BioBuilding: Synthetic Biology for Students: Lab 5: Difference between revisions
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#Replace the lid of the petri dish and incubate the plate media-side UP in an incubator (room temp or 30° for 2 days). | #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. | #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 1B: Measuring with TLC variations in vitamin production==== | |||
It seems like the different colored yeast strains are probably making different amounts of the vitamin A precursor molecules and we'd like a quick, easy way to know for sure. Though it's not the most precise method, we'll be using thin layer chromatography (TLC) since it's a handy way to see qualitative differences between the carotenoids being made by the different colored yeast. TLC separates complex mixtures of chemicals based on how fast they move through a solid matrix (in our case silica). The matrix can be painted onto a solid support slide (paper or glass or aluminum foil) and the material you want ot analyze gets moved through the matrix by a solvent as it "wicks" from one end of the slide. For comparison we'll use store-bought vitamin A that's sold as a dietary supplement. | |||
====Part 1C: Identifying the genetic variant with PCR==== | |||
====Part 2: PCR==== | ====Part 2: PCR==== |
Revision as of 10:16, 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 1B: Measuring with TLC variations in vitamin productionIt seems like the different colored yeast strains are probably making different amounts of the vitamin A precursor molecules and we'd like a quick, easy way to know for sure. Though it's not the most precise method, we'll be using thin layer chromatography (TLC) since it's a handy way to see qualitative differences between the carotenoids being made by the different colored yeast. TLC separates complex mixtures of chemicals based on how fast they move through a solid matrix (in our case silica). The matrix can be painted onto a solid support slide (paper or glass or aluminum foil) and the material you want ot analyze gets moved through the matrix by a solvent as it "wicks" from one end of the slide. For comparison we'll use store-bought vitamin A that's sold as a dietary supplement. Part 1C: Identifying the genetic variant with PCRPart 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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