BioBuilding: Synthetic Biology for Students: Lab 5: Difference between revisions
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#Finally, there are many regulatory and human practice questions to ask if you are thinking of moving a genetically engineered organism out of the lab and into the world. If you're feeling entrepreneurial and would like to explore the questions related to the interplay between science and society, jump to Part 3 in this procedure. | #Finally, there are many regulatory and human practice questions to ask if you are thinking of moving a genetically engineered organism out of the lab and into the world. If you're feeling entrepreneurial and would like to explore the questions related to the interplay between science and society, jump to Part 3 in this procedure. | ||
==Procedure== | ==Procedure== | ||
====Part | ===Part 1: Discovering the reason for the strain's genetic instability=== | ||
====Part 1A: Characterizing the genetic variability==== | |||
#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? | ||
=====How to restreak cells===== | =====How to restreak cells===== | ||
A video showing you how to restreak cells is [http://youtu.be/bfKUUShF2-M here.] | [[Image:RestreakingCells.png|thumb| 350px]] A video showing you how to restreak cells is [http://youtu.be/bfKUUShF2-M here.] | ||
#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. | #Label your new petri dish with your initials, today’s date, the kind of media in the petri dish (YPD) and the strain that you’ll be restreaking onto it. | ||
#Start by dabbing the flat end of a toothpick onto a colony of yeast | #Start by dabbing the flat end of a toothpick onto a colony of yeast that you want to restreak. The colony should be well isolated from the others and uniform in appearance. | ||
#Transfer the cells from that toothpick by | #Transfer the cells from that toothpick by lightly 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. | ||
#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. | #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. | ||
#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. | #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. | ||
#Replace the lid of the petri dish and incubate the plate media-side UP in an incubator ( | #Replace the lid of the petri dish and incubate the plate media-side UP in an incubator (room temp or 30° for 2 days). | ||
#When you're back to examine the cells, make sure you not only notice variations in the number of colonies and how they are growing on the plate, but also the color of the colonies and how many of each type you see. If you'd like to explore the questions of stability further you can ask if a colony of one color always stays that color (e.g. does a red colony always give rise to red). You could also ask if there are experimental conditions you can control that affect the variation you see. | |||
====Part 2: PCR==== | ====Part 2: PCR==== |
Revision as of 09:52, 23 February 2013
Eau That Smell Lab |
Lab 5: Golden Bread
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 countryObjectivesBy the conclusion of this laboratory investigation, the student will be able to:
IntroductionOne goal in the synthetic biology community is to convert scientific discoveries into practical solutions that meet real world needs. The world’s needs are many -- our population is aging, we’re putting increased pressures on our environment and there are widening economic inequalities -- but biology is a challenging material to work with. Our understanding of nature is incomplete and evolving. Our tools for engineering it are primitive. Biology is not perfectly predictable. And as a society we’re often awkward or misguided when we interface with emerging technologies. We’d like to use our powers for good, to benefit all people and the planet, but what a complex challenge that is! Background on Vitamin A production"Nature is a masterful and prolific chemist" [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 a carotenoid, a member of the isoprenoid family of chemicals that is responsible for many of the vibrant colors seen in plants and animals. Nature makes it look easy! 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 color of the carotenoids is directly related to their structure, in particular the number of conjugated double bonds. A minimum of 7 conjugated bonds is needed for any color so cis-phytoene with only 3 is colorless while trans-neurosporene with 9 is yellow, and lycopene with 11 is red. 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. The Science and Engineering of Golden BreadXanthophyllomyces dendrorhous is a naturally red fungi that grows on tree stumps and other places. It's red because it can make its own carotenoids but it's not a particularly useful fungi in the lab or in industry. A much more useful yeast is Saccharomyces cerevisiae. That's the fungi also known as baker's yeast since it can be used to bake bread or brew beer. Based on how much Wonderbread and Budweiser is made each year, it seems like this S. cerevisiae would be a better chassis choice for large scale production efforts. So the reasonably simple idea to move the genes over was first published by van Ooyen in 2007 pdf is here and then developed further by the 2011 iGEM team from Jef Boeke's lab at Johns Hopkins, iGEM 2011 project. The goal was to transfer the genes that make carotenoids from the red fungi, Xyanthophylomyces, into the strain that we know how to work with, namely S. cerevisiae.There are three enzymes that the red fungi makes which allow it to convert simple molecules into beta-carotene. The genes that encode the enzymes are called crtE, crtI and crtYB. One of the enzymes, encoded by crtE is already made by baker's yeast from the native BTS1 gene. The other genes are needed in a couple of places on the metabolic path from starting material (Farnesyl-PP) to beta-carotene. Then lo and behold: The baker's yeast that has crtI and crtYB and an extra copy of crtE turns out to be bright orange in color...a great indication that it's making b-carotene. But this simple idea turns out to be more complicated (of course!) and before you start baking golden bread to feed people in parts of the world with Vitamin A deficiencies, there are number of things to consider.
ProcedurePart 1: Discovering the reason for the strain's genetic instabilityPart 1A: Characterizing the genetic variability
How to restreak cellsA video showing you how to restreak cells is here.
Part 2: PCR
Part 3: Yeast TransformationPart 4: Measuring Vitamin APart 5: Baking BreadNext dayIn your lab notebook, you will need to construct a data table as shown below. These may be provided. Also be sure to share your data with the BioBuilder community here. Lab ReportI. Introduction
II. Methods
III. Results
IV. Discussion
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