Part:BBa_K2581009
FadD gene with ATC inducible promoter with weak RBS.
The UPF_CRG_Barcelona iGEM team 2018 has created this part which consist of the expression of the own E.Coli acyl-CoA Synthetase gene, fadD. This gene has an essential role in the fatty acid transportation inside the cell, as well as fatty acid activation. This biobrick consists in the assembly of a double terminator which allows forward and reverse terminator (BBa_B0014), a tetR repressible promoter (BBa_R0040), a weak RBS (BBa_B0032) and the the fadD gene (acyl-coA synthetase key for the fatty acid transport inside the cell) obtained of the own E.Coli DH5-alpha strain genome (BBa_K2581016 being a twin of BBa_k861010, part not available).
Methods
In order to study the functionality of our system we characterized the constructs both in palmitic acid (PA) and oleic acid (OA). Bacteria was grown for 3 days in M9 minimal medium with the respective fatty acid, to ensure that cells were using solely LCFA as a carbon source . Additionally, the supernatant was also collected and LCFA concentration was determined using cupric-acetate colorimetric (see methods) technique at OD715nm. The same growth assay was performed in LB to infer cell growth in enriched media.
Different levels of fad genes expression were achieved through the use of different strains. Genetic constructs consisting of either FadD or FadL downstream of the tetR repressible promoter were transformed in Top10 (DH5-alpha) and Zn1 E. coli strains. Top 10 constitutively synthesizes tetR, which results in a continuous expression of our construct. Zn1 does not express tetR, thus meaning that our genetic constructs will not be induced in this strain.
Characterization
Our results show that, when fadD is overexpressed, bacterial growth increases both in 0,4 mM PA (Fig 1A) and 2 mM PA (Fig 1B) in relation to control. Moreover, non-induced FadD cells show a lower increase of bacterial growth, compared to the control. On the other hand, when tested with OA medium (Fig 1C and Fig 1D), the difference in growth is higher than in PA medium. As for the LCFA quantificationassay, our results showed that when fadD was overexpressed, OA uptake nearly doubled the uptake of the control (p<0,001) (Fig. 2). However, this increase could not be observed in PA.
Figure 1 | Growth assay comparing induced and non-induced fadD in minimal medium enriched with different concentrations of Oleic Acid (OA) and Palmitic Acid (PA) . Figure A, B, C and D shows OD600nm over time for induced (top10 strain) and not induced (Zn1 strain) fadD in 0,4mM PA, 2mM PA, 0,4mM OA, 2mM OA concentration respectively. Error bars represent the Standard Deviation of the Mean (SEM).
Figure 3 | LCFA uptake in FadD overexpression. Plot showing LCFA uptake when comparing induced (Top10 strain) and not induced (Zn1 strain) fadD gene. Constitutive reporter cell line (top10) was used as a control. Uptake was measured considering the OD715nm difference between the medium at certain concentration of LCFA and the medium with grown bacteria. A concentration of 0,4mM of both PA and OA was used. Cupric-acetate technique was used to quantify LCFA in the medium. LCFA uptake was normalized by the growth of the bacteria, measured at OD600. Error bars represent the Standard Deviation of the Mean (SEM). Statistical significance of the mean was calculated using a paired t-test. P value <0,05, which indicates a statistically significant difference among relevant groups, is designated with an asterisk.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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