Transformation of Bacteria

Student Lab Guide

Biotechnology Teaching Laboratory 631-632-9750

Name: ________________________________ Date of Lab: ______________

Lab partner(s): _________________________________________________________

Station Number: _____________

or RFP

or Red

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NEW TERMS Transformation – the process of an organism taking up DNA from the environment. The

additional DNA is not necessary for survival of the organism but can lead to a

genotype and/or phenotype change. Many plasmids contain genes that are

advantageous to the organism.

Plasmid – a small, circular piece of DNA which contains a few genes. A plasmid is separate

from the chromosome. These genes are not necessary for the survival of the

organism (usually bacteria) but could provide advantageous traits.

Competent – When an organism has the ability to be transformed, take up DNA from the

environment, the organism is considered competent.

Recombinant DNA – A DNA molecule that is made up of segments of DNA from more than

one source.

Arabinose – a monosaccharide that activates the promoter in the Arabinose operon

Terms to be familiar with

Genotype Phenotype

Bacteria

Antibiotic

Resistance

DNA ligase

Chromosome

cDNA

Introns

Exons

Transduction

Conjugation

Restriction Enzymes

Transcription

Translation

Promoter

Operon

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INTRODUCTION:

Bacteria have a single circular chromosome; however, they can also carry other

small circular, self-replicating DNA molecules called plasmids. Plasmids do not carry

genes required for survival under normal conditions, but may have genes that give an

advantage. For example E. coli is normally found living within the intestines of humans and

animals. If the E. coli carries an "invasion" plasmid, it can invade the mucosal cells lining

the intestine. In this example the plasmid allows the bacteria to colonize a new niche.

Plasmids can benefit the bacteria in a variety of ways. Some plasmids carry genes

for making toxins, some carry genes for antibiotic resistance. Antibiotic resistant bacteria

are becoming a major health problem because new antibiotics must be developed to treat

diseases such as skin infections, throat infections and food poisoning. The FDA is

considering changing the rules for antibiotic use on crops and in farm animals to help slow

the emergence of antibiotic resistant bacteria.

We also benefit from bacterial plasmids when they are used in bacteria for genetic

engineering and gene cloning. For example, the human insulin gene is only a small part of

the entire human genome, but it is an important gene for drug development and diabetes

research. Scientists trying to study the gene needed a way to make lots of copies of the

gene. The first step was to make a DNA copy of the mature messenger RNA (cDNA). This

produced a DNA copy of the gene without introns. Researchers used restriction enzymes

and DNA ligase to join the insulin-gene cDNA to a plasmid. The researchers then put the

plasmid into E. coli by transformation. Now they could use bacteria to make lots of copies

of the gene and in this case the bacteria produced insulin to treat diabetics. Many other

human, plant and animal genes have been cloned using this approach.

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Bacteria can pick up DNA from their environment in a variety of ways:

transformation, transduction and conjugation. Each of these introduces new DNA,

thereby changing the genotype of the cell, and may change the phenotype. In this

laboratory exercise you will change the genotype of Escherichia coli (E. coli) by

transformation. The bacteria will gain two new genes one for antibiotic resistance and one

for production of a fluorescent protein.

The plasmids you will be working with carry a gene either a green fluorescent

protein or a red fluorescent protein. A research scientist designed and built these plasmids

using: cDNA of the gene, PCR, plasmid DNA, restriction enzymes and DNA ligase (see Figure

1). In the pGlo plasmid, the Arabinose operon promotor controls expression of the green

fluorescent protein (see Figure 2 and 4). When the sugar Arabinose is present, the

promotor is activated; then the gene is transcribed and message translated into protein. If

the promotor is not active, no protein is made. You may be wondering why anyone would

want glowing E. coli. Recombinant plasmids and fluorescent proteins are often used to

study how transcription is regulated. Regulation of gene transcription by operons in

bacteria is well characterized; regulation of protein expression in mammalian cells is more

complex and less well understood. Remember, each of the nucleated cells of your body

carry the same DNA, but regulation of protein expression lets cells do different tasks.

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FIGURE 1: Making Recombinant DNA

1) Isolate cDNA containing the gene for green fluorescent protein. Today PCR is the most common way to isolate a gene.

2) Cut the DNA with Restriction Enzymes to produce sticky-ends

3) Cut a bacterial plasmid with Restriction Enzymes to produce compatible sticky ends.

4) Use agarose gel electrophoresis to isolate the desired DNA pieces and get rid of the rest.

5) Use DNA ligase to join the cut gene to the cut plasmid. The compatible cohesive ends will help align the pieces correctly.

6) Transform bacteria with the recombinant plasmid.

Gene for Green Fluorescent

Protein GGG

CCCGGG

AAA

AAATTT

Plasmid

CCCGGG

CCC

AAATTT

TTT

Gene for Green

Fluorescent Protein AAA

AAATTT

The plasmid now carries the

Gene for Green Fluorescent

Protein

Recombinant

Plasmid

Cut with

restriction

enzymes Plasmid

CCCGGG

CCC

AAATTT

TTT Plasmid

GGG

CCCGGG

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FIGURE 2: pGlo Plasmid FIGURE 3: pRed Plasmid

pGlo

“plasmid” fluorescent

= Origin of Replication (allows the

plasmid to be copied by the cell)

Ori

Am

pr

Gene for Ampicillin

resistance

GFP

Gene for Green Fluorescent ProteinArabinose promoter

Arap

circular

double-

stranded

DNA

Ara

C

Gene for the

Ara C protein

FIGURE 4: Gene Regulation by the Arabinose Promoter and Arabinose (only on pGlo plasmid)

GFP gene

gene Promoter region

DNA

GFP gene

GFP gene

+ Arabinose

Ara C

GFP gene

RNA

polymerase

transcription

No transcriptionRNA

polymerase

Arabinose

(always on)

(always on)

Gene for Red Fluorescent Protein

pRed

Ori

Ampr

RFP

(always on)

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OVERVIEW OF PROCEDURE:

A) Transformation Procedure

B) Plating Your Bacterial Samples

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PROCEDURE: DON’T FORGET TO USE ASPECTIC TECHNIQUE!

A) TRANSFORMATION PROCEDURE

1) Obtain a chillette which should contain the following three tubes:

Purple Tube Plasmid - Mixture of pGlo & pRed

Yellow Tube Calcium Chloride

White Tube LB Broth

2) Take a blue tube of E.coli bacteria from the ice bucket.

Immediately transfer it to your chillette.

Blue Tube Escherichia coli

3) Now that all four tubes are in your chillette, label the tubes with your initials.

4) With another group, centrifuge the tube of calcium chloride for 10 seconds.

5) Add 10 µL of Calcium (Ca2+) chloride to the bacteria. Gently mix the Calcium with

the bacteria by flicking the tube.

6) Transfer 25 µL of the calcium-treated bacteria to the tube of plasmids. Gently mix

the plasmids with bacteria by flicking the tube.

The purple tube now contains _________________________________________________________.

The blue tube now contains ____________________________________________________________.

7) Place the two tubes containing bacteria and bacteria plus plasmids into the cold

chillette and incubate for 5 minutes.

8) Carry your chillette over to water bath.

9) Heat shock the bacteria by transferring both tubes into the 37 C water bath for 5

minutes. Then return the two tubes of bacteria to the chillette.

10) Add 450 µL of LB to both tubes. Flick to mix.

11) Incubate both tubes in a 37°C water bath for 10 to 15 minutes (Recovery).

12) Transfer the two tubes to the rack on your bench and wait for instructions.

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B) PLATING YOUR BACTERIA SAMPLES

1) Using your experimental design as a guide, label your plates with today’s date,

your initials, what kind of plate it is, and what sample you will put on that plate.

Plates should be labeled on the agar side (not on the cover – in case the covers get

mixed-up).

2) Pipette 100 µL of your two samples on the appropriate plates.

3) Using the yellow spreaders, distribute your sample

evenly across the plate.

4) Leave the plate on the bench for a few minutes to allow the liquid to soak into the

agar.

C) FINISHING UP

1) Clean up your workspace by placing tubes into the scientific waste and the

spreaders into the buckets.

2) After plates dry, invert them and tape your plates in a stack. Then make sure to

wash your hands.

3) Wash hands and clean lab bench with Lysol®

D) BACTERIAL GROWTH AND OBSERVATIONS

1) Your teacher will collect your plates and incubate them for two to three days at

room temperature.

2) After incubation, you will compare growth on your plates. Colonies producing red

fluorescent protein (RFP) will appear pink or reddish in regular light. Colonies

producing green fluorescent protein (GFP) will glow green under black light (long-

wave ultraviolet (UV) light).

3) The plates must be decontaminated before they are thrown away.

Why do you only have two

spreaders?

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DATA ANALYSIS:

1) We have several terms for describing bacterial growth. Discuss the differences

between lawns of bacteria and isolated colonies.

2) Check the growth on your positive and negative control plates. Is the growth as you

expected? Explain why or why not.

3) Can you count individual colonies on your experimental plate? If so, how many

colonies do you have?

4) Check your plates for contamination. How can you recognize contamination?

5) How do plasmids differ from chromosomes?

6) Plasmids occur naturally; how might a plasmid benefit a bacterial cell?

7) How are plasmids used in Biotechnology?

8) In your transformation 10µL of DNA was used. The concentration of the DNA was

10ng/µL. Count the number of transformants (colonies) on your plate. Calculate

the transformation efficiency (# of transformants/ µg DNA) of your experiment.

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DATA ANALYSIS: ANSWER KEY

1) We have several terms for describing bacterial growth. Discuss the differences

between lawns of bacteria and isolated colonies. Lawn a continuous sheet of growth, all the cells plated grow. Isolated colonies only individual cells grow from the original inoculation. Each colony is the descendants of a single transformed cell -genetically they are all alike.

2) Check the growth on your positive and negative control plates. Is the growth as you expected? Explain why or why not. You should have a lawn on the LB plates. If there is no growth the cells may have died during transformation or the plate is not LB. You should have no growth on the LB-amp control, if you have a lawn the ampicillin was no good or the bacteria were ampicillin resistant before transformation. If you have isolated colonies you probably mixed up you with and without plasmid sample tubes.

3) Can you count individual colonies on your experimental plate? If so, how many colonies do you have? This tells you the efficiency of the transformation. You should expect around 100 colonies. Do not count the satellite colonies.

4) Check your plates for contamination. How can you recognize contamination? E. coli makes small glistening whitish grey colonies. Large, fluffy or colored colonies are contaminants.

5) How do plasmids differ from chromosomes? They are not essential for replication or survival under normal conditions.

6) Plasmids occur naturally; how might a plasmid benefit a bacterial cell? Confer new traits which give the cell a growth advantage.

7) How are plasmids used in Biotechnology? To clone genes for research, drug development and genetic engineering.

8) In your transformation 10µL of DNA was used. The concentration of the DNA was 10ng/µL. Count the number of transformants (colonies) on your plate. Calculate the transformation efficiency (# of transformants/ µg DNA) of your experiment. The formula for calculating efficiency is as follows: # colonies on plate / ng of DNA plated x 1000ng/µg Their experiment had total volume of 485µL 25 µL of E. coli, 450 µL LB Broth, 10 µL of DNA. There is 100ng of DNA in 485µL. They plated 100µL of solution, which contains 20.6 ng of DNA.

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Growth and Disposal of Plates containing Ampicillin Resistant E. coli and green or red fluorescent protein.

Growth:

1. Grow plates for two to three days at room temperature (over the weekend is good)

or for 24 to 48 hours at 37C.

2. Plates incubated in room temperature appear to have a stronger expression of the

red fluorescent protein compared to when grown at 37oC. The red fluorescent

protein may be more stable or folds better into its proper conformation at a lower

temperature. Expression of green fluorescent protein is strong at either 37oC or

room temperature.

3. Examine experimental plates using a blacklight, long-wavelength ultra-violet light or

under subdued fluorescent lights. If you store the plates for a few days wrapped in

plastic wrap in the refrigerator the green color will become more obvious.

Disposal:

The best method is to autoclave the plates in an autoclavable waste bag 121C, 10-20

minutes. I have also autoclaved plates in an old metal pan. The plastic hardens to a lump

which you can throw away and pour the molten agar and bacteria corpses into a disposable

container and throw away after the agar hardens. It is messy but it works!

If an autoclave is not available, alternative method is to pour chlorine bleach on top of the

agar. Let the plates soak for one hour and then dispose as normal trash.