Difference between revisions of "Team:UPF CRG Barcelona/Results"

Table 1 | Summary of the parts and experimental conditions used for this section

Figure 2 | Growth assay comparing induced and not induced fadD in enriched medium (LB). Top 10 cells (DH5-alpha) were grown for 16 hours in enriched medium with a PA concentration of 0,4mM. In order 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 are grown in both 0,4mM PA or OA (Fig. 3A and 3C) and 2mM PA or OA (Fig. 3B and 3D). 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, as LCFA can be used as an energetic source.

Figure 3. Growth assay comparing induced and not induced fadL in minimal medium enriched with different concentrations of Oleic Acid (OA) and Palmitic Acid (PA). Figure A shows OD600nm over time for induced (top10 strain) and not induced (Zn1 strain) fadL in 0,4mM PA concentration. Figure B shows OD600nm over time for induced (top10 strain) and not induced (Zn1 strain) fadL in 2mM PA concentration. Figure C shows OD600nm over time for induced (top10 strain) and not induced (Zn1 strain) fadL in 0,4mM OA concentration. Figure D shows OD600nm over time for 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. 4). 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 4 | 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. 5). 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 (Fig. 5).

Figure 5. 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

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

MAGE cycling efficiency:

Estimation of allele replacement frequency (ARF) after each cycle was performed by X-gal blue-white screening. Oligos encoding for two stop codons in the lacZ gene were electroporated in each cycle as a positive control. Recombineered colonies are deficient in the Beta-galactosidase enzyme and therefore appear white when plated on X-gal IPTG containing plates.

Figures 1 & 2 | ARF screening on LB+X-gal+IPTG plates after five mage cycles in EcM2.1 strain. Cultures were plated at 10e-5 (left) and 10e-6 (right).

Cultures where plated after five cycles. ARF was 4.8%, estimated by colony counting. This corresponds to ~1% of colonies incorporating mutations in the lacZ allele in each cycle. Altrouth our ARF was lower than reported (Gallagher 2014), we considered it sufficient to screen for fadR mutants.

MAGE fadR KO screening:

Figure Z: Agarose gel electrophoresis of FadR KO screening through allele specific PCR, after five MAGE cycles. Numbers correspond to screened colonies. 36 colonies were analyzed, only 12 of them are shown in the picture. Top wells correspond to colonies screened with wt specific primers. Bottom line shows 12 same colonies amplified with specific primers for the FadR mutations. Colony 11 shows no amplification for the wt allele (361bp), but has the expected amplicon for FadR knock-outs; 362bp.

After performing five cycles in EcM2.1 MAGE strain using oligos coding for two stop codons in the beginning of FadR ORF, We performed allele specific PCR screening. One primer pair was designed to specifically recognize the WT sequence, while another primer pair was designed to anneal to the mutant sequence. Performing a colony PCR with both primer pairs should lead to a single amplicon, depending on the genotype of the analysed colony, as seen in figure Z. All colonies are amplified with the WT allele except colony number 11, in which amplification with the mutant primer pair appears. This colony was selected as the bona fide mutant of the FadR repressor. A total of 36 colonies were analyzed (only 12 of them shown, all other 24 where WT specific). From this we can make a rough estimation of the efficiency of FadR KO oligo incorporation, which is 2,77%.

fadR optimized screening method:

One of our final composite and biosensor parts; BBa_K2581006 is regulated by FadR repressor, which binds to pfadBA in the absence of LCFA. We hypothesized that FadR KO cells transformed with this construct should have basal expression of RFP. Different red intensities can be seen in EcM2.1 transformed colonies. We speculate that this might be due to the fact that EcM2.1 are not optimal for protein expression since they lack MutS machinery and thus have impaired growth. Red colonies were picked and analysed by allele specific PCR. Of 16 analysed colonies, 4 included the mutation (Figure T) which corresponds to ~25%. Although this screening method does not bypass the need for allele specific PCR screening, it significantly increased the number of positive colonies in our screening, which would allow for a reduced number of cycles when obtaining a FadR KO. Finally, we cultured and re-plated a FadR KO colony (num 9 figure. T) expressing our BBa_K2581006 construct and a fadR negative colony( num. 10 fig. T). As expected, WT colony had a low basal level of RFP expression and fadRKO colonies had much higher RFP expression (fig. X)

Figure x | EcM2.1 strain cycled fadR KO. Cells were transformed with pfadBA-34-RFP(BBa_K2581006).

Figure T | Allele specific PCR screening with red colonies that were transformed with BBa_K2581006. Top gel lanes correspond to colony screening with WT specific FadR primers. 4 of the 16 tested colonies show amplification for the wt allele. The bottom lanes of the gel show colony screening using FadR KO primers. Four colonies (8,9,12,13) tested positive for FadR KO induced by MAGE.

Figure X | Biosensor activation comparison between a FadR KO colony expressing RFP and a wt EcM 2.1 biosensor transformed colony.