - the factor being tested is applied.

– Control plates - the factor being tested is not applied.

Biol 2281, Expt. 8


Spring 2016 5

Results of Transformation- normal room lighting

LB/Amp/Ara, +pGLO LB/Amp, +pGLO

LB, -pGLO LB/Amp, -pGLO

Results of Transformation- ultraviolet light

LB/Amp/Ara, +pGLO LB/Amp, +pGLO

LB/Amp, -pGLO LB, -pGLO

Data analysis

• Transformation efficiency = Total number of Transformants on LB/Amp plate

Amount of plasmid DNA (in μg) used in transformation

Read procedures

Study the steps of calculation on page 7

Turn in Part-(a) in Data analysis (p 10) for grading 30 minutes before

the end of your session in the lab.

Quiz samples

• What are the three main steps in procedures of transformation?

• What methods can be used to make cells permeable to plasmid DNA?

• Describe the important characteristics of the pGLO plasmid and host cells in this experiment.

• Which is the selectable media for transformants and which media will produce glowing colonies? Why?

experiment 8: bacterial transformationbiol 2281, spring 2016 e8: bacterial transformation...

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