BioBuilding: Synthetic Biology for Students: Lab 5

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*[[Media:Alex B. Berezow- Embracing the Promise of GMOs - WSJ.pdf| WSJ article on GMOs]]
*[[Media:Alex B. Berezow- Embracing the Promise of GMOs - WSJ.pdf| WSJ article on GMOs]]
*[[Media:CowTippingPoint.pdf| Public benefits to Science as UN Declaration of Human Rights]]
*[[Media:CowTippingPoint.pdf| Public benefits to Science as UN Declaration of Human Rights]]
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*[[http://www.open.edu/openlearn/science-maths-technology/science/biology/gene-manipulation-plants/content-section-4.1| plant genetic manipulation]]
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*[[http://www.open.edu/openlearn/science-maths-technology/science/biology/gene-manipulation-plants/content-section-4.1 plant genetic manipulation]]
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*[[http://www.goldenrice.org/Content3-Why/why3_FAQ.php| Golden Rice]]
+
*[[http://www.goldenrice.org/Content3-Why/why3_FAQ.php Golden Rice]]
=====Who decides? FDA, EPA=====
=====Who decides? FDA, EPA=====

Revision as of 16:15, 26 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 of 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
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.

  1. 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.
  2. 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.
  3. 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

  1. Yeast will arrive on a YPD plate to grow at 30°C or room temp and stored at room temp or in the fridge.
  2. 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.
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. Replace the lid of the petri dish and incubate the plate media-side UP in an incubator (room temp or 30° for 2 days).
  7. 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 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 stock

For 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 lysate

Since 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.]

  1. Mix the starting liquid overnight culture so it’s homogeneous. This can be done by swirling the culture if it’s in a flask, by inverting if the cap is tight, by flicking a small tube, or by vortexing if there is a vortex available.
  2. Place a tip on your pipetman and move 500 ul of the culture to an eppendorf tube.
  3. Using an eppendorf tube with end ½ trimmed off at the 100 ul mark, measure 100 ul of [small| glass beads] and add them to the 500 ul of yeast you measured out.
  4. Close the eppendorf tube.
  5. Vortex the mixture of yeast and glass beads, keeping the tube on the vortex at full speed for 15 seconds and then letting the solution “rest” for 15 seconds. This rest is needed to keep the temperature of the yeast/beads mixture from overheating.
  6. Repeat the vortexing and resting series a total of 4 times so the yeast will have been mixed with the beads for a full minute.
  7. 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.
  8. 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 [such as these] and a video of this procedure is [here.]

  1. Begin by putting on gloves to protect the TLC sheets from any oils or pigments on your hands
  2. 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.
  3. Cut the sheet to size.
  4. 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.
  5. Spot 3 ul of Vitamin A about 1/3 of the way across the line you drew at the bottom of the slide.
  6. Spot 3 ul of Yeast extract about 2/3 of the way across the line you drew at the bottom of the slide.
  7. 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.
  8. 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.
  9. Replace the lid on the 50 ml conical tube and allow the TLC to proceed undisturbed for 20 or 30 minutes.
  10. Using a gloved hand, remove the slide from the conical tube.
  11. 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

We don't know why the Vitamin A producing yeast system we are working with gives colonies of different colors. Perhaps some of the genes needed for vitamin production are no longer present. The strain we started with is thought to have at least one copy of all 3 genes needed for beta-carotene production. According to the original publication, these genes (crtYB, crtI and crtE) were integrated into the yeast genome using another gene (URA3) as a selectable marker. A second copy of crtI was integrated using LEU2 as a selectable marker.

We can look for the presence of these genes using the polymerase chain reaction ("PCR"). A nicely animated review of this technique is shown [here.] We will use short DNA pieces called primers to look for variations in crtYB. The primers are bases on the sequence of [BBa_K530000,] the version of the crtYB gene that was deposited in the [Registry of Standard Biological Parts.] A sequence file for this gene is here. The gene came from the Xanthophyllomyces dendrorhous mRNA sequence of crtYB [that is here.] Based on these links, what is the length of the crtYB product you are expecting from the PCR experiments?

PCR

We can expect no PCR product if we try to amplify crtYB from a strain that lacks the crtYB gene. Since a negative result could mean that the reactions themselves were not set up properly, we'll have to include a positive control and amplify crtYB from some plasmid DNA in a second reaction. We'll also want to compare colonies of different colors to look for different amounts of PCR product and how that might related to the colony colors. Can you anticipate any of the results? Perhaps you can make sketch of the agarose gel results you'll expect in advance of trying the experiment. That way you can more easily compare what you actually discover to what you predicted at the outset.
1. Move an [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)
2. Thaw primer pair NO302 and NO303. These amplify the crtYB gene ORF. Their sequences are:

  • NO302 = crtYB-ORF-F
    • 5'- ATG ACG GCT CTC GCA TAT TAC CAG ATC CAT CTG ATC TAT ACT CTC CC -3'
  • NO303 = crtYB-ORF-R
    • 5'- TTA CTG CCC TTC CCA TCC GCT CAT GAC CAC ACT CAA GAC TTT CCG TAC -3'

3. 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.
4. 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

5. Add 5 ul loading dye to each sample
6. 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 2: Engineering reliability

A person who wears both a belt and suspenders may be WAY too worried about his pants falling down, but for system engineers, such redundancies are commonly used to avoid catastrophic failures. Car manufacturers didn't take out seat belts once air bags were invented. And in biology, many cells are diploids since haploids are more prone to suffer (i.e. die!) from deleterious mutations.
One way to improve the reliability of the Vitamin A yeast system would be to engineer redundancy of the component most prone to failure. We've chosen to focus on crtYB here but could use the same strategy for any of the system's components. No matter what gene you choose, though, take care not to make more problems for the cell. If you were to introduce an absolutely identical copy of that gene, then it's likely to undergo recombination (think about the crossing over events in sister chromatids that you learned about when you studied mitosis and meiosis). We can help the cell avoid this problem by codon shuffling the duplicate copy, as you'll see below.

Part 2A: Designing a redundant version of crtYB
workflow from DNA2.0
workflow from DNA2.0
With these steps, you'll see how the crtYB gene was recoded using degeneracies in the standard genetic code. With conservative substitutions, we kept the amino acid code the same but changed the DNA sequence enough to inhibit homologous recombination -- at least that's the hope! We call the new copy " crtYB' " with the "prime" indicating a codon shuffled version of the original sequence.
  • If you'd like to see what we did, start by importing the existing sequence file for crtYB into a plasmid editing tool such as [Ape].
  • Import the primer sequences into new Ape (or other DNA plasmid) files.
    • NO302 = crtYB-ORF-F
      • 5'- ATG ACG GCT CTC GCA TAT TAC CAG ATC CAT CTG ATC TAT ACT CTC CC -3'
    • NO303 = crtYB-ORF-R
      • 5'- TTA CTG CCC TTC CCA TCC GCT CAT GAC CAC ACT CAA GAC TTT CCG TAC -3'
  • Align the primers to the crtYB sequences (this is done by selecting the "align sequences" option from the drop down menu in ApE). We wanted to keep these sequences identical to the original sequence so we could amplify the new copy of the crtYB gene with the same primer pair.
  • We decide to make your new copy of crtYB into a standard biological part so it might be entered into the [Registry of Standard Biological Parts.] To make it a standard part, we identified which restriction sites are NOT permitted in the new sequence. [Here] is a video tutorial for how to do this with DNA2.0's software, if you'd like to try it yourself.
  • We next sent a note to the gene design experts at a company called "DNA 2.0" that said:
  • "Please codon randomize this sequence after bp 47 and until bp 1975. We want there to be no sequences for EcoRI, PstI, SpeI, XbaI restriction sites. We'd like the final gent to be controlled with a constitutive yeast promoter and cloned into the vector, pRS414."

After some back and forth and some further refinements, the crtYB' DNA arrived in the mail about 4 weeks after the initial request was made. The sequence of the crtYB' gene is linked here. Using the programs below from [NCBI] and [NEB,] we double checked that the DNA was, in fact, different but the amino acid sequence was, in fact, identical. We further checked that the restriction enzyme recognition sites that we wanted excluded from the sequence were really missing. Here are the programs we used to do that:

Part 2B: Transforming yeast with crtYB'

S. cerevisiae does not naturally uptake new DNA from its environment but can be made competent by chemical treatment. These instructions are written for a kit sold by [Q-biogene] to prepare competent cells. The contents of the kit are proprietary but the protocol seems most like ones for chemically competent cells
Unlike transformations that you might be familiar with using bacteria, yeast that have been transformed are selected for using "drop out" media. In our case, the Vitamin A producing yeast also have a defect in a gene for tryptophan biosynthesis. If we grow the yeast on "rich" media, like YPD, there is enough tryptophan provided by the media for the cells to grow. If we grow the yeast on media that lacks tryptophan (called "SC-trp," where SC stands for synthetic complete), then the Vitamin A producing yeast will not live. Finally, if we transform our yeast with a plasmid like [pRS414] or a version of pRS414 that also carries the crtYB' gene, then cells with the plasmid will grow and we can test them for Vitamin A production.

  1. For each transformation you want to perform (positive control, negative control, experimental), begin by swirling a toothpick full of Vitamin A producing yeast into 500 ul of water in an eppendorf tube.
  2. Harvest the yeast by spinning the eppendorf for 30 seconds in a microfuge.
  3. Remove the supernatant from each pellet by aspirating or pipeting it away into a waste beaker with some 10% bleach in it. You do not have to remove every drop of the supernatant.
  4. Wash each pellet of cells by resuspending them 500 ul of "wash solution" (most likely just sterile water!) from the kit.
  5. Harvest the cells in a microfuge, spinning 30 seconds at full speed.
  6. Aspirate or remove the supernatant as before.
  7. Resuspend each pellet in 50 ul of "competent solution" (most likely lithium acetate and DTT which permeabilizes the yeast through an unknown mechanism). Unlike chemically competent bacteria, competent yeast are not "fragile" in this state and can remain at room temperature.
  8. Add 5 ul of just water one eppendorf and label the top appropriately. This should serve as your "no DNA," negative control. Flick the tube to mix the contents.
  9. Add 5 ul of pRS414 DNA (50 ng) to another eppendorf and label appropriately. This plasmid bears a yeast origin of replication and a TRP1 gene and will serve as a positive control for transformation.
  10. Add 5 ul of your pRS414+crtYB' DNA (50 ng) to another eppendorf and label appropriately. This is your experimental sample.
  11. To each tube add 500 ul "transformation solution" to your cells. This material, most likely polyethylene glycol ("PEG" aka antifreeze) is thick and goopy and is included in transformation protocols to help deliver the DNA into the yeast. Use your P1000 to pipet the yeast and the "transformation solution" and vortex the tube to make an even suspension.
  12. Incubate the tubes at 30° for approximately one hour, along with the needed number of SC-trp petri dishes, with their lids ajar if there is moisture on their surface. During this hour you can periodically "flick" your tubes to mix the contents, this will help keep the cells from settling to the bottom.
  13. After at least an hour (longer is OK too), flick the tubes to mix the contents and then spread 250 ul of each mixture on your SC-trp dishes.
  14. Incubate your petri dishes, media-side up, at room temp or in a 30° incubator for 2 days.
  15. After you return to collect your data, determine the number and color of colonies on each dish. You can undertake the same analysis that you did in Part1 for the starting strain but will have to grow your colonies on SC-trp instead of YPD to maintain the plasmid in the yeast.
Part 2C: Considering alternative strategies for reliability

As a thought exercise, research and then consider how you might implement a "kill switch" as a way to keep the Vitamin A producing yeast stable.

Part 3: Producing Golden Bread®

A handful of genes, a cup of flour and voila-- Golden Bread®! Sounds like a million dollar idea, and one that could help address world hunger. But not so fast: what does it really take to bring this clever food product to market. Could a successful company be built around this technology?

Imagine you’ve just pitched this idea for a nutritionally-enhanced bread to a venture capital firm and a panel of angel investors. These are groups or individuals who invest their money in early stage initiatives, with expectations that they’ll make many times their investment in returns. These investors are looking for the next “Facebook.” Happily, they thought that genetically engineered yeast capable of baking vitamins into bread was an attractive idea. They were impressed by the fluffy and bright orange loaves of bread you brought. They looked yummy, even with a hearty dose of beta-carotene. They’d like you to compete with other biotech groups for next month’s round of $100,000 seed funding, but there’s a lot of work to do if you’re going to make a successful company from this synthetic organism.

Here are some starter ideas for your work on this aspect of the module. Perhaps as a class you'll work through these or perhaps they'll be assigned as a project or report. Either way, if you have ideas to share, there are places on the BioBuilder website to do that!

Should we engineer food? GMO debate
Who decides? FDA, EPA
  • Regulatory bodies
  • Funding for research
  • Testing for safety and efficacy
How to make $: Business models
  • Cost/benefit analysis
  • Lowering costs
How to market? Logo design

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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