20.109(F15):Growth of phage materials (Day1)

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20.109(F15): Laboratory Fundamentals of Biological Engineering

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Contents

Introduction

Abalone shell
Abalone shell
Diatoms
Diatoms

The accomplishments of the natural world can inspire us to great engineering feats. Biomineralization is one particularly impressive trick nature pulls off. Vertebrates, invertebrates and plants all have ways to precisely position inorganic substrates into crystalline order. For example, calcium carbonate will form unstructured dust in the absence of genetically-programmed organizers, but the same material can be made into the hard and luminous shells of sea creatures. Similarly, diatoms organize silicon dioxide into intricate patterns that manufacturers of electronic components can’t begin to approach. In one more instance, bacteria align iron inside their cytoplasm to form magnetic rods on the submicron scale. These feats are accomplished without harsh chemicals, without extreme temperatures, and without noxious wastes that poison the nests of the organisms themselves. Humans have a lot to learn from nature’s successes. In the upcoming weeks we’ll use a virus that infects bacteria, namely the bacteriophage M13, and we'll rely on the self-assembling coat of this virus to template gold and silver nanowires. The interaction of metals with a protein on the phage coat yields nanoscale-particles with useful energetic properties, as we’ll see.

About M13

The bacteriophage M13 is a member of the filamentous phage family. It has a long (~900 nm), narrow (~20 nm) protein coat that encases a small (~6.4 kb) single stranded DNA genome. The genome encodes 11 proteins, five of which are exposed on the phage’s protein coat and six of which are involved in phage maturation inside its E. coli host. The phage coat is primarily assembled from a 50 amino acid protein called pVIII (or p8), which is sensibly enough encoded by gene VIII (or g8) in the phage genome. For a wild type M13 particle, it takes about ~2700 copies of p8 to make the ~900 nm long coat. The coat's dimensions are flexible though and the number of p8 copies adjusts to accommodate the size of the single stranded genome it packages. For example, when the phage genome was mutated to reduce its number of DNA bases (from 6.4 kb to 221 bp) [2], then the p8 coat “shrink wrapped" around the reduced genome, decreasing the number of p8 copies to less than 100. Electron micrographs of the resulting “microphage” and its wild type parent are shown below (image courtesy of Esther Bullitt, Boston University School of Medicine), where the black bar in each image is 50 nm long. And what about the upper limit to the length of the phage particle? Anecdotally, viable phage seems to top out at approximately twice the natural DNA content. However, deletion of a phage protein (p3) prevents full escape from the host E. coli, and phages that are 10-20X the normal length with several copies of the phage genome can be seen shedding from the E. coli host.

Electron micrographs of microphage described by Specthrie et al. [[1]], images courtesy of Esther Bullitt
Electron micrographs of microphage described by Specthrie et al. [[1]], images courtesy of Esther Bullitt

E. coli shedding M13 with p3 mutation, image courtesy of M. Russel and schematic of M13 genome, image courtesy of M. Blaber
E. coli shedding M13 with p3 mutation, image courtesy of M. Russel and schematic of M13 genome, image courtesy of M. Blaber

Phage life-cycle

The general stages to a viral life cycle are: infection, replication of the viral genome, assembly of new viral particles and then release of the progeny particles from the host. Filamentous phages use a protein at their tip, namely p3, to contact a bacterial structure known as the F pilus to infect E. coli. The phage genome is then transferred through the pilus to the cytoplasm of the bacterial cell where resident proteins convert the single stranded DNA genome to a double stranded replicative form (“RF”). This DNA then serves as a template for expression of the phage genes and produces new phage particles that shed off the surface of the infected cell. Other phage are known to lyse their host cells but in the case of M13 and E. coli, they co-exist in a balanced way, allowing the growth of both host and virus, though the infection does slow down the doubling time of the E. coli (as we'll see later today).

Phage display

Phage display
Phage display

Phage display has been used for decades as a tool for discovery. This technique exploits natural selection and identifies functional peptide sequences that can be fused to the phage coat. Most often it’s the p3 protein at the phage tip that is used for phage display because, despite the limited number of displayed peptides per phage (on the order of 5), there is enough flexibility to accommodate peptides of 20 to 30 amino acids. The other protein used for phage display, p8, is present at a much higher copy number per phage (on the order of 2700) but it has limited flexibilty. The semi-crystalline packing of p8 on the phage coat restricts fusions to only 4 to 6 neutral or negatively charged amino acids. For scientists who can tolerate a mix of p8 proteins on the phage coat for their work, there are phage-display variations that mix and match fusion and wild-type proteins on a phage coat, but for those who want phage of a particular form, the options are limited.

Nonetheless, peptides with remarkably diverse functions have been isolated with phage display. Once the fusion site is chosen, a library of sequences encoding random peptides can be synthesized and cloned. In this way a pool of phages, each with different fusions, can be made. Finally, the phage pool can be screened for interesting behaviors or properties. Peptide-fusion proteins to p8 or p3 that include stop codons or intolerable sequences are largely lost from the population after the first round of “panning.” Other phages can bind to a substance of interest or show enzyme activity or glow green…, these remain and can be directly isolated from the pool or further enriched by a second, third, fourth round of panning. Ultimately anywhere from 10 to 1000 candidate sequences may remain from a starting pool of 1 billion.

Despite phage display techniques being available for more than a generation, this tool has been applied only recently to the search for novel materials. Largely it’s been Angela Belcher and her lab who highlighted and then demonstrated the usefulness of this search tool for finding peptides that interact with materials to meet human needs. That M13 could interact with inorganic materials could not have been predicted from the original genetic studies on the phage, but there was also no one who had tried it! Phage that can bind to cobalt oxide, gold, iridium and indium tin oxide are all in-hand thanks to their work (e.g see reference [3]). Today you will harvest and titer a phage that can bind to gold since it can be used to build nanowires next time.

Protocols

In advance of this lab, a bacterial host (XL1-blue) was infected with the modified M13 phage called "8#9." The p8 modification (=VSGSSPDS) was isolated from a library of p8 mutants that enable the phage to bind thin gold films PMID: 16178252. Today you will harvest the phage from the supernatant of the infected bacterial culture and setup the biotemplating reaction.

Part 1: Phage purification

  1. Divide the overnight culture (~50 mL volume) into 2 Oak Ridge Centrifuge tubes.
  2. Label with your group color using tape (not the small circles; these can come off in the rotor)
  3. Spin at 10,000 rpm for 10 minutes (you will be shown where down the hall you can find a centrifuge to spin this volume).
  4. Transfer the supernatant to a 50 mL conical tube and then clean your two Oak Ridge tubes by adding some 10% bleach and some water to the pellets, allowing the solutions to sit in the sink for 5 minutes and then washing the tubes with copious amounts of distilled water from the tap.
  5. Once the Oak Ridge tubes are clean, split the supernatant that is in the conical tube between the two Oak Ridge tubes. The transfer should be done with a plastic pipet and a bulb so you can measure the volume of supernatant.
  6. Add a 1/4th that volume of 20% PEG-8000/2.5M NaCl solution.
  7. Invert to mix then incubate on ice 60 minutes.
    • Proceed to Part 2 during this incubation.
  8. Spin at 15,000 rpm for 15 minutes. A white pellet may be visible... these are your precipitated phage. If you can't see a pellet keep going, but be aware of where the pellet you can't see is in the tube and don't scrape a tip against it or you will accidentally remove it.
  9. Remove the supernatant by pouring most down the sink and the rest with aspiration (carefully so as not to disturb the pellet).
  10. Resuspend the pellet in 3 mL sterile water. This is best done by adding 3 mL of H2O to one of the Oak Ridge tubes, washing the water up and down the side of the tube with the phage pellet, and then moving the 3 mL of phage solution to the second tube and dissolving that pellet as well by washing the water up and down the side of the tube.
  11. Split the phage solution between three eppendorf tubes.
  12. Add a 1/4th volume of 20% PEG-8000/2.5M NaCl solution.
  13. Invert to mix. Then incubate on ice for 15 minutes.
  14. Spin the tubes full speed in a microfuge for 10 minutes.
  15. Aspirate the supernatant and resuspend the pellets (if you can see them) in 1.5 mL TBS - using 0.5 mL to resuspend each pellet and then pooling the volumes into a larger (2 mL) microfuge tube. This is your phage stock (yay!).
  16. If the solution looks at all cloudy, spin in a room temperature microfuge for 1 minute more and move supernatant with the phage to a new tube.

Part 2: Synthesis of gold and silver substrates

You should wear your lab coat, your safety glasses and gloves throughout the preparation of these materials for your chemical synthesis.

CTAB

Each group should make 20 mL of a 0.1 M solution of hexadecyltrimethylammonium bromide ("CTAB"). The molecular weight of CTAB is 364.45 Da and the solution can be prepared in distilled water in a 50 mL conical tube. You'll have to leave the conical tube in the 37 °C incubator overnight to fully dissolve the CTAB.

Solutions to share

Your group will be responsible for making one of the following solutions that the class will use in the synthesis of nanowires. Pool the volume you make with a second group making the same solution if there is another group assigned this task.

  • 10 mL of 10 mM hydrated hydrogen tetrachloroaurate (HAuCl4•3H2O)
    • The formula weight of HAuCl4•3H2O is 393.83
    • Prepare this solution in a 15 mL conical tube with distilled water
    • WRAP IN FOIL to inhibit the photoreactivity of the material
  • 10 mL of 10 mM silver nitrate (AgNO3)
    • The formula weight of AgNO3 is 169.87
    • Prepare this solution in a 15 mL conical tube with distilled water
    • WRAP IN FOIL to inhibit the photoreactivity of the material
  • 10 mL of 0.1 M ascorbic acid
    • The formula weight of ascorbic acid is 176.13
    • Prepare this solution in a 15 mL conical tube with distilled water
  • 50 mL of 2.5 M NaCl
    • The formula weight of NaCl is 58.44
    • Prepare this solution in a 50 mL conical tube with distilled water

Part 3: Measuring concentration of phage

With this technique you will calculate the concentration of phage in your stock using the spectrophotometer. This method can approximate the number of phage based on the ability of the virions to absorb ultraviolet light. The number of phage is calculated by the formula:
Number of phage particles/ml = (6x10^16)*(A269 - A320)/(#DNA Bases in the genome of the phage)

where

  • the molar extinction coefficient of the phage and the average size of a DNA base are used collected into the constant
  • the absorbance at 269 nm reflects the protein and DNA content in the solution
  • the absorbance at 320 nm corrects for the naturally high baseline value of the solution
  • the number of DNA bases in DSPH is ~7220.

This method for titering the phage stock is less informative than the traditional plaque method (known as titering) since materials other than phage might be contributing to the absorbance readings. Thus, the number of infectious particles isn't truly known. Since infectivity is not critical for the synthesis of SWNT-TiO2 nanowires,however, we will be using spectrophotometry only.

  1. Dilute the phage stock you have 1:10 by adding 70 ul of the phage to 630 ul of TBS, vortex to mix and then move this solution to a quartz (not plastic!) cuvette.
    • A few things to be aware of when using quartz cuvettes:
      • They are very expensive.
      • The lab has very few.
      • When you are done using your cuvette, you should carefully clean it by shaking out the contents into the sink and rinsing it once with 70% EtOH, then two times with water. Quartz cuvettes get most of their chips and cracks when someone is shaking out the contents since it is so easy for the cuvette to slip from wet fingers or be hit against the sink. Don’t let this happen to you.
  2. Read the absorbances of your phage dilution at 269 and 320, using TBS in a second quartz cuvette to blank the spectrophotometer at each wavelength.
  3. Calculate the number of phage particles/ml using the formula shown above.

Part 4: Biotemplate the gold and silver on the phage

  1. In a 50 mL falcon tube, add 15 mL CTAB (a cationic surfactant), 10 mL water, 10 mL of phage (3.5 x 107 phage particles/μL), and vortex about 5 seconds.
  2. Add 1ml of Au+ and invert one time exactly.
  3. Place the tube, lying flat, on the orbital shaker in the hood (speed = 50) and rock at room temperature for two hours.
  4. Add 300 μL ascorbic acid (a reducing agent), making sure to place the tip of your pipetman under the surface of the solution when you add the ascorbic acid to the nanowires. Do not vortex and do not invert.
  5. Add the volume of Ag+ you and your partner have been assigned (10, 33 or 50% - sign up on talk page), again adding the solution of silver with the tip of your pipetman submerged in the nanowire solution. Do not vortex. Instead invert 3 times exactly.
  6. Incubate by taping the tube horizontally on the desk (static) until the next lab period.
  7. Next time you'll collect the nanowires with centrifugation and prepare a copper grid using your sample. On the final day of the module you will visualize your nanowires by TEM.

Reagents list

Overnight of ER2267 MSDS
20% PEG 8000, 2.5 M NaCl
LB

  • 10 g tryptone
  • 5 g yeast extract
  • 10 g NaCl per liter
  • 20 g of agar for plates.
  • Autoclaved 30 minutes with stirbar. Poured when ~55 °C. Plates dried on bench and store in sleeves in 4 °C.

TBS

  • 50 mM Tris
  • 150 mM NaCl
  • pH 7.6

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