Protocol for SSC (Simultaneous Saccharification and Catabolism)

Written by Lisa Haney and Paul Scott

 

Overview

This assay uses strains of E. coli that have been engineered to metabolize 5 and 6 carbon sugars to quantify the sugars produced by hydrolysis of corn stover.  When grown in carbon-limited media supplemented with stover hydrolysis products, these bacteria act as a sugar biosensor.  Bacterial growth is proportional to the sugars available for metabolism and is monitored by fluorescence of the bacteria.  Sugars produced by chemical or enzymatic hydrolysis or a combination of the two can be quantified with this method.  Chemical hydrolysis procedures can be monitored by addition of buffered bacterial media to the hydrolysate followed by bacterial growth and quantitation.  Enzymatic hydrolysis can be monitored by running the hydrolytic reaction concomitantly with bacterial growth in a solution containing nutrients required for bacterial growth at the optimal pH for the hydrolytic enzymes.  Two reactions are run with each sample, one with a fluorescent strain of the bacteria (the experimental sample) and the other with a non-fluorescent strain of bacteria (the control sample).   By measuring the difference in fluorescence between these two reactions, variations in intrinsic fluorescence between samples are accounted for.  The level of fluorescence is converted to the amount of sugars in the reaction with a standard curve based on known levels of sugars.  For this sugar standard, we normally use a mixture of Xylose and Glucose (37.5:62.5) that approximates the expected composition of the stover hydrolysis products.

 

Day 1:

 

  1. Prepare 4 liquid cultures of bacteria to inoculate Day 2 bacterial media tomorrow.
  2. Label two 14mL falcon tubes and indicate bacterial strain:
    1. 1840µL of 0.5% sulfuric acid + 6160µL Day 1 bacterial media + 1 colony of crp*-gfp bacteria  (make sure to select a GFP+ colony)
    2. 1840µL of 0.5% sulfuric acid + 6160µL Day 1 bacterial media + 1 colony of crp*-gfp- bacteria
  3. Incubate/shake at 37 C and 225 rpm for 20 hours.

 

Day 2:

 

  1. Label 2 sterile 14mL falcon tubes* for each stover sample to be assayed.  (Indicate that the first tube is the control and the second tube is the experimental).

*Sterile glass test tubes can be substituted.

  1. Also label 2 sterile 14mL falcon tubes for each of the 5 mixed sugar standards to be assayed.  (Indicate that the first tube is the control and the second tube is the experimental).

 

Mixed Sugar Standard

% Sugar

1

0 %

2

5%

3

10%

4

15%

5

20%

 

Note: See “Sugar Standards” in the Solutions section of this protocol for proper dilutions.

 

  1. Weigh 24.8mg to 25.2mg of stover sample into the indicated sterile 14mL falcon tube.
  2. Add 1150µL of 0.5% sulfuric acid to each tube containing stover.
  3. Add 1150µL of 0.5% sulfuric acid to each of the standard tubes.
  4. Arrange tubes as follows in oven to avoid tube melting.  Use autoclavable test tube rack that holds 40 tubes.  (Bel-Art product #F187450002).  Place tubes in positions 1 and 2, 5 and 6, 9 and 10 in rows 1 and 3, and positions 3 and 4, 7 and 8 in rows 2 and 4).  (The positioning may not be necessary with the sterile glass test tubes).
  5. Incubate falcon tubes in oven for 1 hour at 100 C. 
  6. Allow tubes to cool for 30 min. 
    1. During this time, make the Day 2 bacterial media.  The rest of the steps should be done using sterile technique.
  7. Add 3850µL of crp*-gfp- bacterial media inoculum to each control sample tube.
  8. Add  3850µL of crp*-gfp bacterial media inoculum to each experimental sample tube.
  9. Add 3750µL of crp*-gfp- bacterial media inoculum to each control standard tube.
  10. Add  3750µL of crp*-gfp bacterial media inoculum to each experimental standard tube.
  11. Add 100µL of mixed sugar standards of the percentages indicated in the table above to the appropriate standard tube.  (Add 100µL sterile water to each 0% standard tube).
  12. Add 25µL of enzyme to each tube containing stover.
  13. Incubate/shake at 37 C and 225rpm for 20 hours.

 

Day 3:

 

  1. After incubation, let the bacterial suspension sit for 10 min. to allow the stover to settle to the bottom.
  2. Pipette 150µL of the suspension into a 96-well, black fluorescence plate.
  3. Shake plate on plate shaker at 400rpm for 1-2 min.
  4. Read the fluorescence of GFP: Excitation wavelength: 485nm

              Emission wavelength: 535nm

 

Solutions:

 

25mL of Day 1 bacterial media (for liquid inoculum on Day 1):

18.75mL sterile water

10mL 5x salts

993.2µL 20% mixed sugar standard (37.5 xyl: 62.5 glc)

196.6µL 1M MgSO4

5µL 1M CaCl2

250µL Kanamycin at 10mg/mL

25µL thiamin at 10%

 

160mL of Day 2 bacterial media (for SSC reactions on Day 2):

In two separate autoclaved flasks labeled “bacterial media with crp*-gfp” or “bacterial media with crp*-gfp-”:

120mL sterile water

64mL 5x salts

1274.7µL 1M MgSO4

31.8µL 1M CaCl2

320µL thiamin at 10%

1.6mL Kanamycin at 10mg/mL

6.4mL crp*-gfp or crp*-gfp- liquid culture (grown overnight at 37C)

 

1L of 5x salts (used to make bacterial media)

64.0g NaHPO4*7H2O

15.0g KH2PO4

2.5g NaCl

5.0g NH4Cl

Add ddH2O to 1L

Autoclave and store at room temperature

 

Sugar Standards

(Use sterile technique when making sugar standards)

Store standards at -20 C.

 

Glucose (used to make mixed sugars)

20% glucose: 6g glucose/30mL ddH2O (filter sterilize)

5% glucose: 2.5mL 20% glucose/7.5mL autoclaved water

10% glucose: 5mL 20% glucose/5mL autoclaved water

15% glucose: 7.5mL 20% glucose/2.5mL autoclaved water

 

Xylose (used to make mixed sugars)

20% xylose: 6g xylose/30mL ddH2O (filter sterilize)

5% xylose: 2.5mL 20% xylose/7.5mL autoclaved water

10% xylose: 5mL 20% xylose/5mL autoclaved water

15% xylose: 7.5mL 20% xylose/2.5mL autoclaved water

 

Mixed Sugar Standards (used as standards in the assay)

5% sugar: 3.75mL 5% xylose/6.25mL 5% glucose

10% sugar: 3.75mL 10% xylose/6.25mL 10% glucose

15% sugar: 3.75mL 15% xylose/6.25mL 15% glucose

20% sugar: 3.75mL 20% xylose/6.25mL 20% glucose

 

Enzyme

1mL GC 220: 1mL Multifect Xylanase

(Both enzymes are from Genencor International®)

 

Bacterial Strains:

The following strains differ by a 1 bp change in the GFP gene resulting in a non-functional GFP gene in the crp*-gfp- strain:

 

crp*-gfp- (used for control samples)

Relevant genotypes:  thi-, CRP*, pProbe-gfp-: Kanr, GFP-

 

crp*-gfp (used for experimental samples)

Relevant genotypes:  thi-, CRP*, pProbe-gfp: Kanr, GFP

 

Maintaining E. coli strains crp*-gfp and crp*-gfp-:

These E. coli strains are distributed as stab cultures of LB (Luria-Burtani) agar with 50mg/mL Kanamycin.  Since these strains are thi-, cultures can be tested on media with and without thiamin to verify the genotypes of the strains.  These E. coli strains can be maintained on LB agar plates with 50mg/mL Kanamycin.