20.109(F07): Growth of phage materials

Introduction

Abalone shell

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. Even more amazing, 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 biological character from earlier in the term, namely M13, and rely on the self-assembling coat to template a crystal of iridium oxide. The interaction of iridium with the p8 protein of the phage coat yields nanoscale-particles with useful optic properties, as we’ll see (literally).

Thinking back to the work we've done so far this term, there seems to be nothing in our initial studies of M13 that would lead us to believe the phage could interact with inorganic materials. As far as its natural history goes, it's unlikely this phage would have run into lots of iridium in its home of the sea. It seems even less likely that the phage might, by happy coincidence, bind this metal. It's in fact through some clever human intervention that the a phage with such an unusual property was found.

Protocols

In advance of this lab, a bacterial host (namely XL1-blue) was infected with the modified M13 phage called "3-12." Though the precise details of the 3-12 modification are still unpublished, it was isolated from a screen for changes in the M13 p8 that enable the phage to bind a metal (Iridium). Today you will harvest the phage from the supernatant of the infected bacterial culture and then titer it.

Part 1: Phage purification

1. Spin 2 eppendorf tubes with infected cells in a room temperature microfuge, 1 minute at full speed.
2. Remove the supernatant to clean fresh eppendorf tubes.
3. Use your P1000 to measure the volume, then add a 1/6th volume of 20% PEG-8000/2.5M NaCl solution.
4. Invert to mix then incubate on ice 60 minutes.
5. Spin in a room temperature microfuge, 15 minutes at full speed. A white pellet should be visible...these are your precipitated phage.
6. Remove the supernatant by aspiration (carefully so as not to disturb the pellet) or using your P1000.
7. Spin the tubes 1 minute more to pellet any droplets stuck to the walls of the tube and use your P200 to remove the last drops of liquid from the pellet.
8. Resuspend the pellet in 100 ul sterile H2O, pool the volumes of both eppendorf tubes into one and add a 1/6th volume of 20% PEG-8000/2.5M NaCl solution.
9. Invert to mix then incubate on ice, 15 minutes.
10. Balance your sample against that of another group, or against an eppendorf with water. Spin in a room temperature microfuge, 10 minutes at full speed.
11. Aspirate the supernatant and resuspend the pellet in 100 ul sterile H2O.
12. If the solution looks at all cloudy, spin in a room temperature 1 minute more and move supernatant with the phage to a new eppendorf tube.

Part 2: Preparation of Ir(III)Cl3

A solution of 25mM IrCl3 was prepared by dissolving 1.7 g in 200 ml H20. You will adjust the pH of an aliquot to somewhere in between 7.0 and 7.5, and then store the solution in the dark until next time. This "rest" period between pH-ing and usefulness seems necessary, though the reasons for it are unclear. One idea is that the water structures around the iridium ions in a way that's important for their deposition on the phage, a process you'll work on next time.

1. Move ~4 ml of the 25 mM IrCl3 solution from the stock bottle to a 15 ml falcon tube. The volume you pH can be approximate, estimating the amount using the markings on the side of the tube.
2. Measure the starting pH by dribbling a small amount on pH paper. A pasteur pipet and bulb work well to transfer the liquid to the paper, or your P200 set to 50 ul. Do not dip the paper into the solution.
3. Add one drop of 1N NaOH to your aliquot of IrCl3. Invert the tube to mix and check pH again using pH paper.
4. If you're still well below pH 7 then add another drop of 1N NaOH. If you're close, dilute the 1N to 0.1N with water and add a drop of the dilute base. If you've overshot the range and are at 10 or above, then add 500 ul of un-pH'd IrCl3 to your solution.
5. Recheck with pH paper.
6. When the pH paper tells you that the pH is close, use the pH meter to know precisely what the pH of your solution is. Add this pH value to the table of experimental variables. If you've forgotten how to use the pH meter, ask one of the teaching faculty.
7. Store your pH'd solution of IrCl3 wrapped in foil in the dark until next lab.

Part 3: Titering phage

This protocol is identical to one you performed earlier in the term. You should dilute the phage you've prepared today so the final concentrations of phage are 10^6, 10^8, and 10^10th less concentrated than your stock. In addition to 10 ul of these three dilutions, you should include a "no phage" sample.

Before you leave you should place your petri dishes in the 37° incubator and give the phage stock to the teaching faculty, who will store it at 4° for you until next time.

DONE!

For next time

1. The primary assignment for this experimental module will be for you to develop a research proposal and present your idea to the class. For next time, please describe five recent findings that might define an interesting research question. You should hand in a 3-5 sentence description of each topic and list the reference that led you to each item. The topics you pick can be related to any aspect of the class, i.e. DNA, protein, systems or bio-material engineering. During lab next time, you and your partner will review the topics and narrow your choices, identifying one or perhaps two topics for further research.
2. electronic paper q

Reagents list

25 mM IrCl3

• 1.7 g dissolved in 200 ml H2O

1N NaOH