Composite

Part:BBa_K2924019

Designed by: Mirko Kraus   Group: iGEM19_Duesseldorf   (2019-10-14)


Promoter AR with the reporter gene sfGFP

Long-chain fatty acid sensitive promoter PAR expressing sfGFP. This part is an improvement of BBa_K2581012.

Usage and Biology

This year the iGEM team from Düsseldorf worked on the improvement of an existing part (BBa_K2581012) to create a more efficient version. The original part is a fatty acid acyl-CoA biosensor with red fluorescent protein (RFP) (BBa_E1010) as a reporter gene. This part uses the synthetic PAR from a previous publication1 and so the biosensor is sensitive for long chain fatty acids (LCFA). The previous team performed measurements with this part with different concentrations of palmitic acid (C16:0). These concentrations were 0.4 mM and 1 mM and a 0 mM control2.

As an improvement for this part, we chose to add a stronger, more efficient reporter gene. On the one hand, the superfolder Green fluorescent protein sfGFP (BBa_I746916) from Aequorea victoria as a reporter gene was used, because sfGFP is, like RFP, a common fluorescent protein. In contrast to RFP, sfGFP has some improved folding characteristics for faster signal output.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 291


Characterization

Fig.1: Promoter AR with reporter gene RFP and sfGFP in a pBb backbone.3 The backbone has a chloramphenicol resistance and a medium copy ori pBBR1. EcoRI and XbaI were used as restriction enzymes.


The constructs were cloned into a variant of the pBbB6c3 medium copy number backbone which lacked the lac-promoter, with the restriction sites EcoRI and XhoI. This backbone has the antibiotic resistance chloramphenicol and the origin of replication (ori) pBBR1. The Escherichia coli strain Top10F was transformed with the different biosensor plasmid variants, as seen in Fig. 1.

The transformation of the organism was validated by colony PCR. In some cases, successful transformation could quickly be detected by the different colors of the colonies on the plate and as a pellet.
The fluorescent protein RFP and sfGFP could be seen under blue light.

Fig. 2: Agar plate of Escherichia coli Top10F with the construct pBbB6c + PARsfGFP under blue light. The sfGFP is excited and emits a light yellow/green color.

The positive clones were used for experiments and were grown in LB-medium over night. The fatty acid stocks for the experiments were prepared. All of the fatty acids were dissolved in ethanol with the exception of butyric acid, which was dissolved in water. The stock solutions of the fatty acids are shown in Table 1.
Tab.1: Stock solutions for different fatty acids

Fatty acid Chain length MW [g/mol] Solvent Solubility Stock concentration
Butyric acid C4:0 88.11 Water 60 g/L 200 mM
Capric acid C10:0 172.26 Ethanol 30 g/L 100 mM
Lauric acid C12:0 200.32 Ethanol 20 g/L 100 mM
Myristic acid C14:0 228.37 Ethanol 15 g/L 75 mM
Palmitic acid C16:0 256.42 Ethanol 20 g/L 75 mM
Stearic acid C18:0 284.48 Ethanol 20 g/L 50 mM
Oleic acid C18:1 282.46 Ethanol 100 g/L 200 mM

The absorption of the over night cultures was measured and the cultures were used to inoculate fresh LB medium with an OD600 of 0.05. The cultures were induced with different concentrations of fatty acids. First preliminary experiments were carried out with palmitic acid at final concentrations of 0.4 mM, 1 mM and a control without fatty acids. For better dissolving of the hydrophobic fatty acids in LB-medium, 0.5 % v/v Tergitol-NP-40 was added to the medium. The induced samples were transferred to a 24 well plate and the plate was incubated over night in a 37 °C incubator at 250 rpm. A detailed protocol can be found here. After nearly 16 hours, the samples were taken out of the incubator and distributed on a 96 well plate in 200 µL aliquots. To account for biological heterogeneity and technical errors, three biological replicates were measured in three technical replicates each.

The samples were analysed in a plate reader (ClarioStar). The absorption was measured at the wavelengths 588 nm, 600 nm and 750 nm. The fluorescence was measured at the excitation wavelengths 470 nm and emission 515 nm for sfGFP and the excitation wavelengths 488 nm and emission 588 nm for RFP.

This exact plate reader protocol was used for all further experiments to be able to compare values later on. For each dose-response experiment, seven different fatty acid concentrations were prepared and one negative control was included for every new plate. An empty vector control (EVC) was also supplemented with fatty acids at different concentrations and measured. All experimental details are listed in table 2.
Table 2: List of all part improvement experiments. Concentrations are listed as final concentrations used for induction of the cultures.

Construct Fatty acid Concentrations
PAR:RFP Lauric acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:RFP Myristic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:RFP Palmitic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:RFP Stearic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:sfGFP Lauric acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:sfGFP Myristic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:sfGFP Palmitic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
PAR:sfGFP Stearic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Lauric acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Myristic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Palmitic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM
EVC Stearic acid 0 mM; 0.01 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1 mM

The previous part from UPF_CRG_Barcelona contains RFP as the reporter gene for palmitic acid. We repeated their measurement for palmitic acid; in addition, we included more concentrations. Other fatty acids of different length were added to the measurement as well.

Fig.3: Response of PAR:RFP (red) to different concentrations of various fatty acids compared to an empty vector control (gray). Plot A presents the response for lauric acid, plot B for myristic acid, plot C for palmitic acid and plot D for stearic acid.
Fig.4: Overview (A) and at detailed (B) image from RFP in an Escherichia coli strain after being induced with 1 mM palmitic acid. The RFP can be detected in the whole cytosol.


By adding fatty acids to the culture medium, a dose-dependent increase of fluorescence, consistent with the results shown by UPF_CRG_Barcelona, was expected, while the empty vector control (EVC) should remain at a basal level.
Fig. 3 shows that by addition of fatty acids to the culture medium, the fluorescence increases in a dose-dependent fashion compared to the EVC. The most clear result could be shown with the fatty acids palmitic acid (C16:0) and stearic acid (C18:0). By adding myristic acid (C14:0) to the culture medium, fluorescence of the reporter gene RFP was increased, but the fluorescence of the EVC increased slightly as well. These results indicate that the promoter is more specific to the chain lengths C16:0 and C18:0 than to the chain lengths C12:0 and C14:0. The E. coli cells expressing RFP were also monitored with confocal fluorescence microscopy to visualize the intracellular localization of RFP in the cells. The RFP appears to be located in the cytosol instead of being bound to a membrane (Fig. 4).


Next, the exact same experiment was repeated for our improved part, PAR:sfGFP.

Fig. 5 shows the same dose-response experiments for the new part, PAR:sfGFP. The graphs show a significant increase in fluorescence compared to the original part of Barcelona with RFP. Fluorescence is strongest in the experiment with stearic acid (C18:0), but in the other experiments, a large increase in fluorescence can also be seen. For example, by addition of the fatty acid palmitic acid (C16:0), a two-fold increase of fluorescence from 0.01 mM to 1 mM could be observed. The controls also show better results. The fluorescence of the samples increased only minimally with increasing concentrations of fatty acids, with only a neglectable change in fluorescence compared to sfGFP, indicating some kind of excitation resulting directly from some of the fatty acids in the case of RFP-exciting wavelengths, instead of from the reporter protein. This is not the case for excitation wavelengths relevant for sfGFP, further supporting the improvement of our part. For induction with lauric acid (C12:0), the results appear more erratic at higher concentrations , which is probably due to a decreasing specificity of the promoter AR to the fatty acids - this result is also consistent with the data measured for PAR:RFP.

The E. coli cells expressing sfGFP were also monitored with confocal fluorescence microscopy to visualize the localization of sfGFP in the cells. As for RFP, sfGFP is also not bound to the membrane, but is localized in the cytosol (Fig. 6).

Fig.5: Response of PAR:sfGFP (green) to different concentrations of various fatty acids compared to an empty vector control (gray). Plot A shows the response for lauric acid, plot B for myristic acid, plot C for palmitic acid and plot D for stearic acid.
Fig.6: Overview (A) and at detailed (B) image from sfGFP in an Escherichia coli strain after being induced with 1 mM palmitic acid. The sfGFP can be detected in the whole cytosol.























Conclusion

To summarise the experiments, the created biosensors work for the fatty acids myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0) with every reporter gene. When using lauric acid (C12:0), the results become more inaccurate. A dose response can still be observed, but it is not as strong or linear as with the other fatty acids.
Overall, we could show that using sfGFP instead of RFP as a reporter gene significantly improved the results we obtained. We were able to record higher fold changes for PAR:sfGFP; we believe the reason for this is the faster folding characteristics of sfGFP. This is an important consideration, especially when working with fast growing organisms such as E. coli.

A second point we were able to improve was the background fluorescence. For some fatty acid chain lengths, EVC fluorescence appears to increase with increasing inducer concentration in the case of PAR:RFP. This is likely due to some sort of autofluorescence effect from the inducer, but not the genetic construct itself. The effect is significantly reduced at wavelengths relevant for sfGFP measurements, further leading to more specific output signals which significantly improves fatty acid quantification via this biosensor.

References

1: Fuzhong Zhang, James M Carothers, Jay D Keasling. “Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids” Nature Biotechnology volume 30, pages 354–359 (2012)

2: http://2018.igem.org/Team:UPF_CRG_Barcelona/Improve

3: Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. ”BglBrick vectors and datasheets: A synthetic biology platform for gene expression.” J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410

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