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InvisibleLanaV
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How to make glow-in-the-dark bacteria
    #560335 - 02/23/02 03:07 AM (14 years, 9 months ago)

Thought some of you would find this NEAT :smile:

Lana

http://biotech.biology.arizona.edu/labs/Bacteria_glow_rapid_teach.html

What is genetic engineering? How to make glow-in-the-dark bacteria (Rapid colony transformation)- Teacher Guide
 
Photos by Faith Carter

What is genetic engineering, and how is this technique used? Students perform a genetic engineering experiment using bacterial transformation to introduce a bioluminescence gene into E. coli. Students transform E. coli with a plasmid that contains the lux bioluminescence gene to produce bacteria that glow in the dark. This activity helps students understand what genes do and how they can be manipulated by genetic engineering.

Although this method does not produce competent cells with high tranformation efficiency, this activity is much simpler to set up and requires less special equipment than the traditional method of preparing competent cells.

Classroom time needed for this lab:

two class periods (~50 minutes each)
Setting up the classroom:

Distribute for each pair of students:

2 microcentrifuge tubes containing 200 ul calcium chloride (50 mM).
Plate of E. coli.
1 microcentrifuge tube (0.5 or 0.65 ml size) containing 5 ul pUC18 DNA (10 ng/ul)
1 microcentrifuge tube (0.5 or 0.65 ml size) containing 5 ul pLux DNA (10 ng/ul)
3 ml LB broth in sterile tube
2 LB agar + ampicillin plates
Graduate micropipets with tips
2 sterile 1 ml disposable transfer pipets
2 sterile 0.3 ml disposable transfer pipets
Container of ice
Permanent marker
Sterile cotton swabs
Have a 370C incubator (for growing bacteria) and 370C water bath ready (for heat shock). If a water bath is not available, an incubator could be used for the heat shock step, but this is less accurate. You will need large waste beakers at the lab stations for disposing tips, transfer pipets, and cotton swabs. A biological waste bag is also needed for disposal of bacterial plates.

Be sure to prepare a plate of E. coli on LB agar for each group.

You will need to prepare the following materials (recipes to follow):

Ampicillin (100 mg/ml)
LB agar + ampicillin plates
LB agar plates
LB broth
Sterile toothpicks and flasks
Sterile 50 mM calcium chloride
During the lab:

Make sure that all materials contaminated with bacteria are disposed properly. After the plates are incubated overnight, you can store the plates containing bacteria at room temperature for a day or so until the class meets again. If any more time required, store the bacterial plates in the refrigerator.

Equipment Supplies
Magnetic stirrer LB agar mix (Life Technologies cat. no. 22700-025.)
Autoclave or pressure cooker LB mix (Life Technologies cat. no. 12780-052)
Bunsen burner Starter plate of E. coli (strain DH5)
370C Shaker Calcium chloride (anhydrous or dihydrate)
370C Incubator Ampicillin (sodium salt)
Clinical centrifuge Sterile filter (0.45 or 0.22 micron, Nalgene or Corning)
Deionized or distilled water
Aluminum foil
Petri dishes
Toothpicks
Cotton swabs
15 ml centrifuge tubes
Microcentrifuge tubes (0.5 or 0.65 ml)
Microcentrifuge tubes (1.5 ml)
Sterile pipets (5 or 10 ml)

Preparing LB agar plates with ampicillin
Ampicillin (100 mg/ml solution)

Dissolve 5 g of ampicillin (sodium salt, MW = 371.40) in 50 ml of deionized or distilled water.


Prewash a 0.45- or 0.22-micron sterile filter by drawing through filter 50 ml of deionized or distilled water. Pass the ampicillin solution through the filter and dispense 10-ml aliquots in sterile capped tubes or bottles. Freeze at -20oC.


LB agar plates with ampicillin

Add 32 g of LB agar mix (Life Technologies) to two liter flask:

Note: If you do not have a two liter flask, you can use 500 ml or 1000 ml flasks. Be sure to adjust the amounts proportionally (i.e., 500 ml of media in 1000 ml flask, 250 ml of media in 500 ml flask).


Add 1 liter of deionized or distilled water and mix to dissolve ingredients, preferably using a magnetic stirrer. The agar in the solution will not dissolve.


Cover the top of the flask with aluminum foil, and autoclave the solution for 20 minutes at 1210C. If you use a pressure cooker, you need to sterilize at 15 lbs pressure for 20 minutes, and temperature should reach 1210C or 2500F.


After autoclaving, swirl LB agar solution to distribute agar evenly.


Let the solution cool just until the flask can be held in your hands (55-600C). If the solution cools too long and begins to solidy, remelt it by briefly autoclaving for 5 minutes or less.


(Omit this step if you are preparing LB + agar without ampicillin.) Add 1 ml of the 100 mg/ml ampicillin to the 1 liter of LB agar solution. Antibiotics are sensitive to heat, so it is important to cool the solution to 55-600C before adding the antibiotic.


Swirl the flask to mix the ampicillin.


Mark sterile culture plates with description of media. If you are using presterilized polystyrene plates, the the plastic bag can be used for storing the poured plates.


To pour media into plates, carefully the lift the lid of the culture plate to pour the solution. Do not set the lid on the lab bench. Quickly pour in agar until the bottom plate is about 1/3 full. Immediately replace the lid. Repeat procedure for each plate, and occasionally flame the top of the flask. Always hold the flask slightly tilted to prevent airborne contaminants from falling into the flask.


Let the agar solidify, and store the plates lidside down overnight to dry the agar and minimize condensation.


Store the plates in a covered container or in the plastic bags in the refrigerator. The ampicillin in the media is effective for 1-2 months when stored in the refrigerator.


Luria-Bertani (LB) broth
Add 20 g of LB broth mix (Life Technologies) to a 2 liter flask.


Note: If you do not have a two liter flask, you can use 500 ml or 1000 ml flasks. Be sure to adjust the amounts of reagents proportionally (i.e., 500 ml of media in 1000 ml flask, 250 ml of media in 500 ml flask).




Add 1 liter of deionized or distilled water and mix to dissolve ingredients, preferably using a magnetic stirrer.


Divide the LB broth among several bottles (for example, 200 ml LB in 250 ml bottles or 100 ml LB in 125 ml bottles). Put on the caps loosely.


Autoclave the solution for 20 minutes at 1210C. If you use a pressure cooker, you need to sterilize at 15 lbs pressure for 20 minutes, and temperature should reach 1210C or 2500F.


Let the solution cool and tighten the caps. The LB broth can be stored at room temperature indefinitely.


Note: If the broth becomes cloudy after storage, this is a sign of microbial contamination, and the broth needs to be disposed. Add bleach to the broth (to a final concentration of ~10%), let stand for 15 minutes or more, then dispose in the sink.




50 mM calcium chloride
Dissolve 5.6 g of anhydrous CaCl2 (m.w. 110.99) or 7.4 g of the dihydrate form (m.w. 146.99) in 180 ml deionized or distilled water.


Add deionized or distilled water to make 200 ml of total solution. This solution, at 500 mM concentration, can be stored in a capped bottle in the refrigerator.


Mix 50 ml of the 500 mM calcium chloride solution with 450 ml deionized or distilled water. The concentration of calcium chloride is now 50 mM.


Dispense the CaCl2 solution (50 mM) into capped glass bottles. Autoclave or use a pressure cooker (20 minutes at 1210C at 15 psi.) with the bottles loosely capped.


After the bottles cool, tighten the bottle caps and store in the refrigerator. The solution will be cooled and ready for making competent cells (see below).


Sterile toothpicks and cotton swabs
Use flat toothpicks (not the pointed ones). Place flat toothpicks in a 50 ml glass beaker, with the wider end of the toothpicks at the bottom of a 50 ml beaker. Cover the beaker with aluminum foil.


Use cotton swabs with a wooden stick. Put cotton swabs in glass beaker, with cotton end at the bottom of beaker. Cover beaker with aluminum foil.


The toothpicks and cotton swabs, can be sterilized by two methods: a) Autoclave the beaker for 20 minutes at 1210C at 15 psi or b) Bake in a oven at 3000F for at least an hour.


Plating bacteria for single colonies
You will need to prepare an E. coli starter plate for each group of students. This procedure will allow students to work with single colonies in their transformation experiment.

Use a permanent marker to label the bottom of an LB agar plate with the date and strain used.


Pick a sterile toothpick from the container. The wider end of the toothpick is used for bacteria; the narrower end is held in your fingers. Remove the lid from the E. coli culture plate with your free hand. Do not place the lid on the lab bench. Hold the lid face down a little bit above the culture plate to prevent contaminants from falling onto the plate. Gently scrape a small mass of bacteria onto the toothpick.


In a small area of the plate, carefully spread the bacteria onto the agar surface. Try not to gouge the agar surface (it helps to hold the toothpick nearly parallel to the plate surface when spreading bacteria). After spreading bacteria, dispose toothpick in a waste container.


Using a fresh sterile toothpick, glide the end of the toothpick two times across the first bacterial streak, and then spread the bacteria on the toothpick on an adjacent section of the plate using a tight zigzag streak. This streak should cover about a third of the agar surface.


Using a fresh sterile toothpick, glide the end of the toothpick two times across the last bacterial streak, and then spread the bacteria on the toothpick on an adjacent third of the agar surface using a tight zigzag streak.


Incubate the plates upside down in a 370C incubator overnight. You should see single colonies on the last series of streaks across the plate.
 


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Offlineuniboner
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Re: How to make glow-in-the-dark bacteria [Re: Lana]
    #560948 - 02/23/02 09:19 PM (14 years, 9 months ago)

quite neat indeed!

Thank you!

peace,
uniboner


--------------------
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OfflineoOjonahOo
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Re: How to make glow-in-the-dark bacteria [Re: uniboner]
    #561396 - 02/24/02 10:40 AM (14 years, 9 months ago)

awesome!
Did you ever see that saturday night live weekend update bit where they were like...

Scientists in a New England Resarch lab have successfully spliced the dna of a jellyfish and monkey making the monkey's skin bioluminsecent." and they showed a picture of the glowing in the dark monkey.

"This was as reported in the New England Journal of Evil."

...man, that was great.


--------------------
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Offlinehomebrew
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Re: How to make glow-in-the-dark bacteria [Re: oOjonahOo]
    #561425 - 02/24/02 11:47 AM (14 years, 9 months ago)

The article on that came out sometime last year, I remember when I read it that the article stated the monkey was not expressing the GFP gene. Has something more come of this?


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Offlinehomebrew
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Re: How to make glow-in-the-dark bacteria [Re: homebrew]
    #561426 - 02/24/02 11:49 AM (14 years, 9 months ago)

oops, didn't see that you said it was from SNL


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OfflineZorba
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Re: How to make glow-in-the-dark bacteria [Re: Lana]
    #561840 - 02/24/02 08:51 PM (14 years, 9 months ago)

I did this in my genetics lab last year. Making E. coli competent to take up plasmids (a small piece of circular DNA containing a couple genes) can be done easily in a few different ways, and was the earliest example of genetic engineering in the early '70s if memory serves me correctly. We visualized the lux+ colonies with UV light and I never knew that they would actually glow in the dark although they did glow brightly under UV. That was a cool lab. It's easy to do if you have the right equipment but I don't know why anyone reading this would want to transform E. coli !!!

Cheers


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InvisibleJoshua
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Registered: 10/27/98
Posts: 5,389
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Re: How to make glow-in-the-dark bacteria [Re: Lana]
    #562030 - 02/25/02 01:15 AM (14 years, 9 months ago)

A similar techique can be used to transform tobacco plants.

I think tobacco companies are looking into a special cigarette that doesn't require "a light":).



Joshua 


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Edited by Joshua (02/25/02 01:41 AM)


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Offlineiangato
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Re: How to make glow-in-the-dark bacteria [Re: Joshua]
    #563133 - 02/26/02 12:39 AM (14 years, 9 months ago)

there are bio-luminescent fungi. i saw some on a site somewhere but i can't remember where it was...... i'll have to check up on that. i want to grow some next to the cubes. they could double as a night-light. adios. peace.


--------------------
a blurry dot dances among the shadows
bends the light
and fizzles into my pink and glowing mind

-ian gato


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OfflineRaiseTheDead
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Re: How to make glow-in-the-dark bacteria [Re: iangato]
    #11931843 - 01/30/10 09:29 PM (6 years, 9 months ago)

Quote:

iangato said:
there are bio-luminescent fungi. i saw some on a site somewhere but i can't remember where it was...... i'll have to check up on that. i want to grow some next to the cubes. they could double as a night-light. adios. peace. 



175g icing sugar


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Invisible99BOOMERMAN
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Re: How to make glow-in-the-dark bacteria [Re: iangato]
    #11952756 - 02/03/10 08:14 AM (6 years, 9 months ago)

Quote:

iangato said:
there are bio-luminescent fungi. i saw some on a site somewhere but i can't remember where it was...... i'll have to check up on that. i want to grow some next to the cubes. they could double as a night-light. adios. peace. 



It's a Panaeolus species,just can't remember,there are a few actually.


--------------------
Anything posted in this thread is just some well thought out bull-shit,which is completely for entertainment purposes only.
                                     
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OfflineMomofo
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Re: How to make glow-in-the-dark bacteria [Re: 99BOOMERMAN]
    #11962262 - 02/04/10 04:42 PM (6 years, 9 months ago)



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Invisible99BOOMERMAN
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Re: How to make glow-in-the-dark bacteria [Re: Momofo]
    #11966046 - 02/05/10 02:38 AM (6 years, 9 months ago)

So beautiful.I too wonder what makes the closed canopy forest the desired habitat,and why they glow.Natural defense or warning,"do not Eat me"??????


--------------------
Anything posted in this thread is just some well thought out bull-shit,which is completely for entertainment purposes only.
                                     
                                      AMU      "Q&A's  Thread"


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