Laboratory Investigations in Microbiology

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Chapter 20: Microbial genetics I: control of gene expression

Even though bacteria such as E. coli have more than 1,000 genes, these are not all continually active. Genes are said to be expressed when their gene product (usually a protein) is being produced by the cell. (Even then, this protein may be in an inactive or active state). However, due to the high cost of synthesizing proteins, a cell can conserve significant energy simply by shutting off unnecessary genes. Enzymes for DNA replication, for example, are only needed during cell division, and flagellar proteins only when cells are motile and planktonic.

In order to regulate the expression of a gene, cells utilize DNA sequences in front of the protein-coding gene sequence (transcribed region). These unique sequences (Fig. 1) are recognized by the enzyme RNA polymerase. Additional DNA sequences may precede or follow the polymerase binding site and allow for the attachment of protein factors that can enhance (transcription factors) or block (repressor protein) transcription.

Transcription of genes in bacteria is known to be affected by oxygen, nutrients, temperature, quorum-sensing molecules, toxins, metals, and many more. In this experiment, we will investigate the control of the prodigiosin pigment-producing genes in Serratia marcescens. Serratia marcescens strain 1722 (Ref. 1) contains a plasmid (pMQ262) inserted into Serratia's pigment-producing operon, placing the pigment-producing genes under control of an arabinose-inducible promoter. Serratia pigmentation is also temperature-sensitive.

 
Materials

Per Lab group

Procedure: first lab period

Arabinose

  1. Inoculate your TSA plate with Serratia 1722 using a cotton swab
  2. Aseptically place 5 paper discs on the agar plate, spaced evenly apart
  3. Pipette 10ul of each solution (CON, ARA 10, ARA 100, GLU 10, GLU 100) onto a cardboard disc. Wait for solutions to be absorbed
  4. Place plates RIGHT-SIDE-UP into bin on the cart for incubation (48 h at 30C)

Temperature

  1. Label 5 TSB tubes as 25C, 29C, 31C, 33C and 35C
  2. Inoculate each test tube with 100 ul of WT Serratia culture
  3. Place tubes into appropriate water baths/incubators for incubation (48 h)
Procedure: Next Lab period

Arabinose

  1. After incubation, observe and describe your plate and the region surrounding each paper disc
  2. Measure the diameter of red pigmentation around each paper disk

Temperature

Reagents: Acid-alcohol (0.02N hydrochloric acid, 95% ethanol)

  1. After incubation, remove tubes from water baths.
  2. Measure the culture density
    1. Pipette 1 ml of distilled water into a plastic cuvette
    2. Set the wavelength to 600 nm and calibrate your spectrophotometer
    3. ADD 0.1 ml of TSB broth from your culture tube, mix gently in the cuvette, and record A600
    4. Dump the cuvette contents into the waste flask, rinse 1x with distilled water
    5. Repeat for each temperature
  3. Extract the red pigment
    1. Pipette 1 ml of each TSB into a separate microcentrifuge tube; label tubes
    2. Centrifuge tubes for 3 min at 13,000 rpm to pellet the cells
    3. Pour off the supernatant (liquid) into the WASTE flask
    4. Add 1 ml of acidified ethanol (wear gloves) to the pellet; mix gently by pipetting up & down a few times
    5. Vortex the mixture an additional 30 - 60 seconds
    6. Centrifuge your tubes for 3 min at 13,000 rpm to pellet the cell debris, leaving a clear supernatant
  4. Measure pigment
    1. Set the wavelength of a spectrophotometer to 534 nm and calibrate your spectrophotometer using 1 ml of acidified water (aH2O)
    2. ADD 0.1 ml of your pigment extract to the 1 ml acidified water; mix gently
    3. Read the absorbance of your culture at this wavelength. Record your data.
    4. Dump the cuvette contents into the waste flask, rinse 1x with distilled water
    5. Repeat this procedure for each of the other 4 temperatures
  5. Calculate the ratio of A534/A600 for each culture.
  6. Plot/graph the data using Excel
References
  1. Khadouri, D.E. and R.M.Q. Shanks. Identification of a methicillin-resistant Staphylococcus aureus inhibitory compound isolated from Serratia marcescens. Res Microbiol. 2013 Oct; 164(8): 821826.
  2. Stella, A. et al. Serratia marcescens cyclic AMP receptor protein controls transcription of EepR, a novel regulator of antimicrobial secondary metabolites. J. Bacteriology 197(15):2468-2478, 2015.

 

 

2003 - 2019 Jos de Ondarza, Ph.D.