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 increases growth when either PA or OA are used as the only carbon source.

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

In order to characterize the functionality of the LCFA biosensors, fluorescent proteins were coupled to them. Top10 (DH5-alpha) E. coli expressing the genetic constructs were grown for 16-20 hours in LB media with different concentrations of palmitic acid (PA). Fluorescence intensity and OD600 were analysed at steady state.

Characterization of the Fatty acid acyl-CoA inducible promoter

To evaluate the function of the pfadBA promoter (BBa_K817002) we designed a reporter system with RFP (BBa_K2581006).

Fig. 6 | E. coli bacteria (DH5-alpha) expressing the pFad34+RFP construct were induced with different concentrations of PA in LB media. Fluorescence was analyzed once it had reached the steady state (13-15h).

Our results show that the baseline fluorescence intensity of the pfadBA34RFP construct is really high. Despite this, an increase of fluorescence intensity was observed for increasing PA induction.

Fluorescence saturation could not be observed in the transfer function due to the fact that medium with higher concentrations than 1 mM PA generated a lot of noise which made impossible to analyse fluorescence at such high concentrations.

Characterization of the Inducible LuxR-pLux engineered device

In order to modulate the expression of the genes under control of the pfadBA promoter for different LCFA concentrations, we designed a pfadBA reporter system inducible by lux-homoserine lactone (HSL).

Fig. 7 | bacteria (DH5-alpha) expressing the pFfadBA_Lux34_plux_32_RFP construct. They were induced with different concentrations of HSL 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 (11-13h). Fluorescence intensity values were normalized by the OD.

Our data indicates that the activation threshold of the lactone inducible pfadBA construct is approximately at 5x10^-9 M of HSL. It saturates at 1x10^-7 M of lactone. Significant fluorescence difference between different PA concentrations is better observed at lactone concentration ranges close to fluorescence saturation.

Characterization of the cI mediated activity

In order to reduce the baseline expression of genes under direct control of the pfadBA promoter we designed two constructs that together would act as a genetic high pass filter. Due to time constraints, we were only able to build the first construct of the designed system (See more). In order to analyse its behaviour we coupled it to a RFP reporter protein.

Fig. 8 | E. coli bacteria (DH5-alpha) expressing the pFadBA+34+CI+ pRM+34+RFP construct were induced with different concentrations of PA in LB media. Fluorescence was analysed once it had reached the steady state(12-14h). Fluorescence was normalised by the OD.

Comparative study between the Inducible LuxR-pLux, the pFadBA and the cI biosensors

Our results indicate that the baseline expression of the pfadBA-CI-prm construct is relatively low. A linear increase of fluorescence intensity is observed for increasing concentrations of PA.

Fig. 9 | Comparison of fluorescence intensity between different pFadBA constructs. Bacteria were grown in LB media with different concentrations of PA (0, 0.4 and 1 mM). Bacteria expressing the pfadBA-plux construct were induced with lactone 1e-7 M. Fluorescence was analysed at steady state. Fluorescence intensity was normalized by the OD.

Fig. 10 | Comparison of fluorescence intensity fold change (induced/non-induced) differences between different pfadBA constructs. pfadBA-plux construct was additionally induced with lactone 1x10^-7.

pfadBA-luxR-plux-RFP construct presented the highest fluorescence intensity in all medium conditions and it showed similar fluorescence fold changes after induction compared to pfadBA alone. On the other hand, pfadBA-CI-pRM-RFP showed little fluorescence compared to the other constructs. Nevertheless, it showed a significant fold change increase compared to the other two constructs.

Characterization of the Improved fatty acid acyl-CoA biosensor

We characterised the improved pFadBA promoter, pAR (BBa_K2581012) coupling it to a RFP reporter gene.

Fig. 11 | E.coli bacteria (DH5-alpha) expressing the constructs were induced with different concentrations of PA in LB media. Fluorescence was analyzed once it had reached the steady state (13-15h). Fluorescence intensity values were normalised by the OD.

Fig. 12 | Comparison of fluorescence intensity fold change (induced/non induced) differences between pFadBA and pAR inducible constructs.

After induction of the pAR promoter with different PA concentrations our results show a significant fluorescence fold change increase compared to the pfadBA promoter. Moreover, our data indicates that the baseline fluorescence of the pAR construct is much lower than that of pfadBA.

Discussion of 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.

Fig. 13 | 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 (Fig. 13). This corresponds to ~1% of colonies incorporating mutations in the lacZ allele in each cycle. Although our ARF was lower than reported [1], we considered it sufficient to screen for fadR mutants.

MAGE fadR KO screening:

Fig. 14 | 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 14. 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 (Fig. 15). 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 (Fig. 15) 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. 16) expressing our BBa_K2581006 construct and a fadR negative colony( num. 10 fig. 16). As expected, WT colony had a low basal level of RFP expression and fadRKO colonies had much higher RFP expression (fig. 17)

Fig. 15 | EcM2.1 strain cycled fadR KO. Cells were transformed with pfadBA-34-RFP(BBa_K2581006).

Fig. 16 | 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.

Fig. 17 | Biosensor activation comparison between a FadR KO colony expressing RFP and a wt EcM 2.1 biosensor transformed colony.

References:

[1] Gallagher R, Li Z, Lewis A, Isaacs F. Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA. Nat Protoc. 2014; 9(10): 2301-2316. Available from: doi: 10.1038/nprot.2014.082.