Revision as of 19:07, 1 November 2017

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Beta Oxidation

Aim

The objective of this part of our project was to genetically engineer E. coli to produce PHB from volatile fatty acids (VFAs) found in human fecal waste. VFAs (acetic acid, propionic acid, butyric acid, and lactic acid) serve as precursors for the synthesis of PHB via the beta-oxidation pathway native to bacterial cells (Reyhanitash, 2017).

Manipulation of the beta-oxidation pathway

In order to synthesize PHB from fatty acids found in human fecal waste, we manipulated the fatty acid beta-oxidation pathway within E. coli. We designed a construct that contains the phaJ4 gene from Pseudomonas putida, encoding for enoyl-CoA hydratase that converts the enoyl-CoA into (R)-hydroxybutyrate (Lu, 2003). Our construct also includes the phaC1 gene from Pseudomonas aeruginosa, encoding the PHA synthase, which converts the (R)-hydroxyacyl-CoA into polyhydroxybutyrate (PHB). To our advantage, this pathway not only uses VFAs but can also use undigested long-chain fatty acids in human fecal waste, thus maximizing the substrates available for PHB synthesis.

Genetic construct

Figure 1: Genetic construct for phaC1J4, including the ribosomal binding sites (BBa_B0034) and the phaJ and phaC genes

Our construct has been designed for translation in E. coli, as it contains the common strong RBS BBa_B0034 in front of each gene, and codons have been optimized. We also ensured that our BioBrick was compatible with all iGEM RFC standards by removing illegal restriction sites. Our construct can be placed under the control of an inducible or constitutive promoter, resulting in controlled or continuous production of PHB.

You can find our phaC1J4 operon (BBa_K2260001) on the iGEM Registry here.

Experiments

Results

Subcultures of E. coli BL21(DE3) were grown in 60 mL of various types media for 24 hours. Cultures were then induced with 0.1 mM IPTG to express phaC1 and phaJ4. Cultures were incubated for 24 hours and OD₆₀₀ was measured. Absorbance was adjusted by diluting cultures with LB so they were all between 0.4-0.6, and nutrients and substrates were added to the flasks to allow for PHB production. The composition of each test culture is shown below:

E. coli BL21(DE3) with pET29B(+)-phaJC in Glucose (positive control)	E. coli BL21(DE3) with pET29B(+) in LB Media (negative control)
10 ml of culture in LB+Kanamycin 7 ml 20% Glucose 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG 20 ml dH₂O	10 ml of culture in LB+Kanamycin "Syn poo" fermented supernatant 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG
E. coli BL21(DE3) with pET29B(+)-phaJC + Fermented "Syn Poo" Supernatant Containing Glucose and VFAs	E. coli BL21(DE3) with pET29B(+)-phaJC + Pure VFAs
10 ml of culture in LB+Kanamycin 10 ml Syn Poo fermented supernatant 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG 23 ml dH₂O	10 ml of culture in LB+Kanamycin VFAs 410 uL propionic acid 118 uL acetic acid 55 uL butyric acid 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG 30 ml dH₂O

Three replicates of each growth condition were performed. Their OD₆₀₀ readings were recorded:

Condition	OD₆₀₀ of replicate 1	OD₆₀₀ of replicate 2	OD₆₀₀ of replicate 3
E. coli BL21(DE3) with pET29B(+) in LB Media (negative control)	0.571	0.531	0.487
E. coli BL21(DE3) with pET29B(+)-phaJC in Glucose (positive control)	0.190	0.195	0.139
E. coli BL21(DE3) with pET29B(+)-phaJC + Pure VFAs	0.140	0.134	0.146
E. coli BL21(DE3) with pET29B(+)-phaJC + Fermented "Syn Poo" Supernatant Containing Glucose and VFAs	0.135	0.107	0.144

After pelleting the cultures, the cells were resuspended in 1 x PBS. The OD₆₀₀ readings were taken:

Condition	OD₆₀₀ of replicate 1	OD₆₀₀ of replicate 2	OD₆₀₀ of replicate 3
E. coli BL21(DE3) with pET29B(+) in LB Media (negative control)	2.659	2.001	2.899
E. coli BL21(DE3) with pET29B(+)-phaJC in Glucose (positive control)	1.934	1.887	1.919
E. coli BL21(DE3) with pET29B(+)-phaJC + Pure VFAs	0.510	0.571	0.532
E. coli BL21(DE3) with pET29B(+)-phaJC + Fermented "Syn Poo" Supernatant Containing Glucose and VFAs	2.533	2.559	2.349

Future Directions

We would like to maximize the amount of PHB produced with our construct by upregulating the genes fadE and fadD upstream of the beta-oxidation cycle. This may produce more substrates for phaJ and phaC to convert into PHB.

We would also like to test out the effects of using a constitutive promoter and compare it to the use of our IPTG-inducible T7 promoter.

Works Cited

Lu, X., Zhang, J., Wu, Q. & Chen, G.Q. (2003) Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) via manipulating the fatty acid beta-oxidation pathway in E. coli. FEMS Microbiol Lett. 221: 97-101

Reyhanitash, E., Kersten, S. & Schuur, B. (2017) Recovery of volatile fatty acids from fermented wastewater by adsorption. ACS sustainable Chemical Engineering. 5: 9176-9184

Rose, C., Parker, A., Jefferson, B. & Cartmell, E. (2015). The characterization of feces and urine: a review of the literature to informed advanced treatment technology. Critical Reviews in Environmental Science Technology. 45: 1827-1879

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