Team:UPF CRG Barcelona/Results

• Overexpression
• Biosensor
• Integration

Background
and design

Overexpression

Biosensor

Integration

Simulation

Results

RESULTS

Overview

Overexpression

Biosensor

Integration

Conclusion

Overview

• When fadL and fadD are overexpressed LCFA uptake increases resulting in a higher growth if LCFA are the only carbon source available.
• pFadBA-luxR-pLux lactone inducible LCFA biosensor can amplify the signal of pfadBA promoter-controlled gene expression.
• pFadBA-cI-pRM LCFA biosensor reduces a lot the baseline expression of genes directly regulated by the pfadBA promoter.
• pAR promoter significantly improves pfadBA function as an off/on switch dependent on the presence of LCFA.
• We used MAGE recombineering to generate a strain missing fadR, in a effort to integrate intake capabilities in to the genome.
• We applied the PfadBA based biosensor to improve screening of recombineered fadR colonies.

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 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 shows OD600nm over time for induced (top10 strain) and not induced (Zn1 strain) fadD in 0,4mM PA concentration. Figure B shows OD600nm over time for induced (top10 strain) and non-induced (Zn1 strain) fadD in 2mM PA concentration. Figure C shows OD600nm over time for induced (top10 strain) and non-induced (Zn1 strain) fadD in 0,4mM OA concentration. Figure D shows OD600nm over time for 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 enriched media (LB) was studied. Expression for induced FadD cell line was tuned with ATc. Tuneable FadD overexpression levels entailed a big metabolic burden (Figure 2). Our results show that when ATc induction increases, growth rate diminishes. This way, our results suggest that even when PA is also available in the media, our system will prefer other carbon sources. However, when LCFA is the only available carbon source, the overexpression of fad genes gives a metabolic advantage reflected in enhanced growth (Figure 1).

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 (Figure 3A and 3C) and 2mM PA or OA (Figure 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) (Figure 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 (Figure 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 (Figure 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

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

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.

Cell	Parts Registry	Construct	Expression	Cellular type
pfadBA Reporter cell	BBa_K2581006	t14_pfadBA_34_RFP	PA inducible	TOP10
pfadBA-Lux Lactone inducible reporter cell	BBa_K2581007	t14_pFadBA_34_luxR_T_32_RFP	PA and HSL inducible	TOP10
pfadBA-prm Reporter cell expressing CI activator	BBa_K2581017	t14_pfadBA_34_CI_prm_34_RFP	PA inducible	TOP10
pAR Reporter cell	BBa_K2581011	t14_pAR_32_RFP	PA inducible	TOP10

Table 2 | Summary of the parts and experimental conditions used for this section

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).

Figure 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).

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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

We have proved that the pFadBa-34-RFP biosensor can detect changes in PA concentration. However, the fluorescence ratio for induced/non-induced cultures is very low due to the fact that the baseline fluorescence intensity is non-negligible . If we expect to use our device as a LCFA quantification system, it is needed to obtain a significant difference in the output signals for different LCFA concentrations. This can be achieved either by amplifying the signal or by increasing the signal fold change between the induction with different LCFA concentrations. We achieved amplification of the output signal by inducing the pfadBA-luxR-plux-RFP with lactone 1e-7 M. On the other hand, we were able to reduce the basality of the previous LCFA biosensors with our pfadBA-CI-pRM construct. By reducing the baseline fluorescence we observed an increased fold change of the output signals between induction with different PA concentrations. Nevertheless, with this construct the fluorescence signal deemed lower than the other constructs due to the fact that the pRM promoter is weak. We think that we could obtain a LCFA reliable quantification system by coupling the two constructs together. The hypothetical system would work as follows: the first part (pfadBA-CI-pRM-luxR) would increase the fold change concentration of luxR and the signal would be processed by the luxR-HSL inducible plux-RFP reporter system in order to amplify the signal. This system would potentially generate enough fluorescence difference between different PA induction concentrations to make it possible to predict the concentration of PA in the medium.

On the other hand, we observed that the pAR promoter has a sharper response after PA induction compared to the pfadBA promoter. Moreover, our data suggests that it saturates at much lower PA concentrations than the pfadBA promoter and it has less leakage. Taking all this together, we can affirm that our newly characterised promoter would work much better as an off/on switch dependent on LCFA than the pfadBA promoter, therefore we have successfully improved a previous iGEM parts registry part.

It’s also important to take into account that we have used RFP as a reporter gene, which has a long expression and degradation time. Consequently, this means a delay when reporting LCFA uptake and the moment when expression stops. This is due to the fact that RFP remains some hours in the medium even if it is no longer being expressed. We suggest that a faster reporter protein, the superfolder GFP, which has a very short expression and degradation time, would be more accurate when measuring the switch ON/OFF of our system.

Integration Results

In order to explore the genomic integration of uptake capabilities for a stable chassis that could be used as a live biotherapeutic, we tested the MAGE technology and developed a FadR Knock-Out mutant.

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.

Figure 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 (Figure 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:

Figure 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 (Figure 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 (Figure 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 Figure 16). As expected, WT colony had a low basal level of RFP expression and fadRKO colonies had much higher RFP expression (Figure 17)

Figure 15 | EcM2.1 strain cycled fadR KO. Cells were transformed with pFadBA_34_RFP(BBa_K2581006).

Figure 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.

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

Discussion of Integration results

We generated a strain missing the fadR using MAGE technology, thus creating a system of overexpression of all the proteins involved in LCFA beta-oxidation, which can be implemented in a biotherapeutic strain. During our screening of mutant colonies, we used the PfadBA biosensor as a way to improve detection of colonies missing fadR expression. Levels of uptake capabilities between overexpression achieved through synthetic constructs and overexpression by the absence fadR remains to be characterized.

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.