20.109(F10): Mod 3 Day 2 Phage nanowires

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20.109(F10): Laboratory Fundamentals of Biological Engineering

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Contents

Phage nanowires

Introduction

Edited from material originally written by the Belcher lab. Special thanks to Mark Allen, and Lieutenant Colonel F. John Burpo.

Coin-type battery
Coin-type battery
The materials prepared in this lab have activity as battery electrodes. The redox properties of the material will determine the operating voltage of the electrode, while other properties of the material will improve capacity (how long the battery will last under a given current load) and rate capability (how quickly the battery can be discharged or charged). Capacity and rate capability can be improved by either making materials very small (nanomaterials) or by incorporating conducting metals into the matrix of the material.

Assembly and testing of the phage-based battery will take place over several sessions. Today's lab will focus on material synthesis. The next lab will be analyzing material by TEM. In the final sessions of the module, dried materials will be formed into electrodes, and finally, these materials will be assembled into a coin type battery and tested. Groups will vary the amount of silver in the gold phage nanowires to see how these variations affect the charge/discharge cycle. Today in lab you will calculate the concentration of phage stock you've prepared and then react it with gold and silver. You will have time while these reactions are going on to work on the research proposal idea you've got started with your lab partner.

Protocols

Part 1: Dilute the 8#9 phage stock

Count the number of plaques on your phage titering plates from last time. Calculate the concentration of phage in your undiluted purified phage sample. Be sure to take into account each dilution when you try this calculation. Express the concentration as PFU/ul and determine the volume of phage needed to make 10 ml of 3.5x10^7 PFU/µl. Make this dilution in TBS.

If you do not have titering plates that can be counted (either too few plaques due to low phage titer or too many plaques due to contamination), try to 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 M13 is ~6400.

This method for titering the phage stock is less informative since materials other than phage might be contributing to the absorbance readings and the number of infectious particles isn't truly known. Since the latter attribute is not critical for the synthesis of gold nanowires, we can give this a go...

  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 2: Template the gold and silver on the phage

  1. In a 50 ml falcon tube, add 15ml CTAB (a cationic surfactant), 10ml water, 10ml of phage (3.5x10^7pfu/µl), and vortex about 5 sec
  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 300µl 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 then wash and visualize them by TEM.

Part 3: Research proposal

Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you’ve gathered. Consider them based on:

  • your interest in the topic
  • the availability of good background information
  • your likelihood of successfully advancing current understanding
  • the possibility of advancing foundational technologies or finding practical applications
  • if your proposal could be carried out in a reasonable amount of time and with non-infinite resources

It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but by the end of today’s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic.

A few ground rules that are 20.109 specific:

  • you should not propose any research question that has been the subject of your UROP or research experience outside of 20.109. This proposal must be original.
  • you should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a ten minute oral presentation.

Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the “yeast rebuild” or the “T7.2” wiki pages on OpenWetWare for examples of research ideas in process. As part of your “for next time assignment” you will have to print out your wiki page specifying your topic, your research goal and at least five helpful references that you’ve read and summarized.

DONE!

For next time

Define your research proposal or exhibit ideas by making a wiki page to collect your thoughts and resources (you can do this on one page with your partner or split the effort and each turn in an individual page). Please start your wiki page name with "20.109(F10): your names and/or topic here"
Keep in mind that your presentation to the class will need:

  • a brief project overview
  • sufficient background information for everyone to understand your proposal
  • a statement of the research problem and goals
  • project details and methods
  • predicted outcomes if everything goes according to plan and if nothing does
  • needed resources to complete the work

You can organize your wiki page along these lines or however you feel is most helpful. Print your user page(s) for next time, making sure it defines your topic, your idea and some references you've collected and summarized.

Reagents list

0.1 M solution of hexadecyltrimethylammonium bromide ("CTAB")
10 mM hydrated hydrogen tetrachloroaurate (HAuCl4•3H2O)
10 mM silver nitrate (AgNO3)

0.1 M ascorbic acid
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