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Revision as of 23:08, 17 October 2018
RESULTS
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 |
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.
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 (Fig. 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 (Fig. 1).
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.
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).
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).
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 |
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).
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).
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.
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.
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.
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
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.
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:
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)
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.