Team:UPF CRG Barcelona/Results
RESULTS
Overview
- When fadL and fadD are overexpressed LCFA uptake increases resulting in a higher growth if LCFA are the only carbon source available.
- In order to characterize the functionality of our system we grew them in enriched medium considering different concentrations of PA. Bacteria was grown for 16-20 hours in enriched medium (LB) with different inductor concentrations.
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Overexpression results
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. This induction can be modulated by adding anhydrotetracycline (ATc) in the medium. We created a cell library expressing the fadD and fadL proteins:
| Cell | Parts Registry | Construct | Expression | Cellular type |
|---|---|---|---|---|
| Constitutive FadD | BBa_K2581009 | T14_ptet_32_FadD | constitutive | TOP10 |
| Inducible FadD | BBa_K2581009 | T14_ptet_32_FadD | tuneable | ZN1 |
| Constitutive FadL | BBa_K2581010 | T14_ptet_32_FadL | constitutive | TOP10 |
| Inducible FadD | BBa_K2581010 | T14_ptet_32_FadL | tuneable | ZN1 |
| Constitutive Reporter | BBa_K2581010 | T14_ptet_32_RFP | constitutive | TOP10 |
Table 1 | Summary of the parts and experimental conditions used for this section
FadD overexpression increases growth when either PA or OA are the only carbon source.
We analyzed the behavior of the constitutive and inducible FadD cell lines with M9 minimal media supplemented with either PA or OA. Constitutive reporter cell line was used as a control. OD measurements were performed at OD 600 nm as an indicative for cell growth for 72 hours.
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.
Figure 2 | Growth assay comparing induced and not induced fadD in enriched medium. Figure A show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadD in 0,4mM PA concentration. Figure B show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadD in 2mM PA concentration. Figure C show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadD in 0,4mM OA concentration. Figure D show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadD in 2mM OA concentration. Error bars represent the Standard Deviation of the Mean (SEM).
Moreover, growth in an enriched media (LB) was studied. Expression of induced FadD cell line was tuned with ATc. Tuneable FadD overexpression levels entailed a big metabolic burden (figure X). Our results show that when ATc induction increases, growth rate diminishes. In this way, our results suggested that even when PA is available in the media, our system will prefer other carbon sources. However, when LCFA are the only ones being present, the overexpression of fad genes gives an advantage reflected in enhanced growth (figure X).
Figure X | Growth assay of induced and not induced fadD in enriched medium. Bacteria was grown for 16 hours in enriched medium with a concentration of 0,4mM Palmitic Acid. All bacteria was top10 (DH5-alpha strain). To induce fadD expression a concentration of 60ng/ml of ATc was added. Error bars represent the Standard Deviation of the Mean (SEM).
FadL overexpression reduces metabolic burden when both PA and OA concentration increases.
We analyzed the behavior of the constitutive and inducible FadL cell lines with M9 minimal media supplemented with either PA or OA. Constitutive reporter cell line was used as a control. OD measurements were performed at OD 600nm as an indicative cell growth for 72 hours.
Here we demonstrate that overexpression of fadL entails a metabolic burden. This is shown in the decrease of the OD600 when induced bacteria is grown in both 0,4mM PA or OA (figure X) and 2mM PA or OA (figure X). When comparing this metabolic burden between the two concentrations it is clear that in the 2mM concentration induced bacteria grow more than in 0,4mM. Thus, this results suggest that FadL entails a metabolic burden even in minimum media but is reduced when LCFA concentration increases.
Figure X. Growth assay of induced and not induced fadL in minimal medium enriched with different concentrations of Oleic Acid (OA) and Palmitic Acid (PA). Figure A show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadL in 0,4mM PA concentration. Figure B show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadL in 2mM PA concentration. Figure C show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadL in 0,4mM OA concentration. Figure D show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadL in 2mM OA concentration. Error bars represent the Standard Deviation of the Mean (SEM).
FadD overexpression increases OA uptake
We analyzed the behavior of the constitutive and inducible FadD cell lines with M9 minimal media supplemented with either PA or OA. Constitutive reporter cell line was used as a control. Supernatant was collected from the medium after 72 hours growing. Cupric-acetate colorimetric technique was performed quantify LCFA concentration (OD715nm).
Our results showed that, when fadD was overexpressed, OA uptake nearly doubled the uptake of non induced bacteria and control (p < 0.001) (Fig. X). However, this increase could not be observed in PA. A possible explanation would be the infeasibility to obtain a proper standard curve for PA using cupric-acetate technique when compared to oleic acid (see methods).
Figure X | 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.
Overexpression of fadL increases OA uptake.
Here to study the LCFA uptake the same experimental procedure was followed as described in FadD. Our results show that, when fadL was induced OA uptake was more than 2-fold higher in relation to non-induced and control bacteria (Fig. X). This increased uptake was only statistically significant in OA when compared to control (p < 0.0001) and not induced bacteria (p < 0.0001). PA media didn’t show significant results (figure X).
Figure X. LCFA uptake in FadL overexpression. Plot showing LCFA uptake when comparing induced (Top10 strain) and not induced (Zn1 strain) fadL 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.
Discussion of overexpression results
Our results demonstrate that overexpression of fadL and fadD in enriched media entails a metabolic burden for the cell. However, when these genes are overexpressed in a minimum media, with LCFA as the unique carbon sources, the metabolic burden is reduced. Furthermore, bacterial growth is even higher between induced and not induced cells. We have demonstrated that induction of fadL and fadD results in a LCFA uptake increase, being statistically significant in OA.
Considering our results, it can be deduced that when fadL and fadD are overexpressed LCFA uptake increases resulting in a higher growth. In this way, we can conclude that LCFA influx is increased in our system, resulting in higher Acyl-CoA concentration inside the cell. Consequently, we have hypothesized that this results in an enhanced expression of the rest of fad genes and therefore in an increase of the LCFA degradation rate. This increase in the LCFA degradation, leads to more metabolic fuel to be used for growth (only when LCFA is the only carbon source available). Therefore, overexpression of fadL and fadD gives the cell a positive advantage that results in enhanced growing when LCFA is the only carbon source.
Biosensor Results
| Cell | Parts Registry | Construct | Expression | Cellular type |
|---|---|---|---|---|
| pfadBA Reporter cell | ####### | pfadBA_34_RFP | PA inducible | TOP10 |
| pfadBA-Lux cell | ####### | pfadBA_34_Lux_34_plux_32_RFP | PA and HSL inducible | TOP10 |
| ##### | ####### | pfadBA_34_CI_prm_34_RFP | PA inducible | TOP10 |
| pAR Reporter cell | ####### | pAR_32_RFPL | PA inducible | TOP10 |
Table X | FALTA PEU DE FOTO
Characterization of the Fatty acid acyl-CoA inducible promoter
To evaluate the function of the pfadBA promoter, we designed a reporter system with RFP. TOP-10 E. coli strain expressing the construct were induced with different concentrations of PA in LB medium. Fluorescence was analyzed once it had reached the steady state.
Our data indicates that the activation threshold of the pfadBA promoter is at 0.2 mM of PA. The baseline fluorescence is really high and we only observed a fold change of 1.5 between the fluorescence of the non induced bacteria and of the maximum induced one (PA 1mM).
We could not see the saturation in the transfer function due to the fact that medium with PA concentrations higher than 1 mM created a lot of noise which made impossible to analyse fluorescence at this high concentrations.
Characterization of the Inducible LuxR-pLux engineered device
In order to modulate the expression of the genes expressed under the pfadBA promoter, we designed a pfadBA reporter system inducible with both LCFA and lactone. TOP 10 bacteria expressing the construct were induced with different concentrations of lactone and 3 different concentrations of PA (0, 0.4 and 1 mM) in LB medium. Fluorescence was analyzed once it had reached the steady state.
Our data indicates that the activation threshold of this lactone inducible pfafba construct is approximately at 5x10-9 M of lactone. It saturates at 1x10-7 M of lactone. Significant fluorescence difference between different PA concentrations is only observed at saturated lactone ranges.
Characterization of the cI mediated activity
Comparative study between the Inducible LuxR-pLux, the pFadBA and the cI biosensors
Characterization of the Improved fatty acid acyl-CoA biosensor
Integration Results
Conclusions