BioBuilding: Synthetic Biology for Students: Lab 5: Difference between revisions
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#Allow the beads to sink to the bottom of the solution and then remove the solution of yeast lysate from the top, using a pipetman and tip and a new eppendorf tube. | #Allow the beads to sink to the bottom of the solution and then remove the solution of yeast lysate from the top, using a pipetman and tip and a new eppendorf tube. | ||
#Label the eppendorf tube that has your yeast lysate with its contents, your initials and today’s date. | #Label the eppendorf tube that has your yeast lysate with its contents, your initials and today’s date. | ||
=====Running the TLC slide===== | |||
This experiment has been performed using TLC slides made from silica [[http://www.lablife.org/p?a=products_show&id=472346 such as these]] and a video of this procedure is [[http://youtu.be/xX4Tesb8TXQ| here.]] | |||
#Begin by putting on gloves to protect the TLC sheets from any oils or pigments on your hands | |||
#Use a microscope slide to measure and a pencil to mark on the white side of the TLC sheet the size of the piece you’ll need to cut. | |||
#Cut the sheet to size. | |||
#Mark the starting line for your materials by measuring 1.5 cm from one of the narrow ends and lightly drawing a line across the white surface of the TLC slide. Do not dig deeply into the material on the surface. | |||
#Spot 3 ul of Vitamin A about 1/3 of the way across the line you drew at the bottom of the slide. | |||
#Spot 3 ul of Yeast extract about 2/3 of the way across the line you drew at the bottom of the slide. | |||
#Allow the spots to dry while you make up the solvent that you’ll use to carry the vitamins up the TLC slide. Mix 9 ml of water with 500 ul EtOH and 500 ul Isopropanol. These can be mixed directly in a 50 ml conical tube. Label the tube with the contents, your initials and today’s date. | |||
#Lean the TLC slide in the 50 ml conical tube, keeping the upper edge of the solvent below the line where you’ve spotted the vitamins. The idea is that the solvent must pass through the vitamins to carry them along as the solvent gets wicked up the slide. | |||
#Replace the lid on the 50 ml conical tube and allow the TLC to proceed undisturbed for 20 or 30 minutes. | |||
#Using a gloved hand, remove the slide from the conical tube. | |||
#Allow the solvent to evaporate and then visualize the extent and the intensity of the materials that have moved along the slide, using a UV lamp to visualize the vitamin. | |||
====Part 1C: Identifying the genetic variant with PCR==== | ====Part 1C: Identifying the genetic variant with PCR==== |
Revision as of 10:37, 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 to 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. Vitamin A stockFor experiments that need a stock of Vitamin A, you will have to snip the end of a capsule off, and squeeze the oily liquid into an eppendorf tube. There will likely be around 100 ul of solution that you can then pipet as needed. Be sure to label your eppendorf tube to note the contents of the tube and the date. A video of this procedure is [here.] Making a yeast lysateSince the vitamins are being made inside the yeast, we'll need a way to open the cells and use the solution that's on their insides (this is called a lysate). You can start with a solution of yeast that you've grown in liquid media (as described below) or you can scoop up some cells on a toothpick and put them into 500 ul of water (in which case you can start with step 3. You'll lyse the cells by vortexing them with small glass beads. A video of this procedure is [here.]
Running the TLC slideThis experiment has been performed using TLC slides made from silica [such as these] and a video of this procedure is [here.]
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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