20.109(F08): Mod 2 Day 2 Yeast transformation

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20.109(F08): Laboratory Fundamentals of Biological Engineering

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Contents

Introduction

Two toddlers, a can of white paint and a new flat screen TV..."what could possibly go wrong?" Microsoft Windows operating systems for web-enabled ATMs..."what could possibly go wrong?" There is no question that optimism is a fundamentally important element of every experiment we do. We work to design solutions; we plan for success not failure. Even difficult projects should be tackled with the idea that they can, should and will work. Attitude alone, however, does not overcome pitfalls and there is much to be gained in applying some of that optimistic energy to answering: "what if...?". And oddly, spending some time being negative before you jump in is likely to improve your chances of a successful outcome since some of the pitfalls we encounter are avoidable if they can be anticipated.

In the laboratory, "controls" help answer the "what if" questions. They are equally or perhaps more important than your experimental samples and often more numerous. If the controls in an experiment haven't worked there is very little point in considering the data you have collected. Even experienced researchers often wish they had included a better or a different control for an experiment since data often leads to more questions, some of which might have been anticipated.

Your experiment today is straightforward: transform your yeast cells with your PCR product and look for cells that can grow on media lacking tryptophan. What if you return to lab next time and saw no yeast colonies growing on any of your petri dishes? What if you return to find yeast covering the plates completely? How can you be sure these are even yeast and not bacterial colonies? You've transformed and transfected cells before...what controls were used for these experiments and can they be applied here?

The negative control helps confirm that any positive experimental data is arising from the experimental sample and not some random event. For example, leaving the DNA out of a transformation reaction is a negative control. No colonies should grow from that sample. If colonies do grow with the negative control then perhaps the petri plates are contaminated, or perhaps the plates are the wrong kind, or perhaps the yeast are already able to grow on those plates without your experimental DNA. The negative control can't distinguish these possible explanations but it can tell you that something needs to be re-worked.

The positive control helps eliminate trivial explanations for failed experiments (these are experiments after all, so you don't really know what the outcome should be....). Transforming plasmid DNA into competent cells is a positive control. If no colonies grow, then something is wrong with the cells or the plates. If cells only grow on your positive control and not on your experimental sample, then the experiment should be refined and repeated.

Even "perfect" results can be misleading and need to be carefully considered. Beyond the negative and positive controls, a good researcher thinks through the experimental sample itself to see if "perfect" data could be explained in more than one way. Today, for example, you will remove an aliquot of your PCR product and transform that DNA into competent yeast. What if you returned to lab next time to find these results:

Sample number of colonies growing on SC-trp
No DNA 0
Plasmid DNA with TRP1 marker 1000
PCR product 1000

It seems like this experiment "worked," really really well, but in fact the number of colonies on the experimental sample is too "good." The PCR product isn't expected to give rise to trp+ cells as often as plasmid DNA can. After all we're relying on recombination of the 39 bases flanking the product to integrate the TRP1 gene. And if the SAGA-subunit deletion makes the cells even a little bit sicker than wild type, the correct product will be even harder to get. So how can this "too perfect" data be explained?

Remember what is in that PCR sample:

  • product (hopefully....in a "real" lab setting you'd check to see that the reactions actually worked before transforming!)
  • leftover primers....those should be degraded by the cell
  • leftover dNTPs....also transparent to the cell once inside
  • leftover Taq...not a problem
  • template DNA...hmmm...

What is to stop the TRP1 template DNA, just another plasmid after all, from giving rise to cells that grow on SC-trp? To solve this problem the PCR product (expected to be around 1500 base pairs) could be purified from the template (around 4 or 5 Kb) using an agarose gel and a Qiagen kit as you did before. A more elegant solution though, is what you will use since the template you were given last time has no yeast origin of replication. Thus the yeast will not be able to copy the plasmid and it will be diluted then lost from your transformed samples. This "trick" has saved some time in the lab but did require some anticipation. Hopefully you can see the value.

Protocols

Part 1: Concentrate your PCR product

Your PCR products are in a volume of 100 ul and we'd like to pool the two reactions that were done in the presence of template and concentrate the material in order to transform all of the product. We'll use a Qiagen column for concentrating the PCR'd materials.

  1. Move the contents of your PCR tubes into full-sized eppendorf tubes and add 5 volumes of PB (e.g. if you moved 100 ul then 5 volumes would be 500 ul). Recall PB is a high salt, low pH buffer that is added so the DNA in your reactions will bind the silica membrane. The salt in PB is guanidine hydrochloride, a chaotropic salt, meaning it will exclude water from the DNA, effectively precipitating it so it will bind the membrane in step 2.
  2. Get a QIAquick column and a collection tube from the teaching faculty then pipet the PCR/PB sample into the top. Microfuge the column in the collection tube for 60 seconds. Remember you must balance your tube in the microfuge. Note: The maximum volume for the columns is ~750 ul so you may have to pass the volume through the column in two spins.
  3. Discard the flow-through in the sink and replace the spin-column in the collection tube. Add 750 ul of PE to the top of the column and spin as before. Qiagen sells PE and does not reveal all its contents, but the solution is at least 80% ethanol which keeps the DNA precipitated and on the membrane but washes the salt away.
  4. Discard the flow-through in the sink and replace the spin-column in the collection tube. Spin for one more minute. Strange as it seems this is a very important step since it removes any residual ethanol from the membrane in the spin-column. If you forget this step the ethanol will elute with your DNA and will inhibit the upcoming transformation.
  5. Trim the cap off two new eppendorf tubes and label the side with the name of sample, your team color and the date. Place the spin-column in the trimmed eppendorf and add 20 ul of EB to the center of the membrane. EB is 10 mM Tris pH 8.5. Since the DNA will solubilize at low salt and high pH, it elutes from the column when EB is added. Lab water can also be used to elute the DNA but it’s worth remembering that the pH is likely to be more acidic than 8.5 and so the company recommends eluting with EB when possible. Allow the column to sit at room temperature for one minute and then spin as before. The material that collects in the bottom of the eppendorf tube is ready to be checked on a gel and also transformed.

Part 2: Competent cells

A culture of NY411 (genotype: MAT(A) his4-917d, lys2-173R2, leu2d1, ura3-52, trp1d63) is growing in a flask. You will prepare these cells for transformation. Like E. coli, S. cerevisiae does not naturally uptake new DNA from its environment but can be made competent by chemical treatment. CaCl2 can work with bacterial cells. Yeast can be made competent by chemical treatments (with Lithium Acetate for example), by enzymatic treatment (with zymolyase for example), with an electrical pulse (called "electroporation"), or biolistically (by using a gun to fire DNA-coated metal particles at the cells). Today you will use a kit sold by Q-biogene to prepare competent NY411. The contents of the kit are proprietary but the protocol seems most like ones for chemically competent cells (this link leads to just one of many similar protocols).

  1. Begin by harvesting 10 ml of log phase NY411 in a 15 ml conical tube. Spin these cells (with a balance) at 3000 rpm for 5 minutes in the clinical centrifuge.
  2. Remove the supernatant by aspirating. You do not have to remove every drop.
  3. Wash the cells with "wash solution" (most likely just sterile water!). You can resuspend the cells in the 15 ml conical in 3 ml of wash solution and then split this volume between three eppendorf tubes.
  4. Harvest the cells in a microfuge (be sure to balance your tubes), spinning 1 minute at full speed.
  5. Aspirate the supernatant.
  6. 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.

Part 3: Transformation

  1. Add 10 ul of your "no template" PCR reaction to one eppendorf and label the top appropriately. This should serve as your "no DNA," negative control. Flick the tube to mix the contents.
  2. Add 10 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.
  3. Add 10 ul of your "plus template" PCR product from last time. This is your experimental sample. You can give the remainder of your PCR products to the teaching faculty who may run them on an agarose gel, depending on the outcome of these transformations.
  4. 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.
  5. Incubate the tubes at 30° for approximately one hour, along with 4 SC-trp petri dishes, with their lids ajar if there is moisture on their surface. During this hour you can:
    • add 2 ul of loading dye to the remaining volume of PCR products and load them on a 1% agarose gel to verify recovery.
    • work on the Materials and Methods section of your upcoming lab report.
    • periodically "flick" your tubes that are incubating at 30° to mix the contents. This will help keep the cells from settling to the bottom of the tube.
  6. 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, plating the experimental transformation twice.
  7. Wrap your plates with your colored labeling tape and incubate them, media-side up in the 30° incubator until next time.

DONE!

For next time

  1. Write the Materials and Methods section for your lab report based on the material we've done so far, namely Primer design, PCR and yeast transformation. Again consult the writing instructions we've provided. Again, you and your lab partner can and should help eachother. When it comes time to write, you must do so on your own. You and your lab partner will hand in individual assignments. And again, please submit this part of the assignment electronically to both nkuldell and astachow AT mit DOT edu.
  2. Read the relevant article by Wu et al, published in Mol Cell in 2004. There is also an associated review article PMID: 15653319 that was written to celebrate and highlight this important work on the structure of the SAGA complex. You might find this review helpful but you are not required to read it.

Below you will find some questions to guide your reading of the Molecular Cell article. You do not have to turn in the answers to these question. You don't even have to answer them all! They are provided to help you decode and understand the most important and aspects of this paper that are relevant to your investigation and your upcoming writing assignment.

Begin by skimming the whole article!
This means read the summary once. Read the first sentence of each paragraph of the introduction. Read the subdivision headings of the Results section. Look at all the figures and their legends. Read the first and last paragraph of the Discussion.

Follow with a more detailed reading of the Introduction:

  • a. Note in two or three words the topic of each paragraph of the introduction.
  • b. What do you think of the first sentence? Is it interesting? Do you agree with what it says? Is it sufficiently broad? Sufficiently focused?
  • c. What about the second paragraph? Is it more detailed and relevant to the investigation than the first? Is the # of polypeptides what you expected?
  • d. In the second paragraph the authors start to break the function of SAGA into functional modules. List them and the subunits involved.
  • e. What is a histone fold?
  • f. If the subject of the second paragraph was functional composition, what is the subject of the third paragraph?
  • g. The fourth paragraph begins to address the genetic and biochemical evidence for how the modules of SAGA fit together. Why is this transition/information included?
  • h. You’ve been told over and over that yeast is a good model system for understanding more complex eukaryotic cells, like human, but what does the fifth paragraph suggest about that belief? Why do the authors spend time describing what’s known about the human SAGA complex?

Next work on the Results:

  • Next time we meet, you and your partner will be randomly assigned a portion of the text to describe to the class but try to understand it all, at least superficially. The following questions are intended to help you think about what you’re reading and do not require long answers.
  • Section 1: A 3d model…
    • a. Read this abstract and a little of the paper itself from the Nature Biotechnology link to get some idea of what TAP-tag purification is.
    • b. Why was TAP-purification of SAGA from an ada1 deletion strain used as control for knowing that EM particles were actually SAGA?
    • c. How many domains were identified in SAGA?
    • d. How many clefts?
  • Section 2: Mapping Taf subcomplex
    • a. Don’t sweat the details of this section. Think more broadly about why a “histone fold” might be found in proteins other than histones and what it means that TAFs which are part of TFIID and SAGA have histone folds.
    • b. Are the TFIID and SAGA complexes similarly shaped? What does this suggest about their function?
    • c. Are the TFIID and SAGA complexes identically shaped? What does this suggest about their function?
    • d. How were antibodies used in this section of the paper?
  • Section 3: Localization of Ada1 and Spt7
    • a. What was the goal of this series of experiments?
    • b. What do the authors learn about the localization and stoichiometry of Spt7 in SAGA?
    • c. What do the authors learn about the localization and stoichiometry of Ada1 in SAGA?
    • d. They describe a SAGA subparticle that can be identified in strains lacking Ada1. What is the significance of this?
  • Section 4: Tra1 and Spt20
    • a. The authors use a myc-tagged Tra1 protein to find out where in SAGA this protein is localized and in the end say “Because of the size of Tra1, it is unclear if the protein resides solely in domain I or extends further throughout the structure.” Can you think of an experiment that would begin to address this uncertainty?
    • b. Based on where Spt20 is found on the complex, does it make sense that it’s not a protein required for structural integrity of the whole complex?
  • Section 5: Spt3 and Gcn5
    • We’re almost at the end of the Results section and the authors have divided it into a presentation of data about
      • 1. The overall structure
      • 2. TAFs and SAGA vs TFIID
      • 3. Ada1 and Spt7
      • 4. Tra1 and Spt20
      • 5. Spt3 and Gcn5
    • Is there other data you’d like to see included? Are there reasons you can think of to explain why the paper is organized this way? Do you think the authors knew when they started the experiments that this is how they’d write the results?

Now for the Discussion section

  • How do the investigators know they’re looking at the whole complex? What if the SAGA complex at it exists in the cell is extremely fragile in one or more places? What might you see by EM? What controls would you run?
  • The homologous human complex called TFTC has some proteins not found in yeast complex. Plus, some of the common proteins are not well conserved at the sequence level (you could BLAST Spt7 if you want to see for yourself). So why bother talking about or even considering the human complex?
  • The scaffold of the TFIID complex is made of TAF pairs (Taf4 and 12, 6 and 9, 10 and 3/8). The scaffold of the SAGA complex is also thought to include TAFs: Taf10 with Spt7 and Taf12 with Ada1, with the latter members of each pair being histone fold proteins like the TAFs. So what? Does this explain why the TAFs are essential for yeast viability? Why isn’t Ada1 or Spt7 essential?
  • Why can’t the authors tell us how many copies of each protein are in the complex and what experiments might be tried to find this out?
  • As engineers, what do you make of the statement:” The positions of SAGA components that are involved in transcriptional regulation…suggest a modular organization that reflects their distinct regulatory activities.” What could you try given nature’s gift of modularity and abstraction here?
  • What is the topic of the last paragraph?

Please come to class next time prepared to discuss the article.

Reagents list

  • Qiagen PCR clean-up kit
  • Q-biogene Transformation kit
  • pRS414 DNA (50 ng)
  • SC-trp (Q-biogene)
    • 1.7g yeast nitrogen base
    • 5g ammonium sulfate
    • 20g dextrose
    • Complete Synthetic Media -TRP
    • 20g agar
  • 1% agarose gel (1X TAE)
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