Optimizing a biosensor with standardized reporter genes
Introduction
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.
Measurement procedures
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 fluorescent protein RFP and sfGFP could be seen under blue light.
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.
Biosensor |
fatty acid |
Final 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 |
Characterization
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 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).
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).
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.
- 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)
- https://2018.igem.org/Team:UPF_CRG_Barcelona/Improve
- 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