20.109 (F07): Phage by design

Introduction

ITO=indium tin oxide

Protocols

Part 1: M13.1

As a class we will be comparing the production of phage (M13K07 and M13.1) from a "standard" lab strain, namely MG1655, and a "minimal" strain from the MDS collection. Four of these minimal strains (MDS12, MDS41, MDS 42, MDS43) are described in a [1] article from Fred Blattner's group. A fifth strain, MDS60, is an unpublished strain with additional genomic deletions. We will make the strains competent for transformation using the protocol described by Inoue, Nojima and Okayama in their 1990 publication.

1. Choose one of the MDS collection to work with, describing in your lab notebook the deletions that the strain includes.
2. Collect 10 ml of log phase cells (MG1655 or an MDS) and move them to a falcon tube.
3. Spin your tube (balanced against another) in the clinical centrifuge, 3000 RPM for five minutes.
4. Pour off the supernatant from the pellet.
5. Resuspend the bacterial pellet in 1 ml ice cold "TB". Move the cells to an eppendorf tube and leave on ice for 10 minutes.
6. Spin the cells in a microfuge for 30 seconds at full speed.
7. Aspirate the supernatant from the pellet.
8. Resuspend the pellet in 150 ul ice cold "TB".
9. Add 10.5 ul DMSO to the suspension of cells and flick the tube to mix.
10. Move 75 ul of cells to a new eppendorf with 5 ul of M13K07 DNA.
11. Move another 75 ul of cells to another new eppendorf with 5 ul of M13.1 DNA.
12. Incubate the three tubes (no DNA, +M13K07, +M13.1) on ice for 5 minutes.
13. Heat shock the three tubes at 42° for 90 seconds exactly.
14. Add 500 ul of LB to each tube and incubate at 37° for at least 30 minutes. Prewarm and dry 3 LB+Kan plates at this time as well.
15. Invert the tubes to mix the contents and spread 200 ul of each transformation mix on an LB+Kan plate.
16. Incubate 37° overnight.
17. Before leaving lab, you should innoculate 3 sterile glass tubes with 3 ml LB+Kan, labelling the top of the tubes with your team color and the contents of the tube (no cells, M13K07 in strainX, M13.1 in strainX). These will be used to grow transformants for you for next time.

Part 2: Pattern for phage deposition

There are two dangerous parts in today's lab. You will be working with an Exact-O knife to carefully cut a pattern onto your slide. These knives are extremely sharp and everyone should show the greatest care in handling these or you will end up at MIT medical. The second danger is the concentrated acid treatment needed to pattern the slides. You must, must, must wear lab coat, closed-toed shoes, gloves (perhaps double gloves), and goggles when you are dealing with the HCl, HNO3 solution.

1. It will not be obvious which of the sides of the slide has ITO once you remove the paper. You should use the multimeter to measure the conductance of each surface. Hold the probes a set distance from one another and measure resistance (in ohms). If there is no load the meter will read "0L." The side of the slide with ITO will yeild a measurement.

   

   multimeter set to measure current rather than resistance as you should be doing

2. Clean the ITO slide by placing it into a 50 ml falcon tube with 40 ml of MeOH. Swish the MeOH over the slide and then let the slide airdry on a Chemwipe on the bench. Do not wipe the ITO surface. Save tube of MeOH for the end of lab.
3. While the slide is drying, decide on your ECD design. What you choose should fit onto the bottom half of the slide, as shown in the figure.. As you think about what image to choose note that the area of your image that you want dark (i.e. visible) should remain under tape and you will cut out the areas that you want clear. Also note that the right edge of the image should remain protected by tape so the current can flow across to the image when we finally hook it up. Print out three copies of your image: one for the slide and one for each of your notebooks.
4. When your slide is dry, place it ITO side up onto the bench and fully cover it with a strip of red 3M tape. Press the air-bubbles out.

   

   
   
5. Cover the end that you'll pattern with double-sided scotch tape and then tape your printed image to the slide.

   

   
   
6. Cut the pattern you've chosen, running the knife-blade back and forth a few times on each cut to insure that all the tapes have been cut.
7. Peel off the paper, scotch-tape and red tape to expose the areas of the slide where the ITO is to be removed (i.e. where no phage will bind, i.e. where no color will form). The silver tweezers in your drawer should be userful for this step. Use the Exact-O knife as needed also.

   

8. Place your slide, pattern down, into the acid wash solution that is gently mixing on a stir plate in the hood. Balance your slide on the edge of the beaker, so the red tape remains outside the acidic solution. It should be possible to balance 2 or 3 slides in each beaker.

   

9. Incubate the slide in the acid solution for 1 hour.
10. Wash the slide by swirling it three times in the 1L beaker of H2O that is in the hood. Be very careful not to drip any of the acid wash solution as you move the slide.
11. Clean the slide by placing it back in the falcon tube with MeOH, then allow the slide to air dry on Chemwipes on the bench.
12. Before fully removing the tape that remains on the slide, verify that the ITO has been removed from the exposed regions. Peel back a small corner of the red tape that remained outside the acid solution. Use the multimeter to compare the conduction when the probes are

	mV
both touching bench top or glass slide
both touching metal
both touching untreated area
both touching treated area
touching treated/untreated area

Part 3: Research Proposal

You should be on your way to becoming an expert on your research topic. In the past few days you've probably read a lot about it and you may feel
(a) like there's too much to read
(b) like you have too many ideas and no way to map or prioritize them
(c) like you don't understand what you're reading
(d) all of the above.

One of the best ways to help frame the problem for yourself is to discuss it with someone new. While your yeast are incubating with the gold slides, you will have time to talk with a person from another lab group. This person will offer you a fresh ear to consider your proposal. Try to describe your research problem to them. Articulate why it's important. Tell them about some recent, relevant data. Describe what you're proposing to do and what the findings from your experiments might reveal. Throughout your discussion, keep careful track of the questions they ask since these will point you to the confusing concepts or fuzzy parts of your explanation or understanding.

Then be a good listener to hear the proposal that they've been working on. Ask lots of questions. No questions are dumb. You are there to offer a naive ear and seek complete explanations. You will have time at the very end of class to reconvene with your own lab partner to hear how their conversation went. Try to identify repeated questions or concerns since these are probably the holes in the project as it stands. You can rework your proposal based on the conversations you've had.

DONE!

For next time

1. Outline your research proposal presentation. Recall that you will need to present
   • a brief project overview (1 slide)
   • sufficient background information for everyone to understand your proposal (1-3 slides)
   • a statement of the research problem and goals (1 slide)
   • project details and methods (3-5 slides)
   • predicted outcomes if everything goes according to plan and if nothing does (3-5 slides)
   • needed resources to complete the work (1 slide)
   • societal impact if all goes well (1 slide)

Suggested numbers of slides are listed here but the number may vary depending on the particulars of your proposal. You will have time to work on the presentation in lab next time as well as ask questions about the format, but you should begin to prepare, in powerpoint or similar presentation program, the materials you need.

Reagents list

Acid wash solution ("aqua regia")

• in 250 ml beaker add 100 ml H2O
• to water add ~45 ml HCl
• to dilute HCl add 15 ml HNO3