Lab 1

Bacterial growth curves

Contents

In this lab you will measure how the growth rate of a strain of Methylobacterium extorquens changes with the expression of formyl-tetrahydrofolate synthetase (FtfL) (see figure 1).

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Figure 1 FtfL is an important part of the main branch point of methanol metabolism.

We’ve knocked out the chromosomal ftfL gene (which encodes the enzyme FtfL) and replaced it with a tetracycline-controlled copy on a plasmid. You will grow the strain under different concentrations of anhydrotetracycline (ATc), a tetracycline derivative that can turn on the tetracycline-controlled transcriptional activation system.

Background

Sterile technique

Sterile technique is an essential skill for any microbiologist. While we owe the discovery of our beloved AM1 to contaminated flask, today we need to learn how to keep our AM1 cultures free of any unwanted bugs. Because things become unsterile when they are exposed to the air or touch a surface, here are some basic practices that will keep your workspace clean and your experiments free of any unwanted microbes:

  • Clean your workspace. These labs are shared by many folks who might not be as hygienic as you are. It’s always a good idea to wipe down your bench surface with ethanol and clean pipettors or other equipment with a Kimwipe soaked in ethanol before use.
  • Use the flame. Another way to prevent contamination is to control the airflow around your workspace. Working near a Bunsen burner helps with sterile technique by causing air to rise, which cuts down on the airborne dander and microbes that would otherwise fall on your experiment. The flame is also necessary for sterilizing metal loops, flaming the openings to glass containers, and for staying warm on cold days.
  • Limit exposure to the open-air or other surfaces. Sterile media and lab ware quickly become unsterile when exposed to the outside world. Limit the time that lids are off, try not to touch the inside of containers with your hands or pipettors, and in general, be conscious of what you’re doing and how things might become contaminated.

Freezing and reviving bacterial cultures

Almost all of the cultures that you grow in this class will start from frozen stocks. Cryopreservation allows us to keep strains constant over time; you can pull them out of the freezer whenever you need for an experiment. In today’s lab, you will learn the proper techniques for inoculating or transferring cultures on flasks and micro-titer plates.

To inoculate a strain from freezer stocks:

  • put freezer tube on ice
  • open, flame, and use the pipette to draw or scrape a bit of culture from the tube
  • inoculate into a fresh media with succinate as the carbon source and all necessary antibiotics
  • return stock to freezer

To transfer a culture:

  • make sure culture has grown up to saturation
  • for a x -fold dilution, take (1)/(x) of culture and inoculate into fresh media with antibiotics.

Growth conditions

The media that we use to grow bacteria can have dramatic consequences on experimental results. In general media needs to:

  • be consistent and reproducible

    We add all necessary trace metals to pure water rather than relying on trace metals from the plumbing.

    This principle is also the reason why we often make a large stock of media before starting a sequence of experiments. By making it only once, we insure that any changes we observe are due to our manipulations and not to differences in our media.

  • supply all necessary growth substrates

    These are usually either succinate or methanol.

  • buffer changes in pH that will happen as a result of growth

  • select for our bacteria

    This usually means that media should have the appropriate concentrations of the antibiotics that our bacteria are resistant to.

We grow bacteria in 50 mL flasks (with 10 mL of media) and in 48-well micro-titer plates (with 630 μL of media per well). Flasks are convenient because they can be flamed and allow for large volumes; plates are excellent for growing lots of number of strains or one strain under lots of conditions.

Bacterial growth

Bacteria grow by doubling. 10 beget 20 beget 40 beget 80… In short, the number of bacteria N at generation g is:

Ng  = 2Ng − 1  = N02g

If we want to describe growth in terms of time t , and rate r , rather than generations, we can invoke calculus:

(dN(t))/(dt) = rN(t)
N(t) = N(0)ert

The growth-rate r (or equivalently 1 ⁄ g ) is our proxy for fitness: its value will help determine which mutations fix and which go extinct. You will measure it by taking readings of optical density over time and fitting the resulting curve to the equation above.

Note

Optical density measures everything in cultures—live cells, dead cells, and miscellaneous cell debris. If the number of viable cells is what you need, then you should calculate the colony forming units per milliliter of culture (cfu/mL). This is calculated by growing dilutions of culture on agar plates and counting the number of colonies.

Protocol

Strain

  • CM4103 ftfL knockout with ftfL on pLC291 (aTc inducible and kanamycin resistant)
  • Dilute 100 fold

Media

  • PIPES buffer with Z3 metal mix
  • 30 mM methanol (1.214 mL / L)
  • 1000X Kanamycin (50 mg/ml)

Plate design

We will test 11 different concentrations of the inducer, aTc:

Level [aTc] ng/mL
1 0
2 0.06
3 0.24
4 0.48
5 1.2
6 2.4
7 4.8
8 7.2
9 8.4
10 12
11 18

In addition to these 11 levels, you’ll include a reference strain (REF) so that we can compare the readings between plates. Also, you will not inoculate the sterile media (M) on the extreme wells of the plates; these wells produce bad readings. In the end, your plates will look like this:

  1 2 3 4 5 6 7 8
A M 1 2 3 4 5 6 M
B M 7 8 9 10 11 REF M
C M 5 6 1 2 3 4 M
D M 11 REF 7 8 9 10 M
E M 3 4 5 6 1 2 M
F M 9 10 11 REF 7 8 M