20.109(F09): Mod 2 Day 1 Testing an engineered biological system: Difference between revisions
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(New page: {{Template:20.109(F09)}} <div style="padding: 10px; width: 640px; border: 5px solid #669999;"> ==Testing an engineered biological system== ===Bacteria as pixels=== ''E. coli'' do not norma...) |
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= | =<center>Testing an engineered biological system</center>= | ||
==Introduction== | |||
===Bacteria as pixels=== | ===Bacteria as pixels=== | ||
''E. coli'' do not normally respond to light but a recent publication [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] describes a combination of genes that lead to light-responsive expression of beta-galactosidase in ''E. coli''. When the cells are grown in the dark, transcription of lacZ is high and the indicator in the media turns black. When the cells are grown in the light, there is very little transcription of the lacZ gene and the media’s natural color (yellowish) shows through. '''How precisely this expression can be controlled (spatially and temporally through different experimental conditions) is what you will investigate during this experimental module.''' | ''E. coli'' do not normally respond to light but a recent publication [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] describes a combination of genes that lead to light-responsive expression of beta-galactosidase in ''E. coli''. When the cells are grown in the dark, transcription of lacZ is high and the indicator in the media turns black. When the cells are grown in the light, there is very little transcription of the lacZ gene and the media’s natural color (yellowish) shows through. '''How precisely this expression can be controlled (spatially and temporally through different experimental conditions) is what you will investigate during this experimental module.''' | ||
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Some media has been prewarming in the 42°C incubator for you. The media is traditional LB supplemented with ferric ammonium citrate and “S-gal,” an artificial substrate for beta-galactosidase. When the enzyme cleaves S-gal in the presence of iron, a black precipitate forms changing the color of the media. The media also has agarose so it will harden as it cools. All manipulations should be with your best sterile technique to avoid contamination of your cultures. | Some media has been prewarming in the 42°C incubator for you. The media is traditional LB supplemented with ferric ammonium citrate and “S-gal,” an artificial substrate for beta-galactosidase. When the enzyme cleaves S-gal in the presence of iron, a black precipitate forms changing the color of the media. The media also has agarose so it will harden as it cools. All manipulations should be with your best sterile technique to avoid contamination of your cultures. | ||
==Protocols== | |||
=== | ===Part 1: Test in solid media=== | ||
# Dilute the overnight culture of cells 1:10. Use LB to dilute the cells and make at least 250 μl of this dilution to use for today's experiment. | # Dilute the overnight culture of cells 1:10. Use LB to dilute the cells and make at least 250 μl of this dilution to use for today's experiment. | ||
# Add antibiotics (50 μl of ampicillin and 50 μl of chloramphenicol) to the prepared media. These will maintain the plasmids in the bacteria as the cells grow. Use the stir plate to mix but do not allow the media to remain at room temperature for long since it will begin to harden as it cools. | # Add antibiotics (50 μl of ampicillin and 50 μl of chloramphenicol) to the prepared media. These will maintain the plasmids in the bacteria as the cells grow. Use the stir plate to mix but do not allow the media to remain at room temperature for long since it will begin to harden as it cools. | ||
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# Pipet approximately 20 ml with a 25 ml pipet into two Petri dishes and allow the media to harden on the bench before moving the plates into the incubator with the yellow hydroponic lamp. One Petri dish should be placed in a box as the “dark” sample and the other can be placed into the incubator as is…media side up of course. | # Pipet approximately 20 ml with a 25 ml pipet into two Petri dishes and allow the media to harden on the bench before moving the plates into the incubator with the yellow hydroponic lamp. One Petri dish should be placed in a box as the “dark” sample and the other can be placed into the incubator as is…media side up of course. | ||
=== | ===Part 2: Test in liquid media=== | ||
# In a 15 ml falcon tube, pipet 10 ml of LB | # In a 15 ml falcon tube, pipet 10 ml of LB | ||
# Add 10 μl of ampicillin and 10 μl of chloramphenicol. | # Add 10 μl of ampicillin and 10 μl of chloramphenicol. | ||
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DONE! | DONE! | ||
==For Next Time== | |||
==Reagents== | |||
Revision as of 06:03, 14 August 2009
FrontPage.jpg)
Testing an engineered biological system
Introduction
Bacteria as pixels
E. coli do not normally respond to light but a recent publication URL describes a combination of genes that lead to light-responsive expression of beta-galactosidase in E. coli. When the cells are grown in the dark, transcription of lacZ is high and the indicator in the media turns black. When the cells are grown in the light, there is very little transcription of the lacZ gene and the media’s natural color (yellowish) shows through. How precisely this expression can be controlled (spatially and temporally through different experimental conditions) is what you will investigate during this experimental module.
For today, you can get used to this system with a simple test. You’ll plate a layer of cells on indicator medium in two dishes, and then incubate one dish in the light and the other in the dark until next time. You expect the dark grown plate to turn a dark color and the light grown plate to stay light. You’ll also grow some liquid cultures under the same light and dark conditions to measure beta-galactosidase activity next time.
Some media has been prewarming in the 42°C incubator for you. The media is traditional LB supplemented with ferric ammonium citrate and “S-gal,” an artificial substrate for beta-galactosidase. When the enzyme cleaves S-gal in the presence of iron, a black precipitate forms changing the color of the media. The media also has agarose so it will harden as it cools. All manipulations should be with your best sterile technique to avoid contamination of your cultures.
Protocols
Part 1: Test in solid media
- Dilute the overnight culture of cells 1:10. Use LB to dilute the cells and make at least 250 μl of this dilution to use for today's experiment.
- Add antibiotics (50 μl of ampicillin and 50 μl of chloramphenicol) to the prepared media. These will maintain the plasmids in the bacteria as the cells grow. Use the stir plate to mix but do not allow the media to remain at room temperature for long since it will begin to harden as it cools.
- Add 200 μl of cells to the media. Stir as before.
- Pipet approximately 20 ml with a 25 ml pipet into two Petri dishes and allow the media to harden on the bench before moving the plates into the incubator with the yellow hydroponic lamp. One Petri dish should be placed in a box as the “dark” sample and the other can be placed into the incubator as is…media side up of course.
Part 2: Test in liquid media
- In a 15 ml falcon tube, pipet 10 ml of LB
- Add 10 μl of ampicillin and 10 μl of chloramphenicol.
- Add 10 μl of your 1:10 dilution of cells. Invert several times to mix.
- Place 5 ml into two Petri dishes.
- Wrap parafilm 3/4ths of the way around each dish to prevent evaporation.
- Add one dish to your “dark” box and place the other in the incubator on the platform rocker under the yellow light.
DONE!
