BioBuilding: Synthetic Biology for Students: Lab 5

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* Compare two engineering solutions to a given problem (redundancy vs kill switches)
* Compare two engineering solutions to a given problem (redundancy vs kill switches)
==Introduction==
==Introduction==
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"Nature is a masterful and prolific chemist" (Microbiol. Mol. Biol. Rev. March 2005 vol. 69 no. 1 51-78, [doi: 10.1128/​MMBR.69.1.51-78.2005] and many laboratories work hard to mimic even the smallest bit of nature's range and skill. In this experiment we'll examine the biosynthesis of carotenoids, a chemical family in the isoprenoid family that's responsible for many of the vibrant colors seen in plants and animals. Nature makes it look easy! Think of the bright orange color of carrots and you're thinking of the carotenoid they make called beta-carotene. There are more than 600 natural carotenoids, playing important  roles in harvesting light for photosynthesis, as anti-oxidants to detoxify reactive species, and as regulators of membrane fluidity. The structure of carotenoids makes them lipophilic so in the lab they're more soluble in organic solvents like acetone than they are in water. We'll exploit this fact when we measure the beta-carotene in a collection of cells that we'll grow.  
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"Nature is a masterful and prolific chemist" [http://mmbr.asm.org/content/69/1/51.short| doi: 10.1128/​MMBR.69.1.51-78.2005] and many laboratories work hard to mimic even the smallest bit of nature's range and skill. In this experiment we'll examine the biosynthesis of carotenoids, a chemical family in the isoprenoid family that's responsible for many of the vibrant colors seen in plants and animals. Nature makes it look easy! Think of the bright orange color of carrots and you're thinking of the carotenoid they make called beta-carotene. There are more than 600 natural carotenoids, playing important  roles in harvesting light for photosynthesis, as anti-oxidants to detoxify reactive species, and as regulators of membrane fluidity. The structure of carotenoids makes them lipophilic so in the lab they're more soluble in organic solvents like acetone than they are in water. We'll exploit this fact when we measure the beta-carotene in a collection of cells that we'll grow. <br>
Plants can make their own carotenoids from scratch, but animals can't so we must eat all we need. That can lead to vitamin deficiencies. In cultures that can't grow many vitamin-rich plants, individuals can develop illnesses related to vitamin deficiency. You may want to consider biotechnology approaches to this issue, including the story of "golden rice" and the social impact of GMOs in the US and in Europe.  
Plants can make their own carotenoids from scratch, but animals can't so we must eat all we need. That can lead to vitamin deficiencies. In cultures that can't grow many vitamin-rich plants, individuals can develop illnesses related to vitamin deficiency. You may want to consider biotechnology approaches to this issue, including the story of "golden rice" and the social impact of GMOs in the US and in Europe.  
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==Procedure==
==Procedure==
====Part 1: Testing Genetic Variability====
====Part 1: Testing Genetic Variability====
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A video showing you how to restreak cells is [http://youtu.be/bfKUUShF2-M here.]
 
#Yeast will arrive on a YPD plate to grow at 30°C or room temp and stored at room temp or in the fridge.  
#Yeast will arrive on a YPD plate to grow at 30°C or room temp and stored at room temp or in the fridge.  
#Identify color variants and restreak onto fresh YPD. Are there differences in the stability of the phenotypes? Are there growth conditions that make the colors more or less stable?  
#Identify color variants and restreak onto fresh YPD. Are there differences in the stability of the phenotypes? Are there growth conditions that make the colors more or less stable?  
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=====How to restreak cells=====
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A video showing you how to restreak cells is [http://youtu.be/bfKUUShF2-M here.]
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#Label your new petri dish with your initials, today’s date, the kind of media in the petri dish and the strain that you’ll be restreaking onto it.
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#Start by dabbing the flat end of a toothpick onto a colony of yeast or bacteria that you want to restreak. The colony should be well isolated from the others and uniform in appearance.
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#Transfer the cells from that toothpick by lightlying touch the toothpick to a spot on the new petri dish that you’d like to grow. Note: you should not break the surface of the agar with any of this procedure, but the results will still be OK, even if you do.
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#With the flat end of a new toothpick, spread out the cells in the dab you made on the new petri dish by drawing your toothpick back and forth through the dab and then along the media in the dish. Do not back up as you draw since you are trying to spread out the cells that are on the toothpick from your one pass through the original dab of cells.
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#With a new toothpick, spread out the cells still further, drawing from the ending line you made with the second toothpick. Again, do not back up as you draw with this third toothpick and try not to break the surface of the media.
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#Replace the lid of the petri dish and incubate the plate media-side UP in an incubator (ideally 37° overnight for bacteria, 30° 2 days for yeast).
   
   
====Part 2: PCR====
====Part 2: PCR====

Revision as of 21:52, 20 February 2013


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Lab 5: Golden Bread

  • Engineering reliability into an unstable genetic system may or may not make it a robust and profitable food source.

Acknowledgments: This lab was developed with materials from the Johns Hopkins 2011 iGEM team, as well as guidance and technical insights from BioBuilder teachers around the country

Objectives

By the conclusion of this laboratory investigation, the student will be able to:

  • Define and properly use synthetic biology terms: chassis, system, device, redundancy
  • Define and properly use molecular genetics terms: PCR, gene expression, codon shuffling.
  • Explain the role of redundancy in synthetic biology and engineering.
  • Conduct PCR and TLC and interpret the results each.
  • Compare two engineering solutions to a given problem (redundancy vs kill switches)

Introduction

"Nature is a masterful and prolific chemist" [http://mmbr.asm.org/content/69/1/51.short

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