20.109(F13): Mod 3 Day 2 Phage nanowires

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20.109(F13): Laboratory Fundamentals of Biological Engineering

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Contents

Fabricating nanocomposites with M13 phage

Today's Goal

In an effort to optimize efficiency in our solar cells, we will test and compare test two sets of nano-composites of titanium dioxide nano wires. One set contains aligned gold nanoparticles, and the other aligned SWNTs, inside the wire core. M13 phage that can create these structures were discovered by using a combinatorial approach of panning and bio-templating.

Introduction

Phage Display - Revisited

Phage display is a technique originally developed in the 1980s that uses affinity selection to identify functional peptide sequences that have been fused to the phage coat. Large peptide inserts must often be fused to the p3 protein 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. On the other hand, only inserts of less than 6-8 residues (primarily neutral or negatively charged) can be tolerated in p8 without inhibiting the formation of new phage particles, due to the semi-crystalline packing of this protein. However, even though it is more restrictive, one major benefit of expressing p8 variants is that the high protein copy number (~2700) leads to a highly multivalent virion. Additional systems have also been developed in which wild type g8 is co-expressed in the infected cell (either from a separate plasmid or an additional gene in the phage genome) such that the resulting phage have both types of p8, and less favorable p8 inserts can still be partially displayed on mature phage.

Phage Display Protocol Courtesy of Dr. Graham Beards
Phage Display Protocol Courtesy of Dr. Graham Beards

Despite these limitations, peptides with remarkably diverse functions have been isolated using phage display. In order to identify an unknown peptide sequence capable of interacting with a material of interest using this technique, one must first decide which coat protein is appropriate for the fusion. Next, a library of sequences encoding random peptides can be synthesized and cloned into the corresponding gene locus, resulting in phage that are identical except for a variable peptide insert appended to the coat protein. Upon washing this pool of phage over virtually any material of interest (carbon nanotubes, whole cells, immobilized antibodies, etc) phage with increased binding affinity can be physically separated from the non-binding phage. Given that each phage in the randomized library houses the genetic information needed replicate its own specific peptide sequence, this subset of binding phage can be amplified in bacteria to regenerate large pools of phage, and the process can be repeated. As more and more stringent washing conditions are imposed upon the subsets of binding phage, the population converges to the strongest binding phage out of the initial library. The peptide or peptide motif responsible for this affinity can be determined by sequencing the phage genome. One caveat is that the amplification step provides a bias towards faster growing phage; thus, strong binding yet slow assembling candidate phage may be inadvertently lost.

Biotemplating

Biotemplating utilizes biological materials to fabricate and/or organize unique nanomaterial structures. A variety of benefits are associated with biotemplated synthesis methods over traditional synthesis methods. From the fabrication standpoint, biotemplating can reduce the environmental impact of materials synthesis. Biotemplating has been shown to enable synthesis of materials at lower temperatures and pressures as well as using milder reagents than traditional synthesis methods. From the stand point of organization, biotemplating can achieve unique structures over broad length scales. These structures are either unattainable or significantly more difficult to create without biotemplating.

For this module, biotemplating of titania on the M13 phage will be explored. For this process, the M13 phage will serve as a nucleation site for the precursor molecule – titanium isopropoxide – to form titania via the following reaction:

Ti{OCH(CH3)2}4 + 2 H2O → TiO2 + 4 (CH3)2CHOH

The above reaction occurs very rapidly and is water sensitive by nature. To compensate, we will perform this reaction at -40 °C to slow down the reaction kinetics, and perform it in 95% ethanol to allow for a more controlled hydrolysis reaction than would occur in excess water.

If you are interested in more information about biotemplating, you may want to consult this review article: Sotiropoulou, Sofia, et al. "Biotemplated Nanostructured Materials." Chemistry of Materials 20.3 (2008): 821-834.

Materials → Device Preview

Eventually, we'll build a solar cell where the photoanode is made from the material that we're synthesizing today. By templating the assembly of the SWNT-or-Au/TiO2 nanocrystal with the M13 phage, the electronic properties of the photoanode are improved.

phage:SWNTs:TiO2 nanocomposite, image from MIT News 04.52.11
phage:SWNTs:TiO2 nanocomposite, image from MIT News 04.52.11

"Improved" here means a few things, which we'll discuss more deeply on Day 4. Briefly, engineers trying to optimize photovoltaic devices want many high mobility electrons inside their devices to efficiently convert the photo-energy input to electrical power output. Gold nanoparticles increase the fraction of collected light that is usable for excitation, while SWNT allow for fast electron movement and reduce dead-end recombination events. Moreover, we will rely on M13 to facilitate electron paths by arranging the SWNTs into a higher-order architecture, thereby improving collection efficiency further. Not only can M13 bundle the SWNTS so they don't clump together (see how the p8 proteins allow the SWNTs to associate in parallel to the phage in the photo), but the viruses also position parts of the SWNTs to the surrounding solution, allowing for a more complete coating with TiO2. The TiO2 nanocrystal shell (like its more expensive silicon cousin) is needed to pass the electrons from the photo-excited dye...which we'll be adding later.

Protocols

Today in lab you will react your SWNT:phage or Au:phage with titanium isopropoxide, harvest a small aliquot to visualize with TEM next time, and then wash the remainder of the nanowires several times – first with ethanol, and then with water. You will have time during these steps to work on the FNT, a first step toward developing a research proposal idea. Next time, you'll share your FNT findings with your partner.

Part 1: React SWNTs:phage with Ti(I-pro)4

Today's lab has some safety hazards and you must work extremely carefully. Lab coats, gloves and goggles are a must when you're at the chemical hood. The reaction of the complexed phage with the titanium will take place in the hood at supercooled temperatures (a bath at ~ -40°C). Once the titanium has been deposited on the surface of the phage, the solution is less hazardous, though you should still treat the materials with care since no reactions run to completion

For SWNT phage only:

  1. Retrieve your dialyzed samples.
  2. Carefully transfer the contents of the dialysis bag to a 15 ml falcon tube. We recommend the following approach: hold the dialysis bag vertically while you remove one of the alligator clips, cover the open end of the tubing with the falcon tube, and then invert the tubing and the falcon tube so the complexed phage solution can be collected.
  3. If there are any nanowires remaining in the dialysis tube after you have emptied the contents, rinse the nanowires out using 1 ml of the NaCl dialysis buffer and add the volume to the 15 ml falcon tube.
  4. Use the markings on the tube to estimate the volume of the nanowires.
  5. Vortex the nanowires if they look clumpy.


Bath set-up
Bath set-up

For all groups:

  1. Chill your complexed phage on ice on your bench until you are ready to react it with the titanium.
  2. Calculate the amount of material necessary for making TiO2 Nano composites. The reaction should take place in a 95% ethanol solution. You have two choices:
    • a. If your phage volume is less than 250 uL total, you may simply use 5 ml of 95% ethanol.
    • b. If your phage volume is > 250 uL total, calculate how much 100% ethanol you would need to add to achieve a 95% solution. Assume that your phage solution is 100% water.
  3. Add the ethanol volume from a. or b. without phage to a 250 mL erlenmyer flask with a stir bar and set aside.
  4. Prepare a super-cooled bath:
    • Mix ethylene glycol and ethanol in a 1:1 volume ratio (make up a total of 100 mL).
    • Place the 250ml Erlenmeyer flask with your ethanol solution (and stir bar) inside an evaporation dish in the fume hood on a stir plate.
    • Fill the evaporation dish with the ethylene glycol and ethanol mixture so as to match the liquid level inside the Erlenmeyer flask.
    • Add ~10 dry ice chunks to the evaporation dish and let sit for 10 min. You can measure the temperature of this mixture, which should be ~ negative 40°C.
  5. Next, calculate how much Ti(I-pro)4 you must add to your phage in order to achieve a 15:1 TiO2:phage ratio. The 15:1 gram ratio is based off of a calculation of the phage surface area to the surface area of the phage covered in TiO2, as the goal is to cover the phage completely with TiO2.
  6. Quantities you need to know:
    • MW of phage = 1.8 x 10^7 g/mol
    • MW of Ti(I-pro)4 = 284.22 g/mol
    • MW of TiO2 = 80 g/mol
    • Concentration of Ti(I-pro)4 = 0.96 g/mL
 Think about the following progression:
 phage particle # --> moles phage --> weight phage --> weight TiO2 --> moles TiO2 = 
 moles Ti(I-pro)4 --> volume of Ti(I-pro)4 to add
  1. Put on a lab coat, gloves, and safety glasses for this next step. Add the calculated amount of Ti(I-pro)4 to the supercooled EtOH. Stir at least 5 minutes.
  2. Add your complexed phage solution to the EtOH:titanium and stir vigorously for 20 minutes.
  3. Remove the Erlenmeyer flask from the bath and place it back on the stir plate. Leave the mixture on the plate until it reaches room temperature. After a maximum of an hour your solution should turn grey (SWNT complexed phage) or white (gold complexed phage) indicating that the reaction has finished.

Part 2: Prepare a grid for TEM

Each group should prepare a TEM grid (so there might be 3 grids for each reaction of phage:SWNTs:TiO2). This will allow for some duplicates to be visualized in case the grid is damaged or different kinds of EM are being performed.

clever trick to keep the stirbar from falling out of your jar when you pour out the solution
clever trick to keep the stirbar from falling out of your jar when you pour out the solution

  1. Transfer the reaction to a 50 ml falcon tube. You can harvest the last few drops but not the stir bar using a larger magnet held to the bottom of the inverted jar (see image)
  2. Sometime before this next step, watch a demo from the teaching faculty, practice using the specialty tweezers, and prepare your grid.
  3. Vortex the nanowires for 1 minute and immediately remove 5 ul of the nanowire suspension to place on the TEM grid that you have balanced in the specialized tweezers. HINT 1: The grid is "sided" and you want the shiny edge side up. If you are uncertain as to which side has the shiny edge, try looking under the dissecting microscope (12X magnification) to find the numeral "1" on the correct side. HINT 2: Treat the grid with care and use the tweezers only on the edge to minimize damaging the delicate mesh.
    TEM grid balanced in tweezers
    TEM grid balanced in tweezers

  4. Allow the nanowires to settle onto the grid undisturbed for 5'. The EtOH will evaporate during this time. You can wick away any residual EtOH by touching the very edge of the grid with a Kimwipe.
  5. Wash the grid by adding 5 uL of 100% EtOH onto the grid. After 30 seconds you can wick away the EtOH.
  6. Wash the grid by adding 5 uL of sterile H2O onto the grid. After 30 seconds you can wick away the water and transfer the grid to a holder to visualize next time. Write down your slot letter/number.

Part 3: Wash your phage:SWNTs:TiO2 or phage:AuNPs:TiO2 nanocomposites

  1. Spin the remaining volume of nanocomposites in the clinical centrifuge at room temperature, 3000 rpm 10 minutes. At the end of this spin you should see dark material collected at the bottom of the tube. This is the material that will serve as the photoanode in our photovolatic device!
  2. Carefully pipette away the supernatant into a fresh 50mL conical tube without disturbing the pellet. Decant this supernatant into a chemical waste bottle in the chemical hood labeled 'Titanium Dioxide'. Resuspend the nanocomposite material in 20 ml dH2O. Spin as before.
  3. Immediately after spinning, invert the capped tube. If the pellet remains stable at the tip of the tube, keep the tube inverted and decant the supernatant into the sink.
  4. If the pellet resuspends after inversion, respin and carefully pipette away as much supernant as possible without perturbing the pellet.
  5. Hand the pellet of phage:SWNTs:TiO2 or phage:AuNPs:TiO2 to the teaching faculty who will begin to prepare the paste needed to build them into the solar cell.

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 could potentially define an interesting research question. You should hand in a 3-5 sentence description of each topic, in your own words, and also formally cite an associated reference from the scientific literature. The topics you pick can be related to any aspect of the class, i.e. DNA, system, or biomaterials 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.
    • Note: for now, you do not have to have a novel research idea sketched out; you simply have to describe five recent examples of existing work. However, you can start to brainstorm how to build off of those topics into something new if you want to get ahead of the game.

Reagents list

  • 100% EtOH
  • Ti(I-pro)4 msds

Equipment

Per group

  • Erlenmeyer flask
  • Glass evaporation dish
  • Rubber stop caps
  • 2 TEM grids

Every other group

  • thermocouple
  • stirplate
  • stir bar

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