20.109(S07): Growth of phage materials

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20.109: Laboratory Fundamentals of Biological Engineering

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Contents

Introduction

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 Science 2006 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 A fifth strain, MDS60, is an unpublished strain with additional genomic deletions.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: M13E4

  1. To infect M13E4 into XL1-blues you should mix 10 ml LB, and 10 ul tet.
  2. Split this between 2 sterile glass tubes.
  3. To one of the tubes add 10 ul M13E4 stock and 2 ul competent XL1-blues.
  4. Label the tubes with your team color and the contents.
  5. Balance them on the 37° roller wheel.
  6. One of the teaching faculty will move them to 4° tomorrow.


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. genome engineering, signal measurements, gene expression engineering 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.

Reagents

  • "TB" from Inoue protocol
    • 10 mM PIPES
    • 55 mM MnCl2
    • 15 mM CaCl2
    • 250 mM KCl
    • filter sterilized
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