In vivo Quantification of Alkanes in Escherichia coli

Figure 1. Schematic representation of AAR-ADO pathway for alkane production

Materials and Reagents

1. 2 ml micro-centrifuge tubes (Tarson, catalog number: 500020 )
2. Culture tubes (55 ml, Borosil, catalog number: 9820U08 )
3. GC vials (Agilent, catalog number: 5190-2280 )
4. Parafilm (Sigma, catalog number: P7793 )
5. 0.2 μm filter (mdi Membrane Technologies, catalog number: SYNN0301MNXX104 )
6. E. coli DH5α strain overexpressing ADO enzyme cloned in pQE30 vector (strain source: Invitrogen, vector source: Qiagen)
7. Hexadecanal (TCI America, catalog number: H1296 )
8. Octadecene (TCI America, catalog number: S0348 )
9. Sodium phosphate dibasic (Na₂HPO₄) (Merck, catalog number: 255793 )
10. Potassium dihydrogen phosphate (KH₂PO₄) (Sigma, catalog number: P5655 )
11. Sodium chloride (NaCl) (Sigma, catalog number: S9888 )
12. Ammonium chloride (NH₄Cl) (Sigma, catalog number: 213330 )
13. Magnesium sulphate heptahydrate (MgSO₄·7H₂O) (Sigma, catalog number: 230391 )
14. Calcium chloride (CaCl₂) (Sigma, catalog number: C4901 )
15. Ferric chloride hexahydrate (FeCl₃·6H₂O) (Sigma, catalog number: 236489 )
16. Zinc chloride (ZnCl₂) (Sigma, catalog number: 746355 )
17. Sodium molybdate dihydrate (Na₂MoO₄·2H₂O) (Sigma, catalog number: 331058 )
18. Copper sulphate (CuSO₄) (Sigma, catalog number: C1297 )
19. Boric acid (H₃BO₃) (Sigma, catalog number: B7901 )
20. Thiamine hydrochloride (Sigma, catalog number: T4625 )
21. Glucose (Sigma, catalog number: G8270 )
22. Bis Tris (Amresco, catalog number: 0715-250G )
23. Triton X-100 (Amresco, catalog number: M143-1L )
24. Ethyl acetate (Merck, catalog number: 1096232500 )
25. IPTG (Sigma, catalog number: I6758 )
26. LB broth (HiMedia, catalog number: M1245 )
27. Ampicillin (Sigma, catalog number: A9393 )
28. M9 salts (see Recipes)
29. M9 modified medium (see Recipes)
    

Equipment

1. Table top centrifuge (Eppendorf, models: 5418R and 5810R)
2. Incubator shaker (Kuhner, model: ISF-1 )
3. GC FID (Agilent, model: 7890 A System , equipped with HP-5 column with catalog no. 19091J-413 )
4. Spectrophotometer (GE Healthcare, model: Ultrospec^TM 2100)
5. Vortex (Sigma, catalog number: Z258423-1EA )
6. Autoclave (Natsteel, model: 24 SR )
   

Procedure

1. In vivo culture
   1. Inoculate E. coli strains harbouring the plasmids carrying ADO gene (Figure 2) from plates streaked and incubated overnight at 37 °C (plates originally streaked from glycerol stocks) in 5 ml LB broth medium with 5 μl of appropriate antibiotics (ampicillin–100 μg/ml used in this study) in 55 ml culture tubes (1 ml of Ampicillin stock of 100 mg/ml was prepared by adding 100 mg of Ampicillin powder in 1 ml of water followed by filter sterilization).
      
      
      Figure 2. Schematic representation of the plasmid involved in alkane pathway construction. Plasmid vector map of pQE_ADO, carrying ado gene in pQE30 plasmid at the restriction sites BamHI/SacI.
      
      
   2. Incubate the samples at 37 °C overnight in an incubator shaker set at 180 rpm.
   3. Use 50 μl (OD₆₀₀ ranging from 3-4) of the primary inoculum grown overnight to inoculate 3 ml secondary culture having M9 modified medium (Recipe 1) supplemented with 2% glucose, 1 mg/L thiamine and 100 μg/ml ampicillin.
   4. Induce the culture with 0.01 mM IPTG (3 μl of 10 mM IPTG to 3 ml culture) at the time of secondary inoculation in M9 medium as the plasmid pQE30 carrying ADO gene is inducible by IPTG.
   5. Add 100 mg/L hexadecanal (dissolved in absolute ethanol) exogenously to the culture medium at the time of secondary culture inoculation and seal the culture tubes with parafilm to prevent any loss of substrate or product due to evaporation (15 μl of 20 mg/ml stock added to 3 ml culture).
   6. Incubate the culture tubes at 30 °C (or higher to test for thermostable enzymes) in an incubator shaker set to 120 rpm for 48 h.
      
      
      
2. Hydrocarbon extraction
   1. Measure the optical density at 600 nm of the samples to estimate the growth before proceeding with extraction of alkanes.
   2. Take equal volumes (0.75 ml) of sample and ethyl acetate (containing 10 mg/L Octadecene added as internal standard–from a 100 ml stock of ethyl acetate containing 1.265 μl of 99% 1-Octadecene) in a 2 ml microcentrifuge tube.
   3. Vortex the samples for 20 min and centrifuge at 15,700 x g for 3 min at room temperature.
   4. Collect the upper fraction and aliquot it into a fresh GC (gas chromatography) vial.
   5. Analyze the samples using gas chromatography (GC).
      
      
      
3. Hydrocarbon analysis
   1. Set the hydrocarbon analysis method on the gas chromatography (GC) system equipped with HP-5 column of 30 m length, 0.32 mm internal diameter and 0.25 mm film thickness. The FID detector is used for analysis. The hydrocarbon analysis method is to be set as follows. The oven temperature programme is set as: initial 100 °C held for 3 min, increase temperature to 250 °C at the rate of 10 °C min^-1 and held at 250 °C for additional 10 min. The total run-time of the programme will be 28 min. The inlet and detector temperature are to be maintained at 150 °C and 280 °C, respectively.
   2. Run pentadecane, hexadecanal, 1-octadecene, and hexadecanol standards of known quantity (Figure 3) prior to sample analysis and use the area under curve to calculate the concentration of metabolites in the test samples.
   3. Run the test samples in GC machine (Figure 4) and quantify the pentadecane content with respect to its standard.

Data analysis

Representative data
For detailed data analysis, please refer to the article of Shakeel et al. (2018). The representative GC chromatograms for all the standards are shown in Figure 3. The area under curve of known quantities of these standards are used to quantify metabolites in the test samples. The representative GC chromatograms for the typical in vivo samples containing alkane are shown in Figure 4. The area under curve of each metabolites is used to calculate their concentrations by comparing it with the area under curve of corresponding standards of known quantities.


Figure 3. GC chromatograms of the standard metabolites. A. Chromatogram of Hexadecanol and 1-Octadecene. B. Chromatogram of Hexadecanal and 1-Octadecene. C. Chromatogram of Pentadecane and 1-Octadecene. The 1-Octadecene was used as an internal standard for checking extraction efficiency, while other metabolites are either substrate (Hexadecanal) or products (Hexadecanol and Pentadecane) observed after the hydrocarbon extraction.


Figure 4. Functionality of ADO protein in recombinant E. coli after addition of exogenous precursor i.e. hexadecanal. A. GC chromatogram of control sample containing hexadecanal and internal standard. B. GC chromatogram of E. coli expressing empty pQE30 plasmid, showing peak of hexadecanal and hexadecanol. C. GC chromatogram of E. coli expressing pQE_ADO, showing formation of end metabolites i.e., pentadecane and hexadecanol.

Recipes

1. M9 modified medium
   Components of M9 modified medium are prepared as follows:
   1. M9 salts
      For 100 ml M9 salts (10x)
      11.32 g Na₂HPO₄
      3 g KH₂PO₄
      1 g NaCl
      2 g NH₄Cl
      Volume made up to 100 ml by adding MQ water and filter sterilized using 0.2 μm filter
   2. Trace element recipe
      1x Trace element composition:
      0.25 g/L MgSO₄·7H₂O
      11 mg/L CaCl₂
      18.71 mg/L FeCl₃·6H₂O
      1.311 mg/L ZnCl₂·4H₂O
      2 mg/L Na₂MoO₄·2H₂O
      1.9 mg/L CuSO₄
      0.5 mg/L H₃BO₃ (Boric acid)
      1 mg/L Thiamine
      All the above components are prepared individually as 100x stock in MQ water and filter sterilized using 0.2 μm filter
   3. 1x Additive composition
      200 mM Bis Tris (5x stock prepared)
      0.1% Triton X-100 (10x stock prepared)
      2% Glucose (10x stock prepared)
      All the above components are prepared individually as in MQ water and filter sterilized using 0.2 μm filter
   
   The 1x constituents from (a), (b) and (c) are mixed sequentially and volume is made up to 100 ml with MQ water autoclaved at 121 °C for 20 min.
   

Acknowledgments

The research was funded by the grants from the Department of Biotechnology, Government of India to SSY with Grant no. BT/PB/Center/03/2011 and BT/IN/INDO-UK/SuBB/21/SSY/2013. The protocol was adapted from the Fatma et al., 2016 where the method was used to screen the E. coli endogenous aldehyde reductase responsible for the conversion of long chain aldehyde to alcohol, and from the Shakeel et al., 2018 where the method was used to screen the thermostable ADO.

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

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