BioBuilding: Synthetic Biology for Students: Lab 5

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PDF of this page 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 One 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. chemical structure of two carotenoids Plants can make their own carotenoids from scratch, but animals can't so we must eat all we need. Think of the bright orange color of carrots and you're thinking of the isoprenoid they make called beta-carotene. Cut beta-carotene in half and add a water molecule and you have Vitamin A ---which is why parents tell their kids to eat their vegetables. And why developing countries that have limited food supplies have high incidents of disease due to vitamin deficiencies. For example, between at least 250,000 children in the developing world go blind each year due to Vitamin A deficiency. It's a huge problem but not a new one. As we start this "Golden Bread" module, you may want to consider existing biotechnology approaches to this issue, including the story of "golden rice" and the social impact of GMOs in the US and in Europe. The Science and Engineering of Golden Bread Xanthophyllomyces 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. First, the baker's yeast seems to be genetically unstable, giving orange colored colonies most of the time but white, yellow and red colored colonies other times. If you would like to explore this challenging question, to try to understand what's causing the instability, jump to Part 1 of this procedure. Second, the ways you might engineer a fix to this instability are many but one possibility is to add some redundancy in a function that's glitchy. If you would like to explore this and other engineering solutions to the instability, jump to Part 2 of 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 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. 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 A video showing you how to restreak cells is here. 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 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 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 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 (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 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 Move Edvotek PCR bead to tube that fits in your PCR machine (or don't move the bead if the tube it comes in fits just fine) Thaw primer pair NO302 and NO303. These amplify the crtYB gene ORF. Prepare lysate: scoop a small colony you'd like to study into 50 ul H2O and microwave for 15 seconds with the lid of the eppendorf closed. Prepare lysate for any yeast you'd like to study. To the bead that's in the PCR tube add 20 ul H2O and then vortex the sample 1 ul of each primer 2 ul of lysed yeast cells or + control DNA that carries crtYB on a plasmid PCR cycle: 95° 2 minutes 95° 20 seconds 50° 20 seconds 72° 2.5 minutes repeat steps 2-4 a total of 35X 72° 10 minutes 4° hold Add 5 ul loading dye to each sample Run 25 ul on a 1% TAE gel with a stain to visualize the bands (Ethidium Bromide or CyberSafe). The gel could run for 20 minutes at 120V. Be sure to load a molecular weight marker on the gel with bands that range from 1 kb to 5 or 8 kb. Part 3: Yeast Transformation Part 4: Measuring Vitamin A Part 5: Baking Bread Next day In 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 Report I. Introduction Provide a brief introduction describing the field of synthetic biology. What is a ___? How does this ___work? How might ____ be useful? Briefly describe the purpose of the lab. What are we trying to do here? Presume that a reader of your lab report has not read the assignment. What is the role of the redundancy? How does redundancy affect the expression of a genetic system? How might synthetic biologists ___? Why is it important to engineer a ___? What are the advantages/concerns of engineering ___? How might we test for the differences that the redundancy that may affect a genetic system? II. Methods You do not have to rewrite the procedure. Explain why you did each step of the protocol. III. Results Present the data tables in clear format. Present drawings of each slide. Describe the results: Describe the appearance of the gel and the TLC plate. Are the bands and spots different? IV. Discussion Draw a conclusion: Do the ____ produce the same results in different chassis? Justify your answer. Analyze the data: Be sure to discuss how each part of the experiment and results adds to your conclusion. Are we sure that the transformation worked? What do the controls that lacked plasmid tell us? Discuss errors and other reasons for data variability. Use your results to explain why it is important for synthetic biologists to fully characterize the chassis used in an engineered system. 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