Our new composite part BBa_K2539550 improves the function of an existing part, BBa_K2092002 (PalcA Promoter). PalcA is an ethanol-induced promoter that also requires the binding of a transcription factor, alcR, in order to be activated. BBa_K2539550 improves the PalcA promoter by adding a constitutively expressed alcR in the same construct, and using a visible reporter GFP. We used this construct to characterize the function of the original part, PalcA promoter alone. Our experiments and results are documented below.
BBa_K2539550:
AlcR and PalcA-Regulated GFP Expression Construct
This construct combines two of our submitted composite parts: a constitutively expressed alcR construct (BBa_K2539300) and a PalcA-regulated GFP expression construct (BBa_K2539500).
PalcA is an inducible promoter (BBa_K2092002), which can be activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001). It was originally isolated from the fungus Aspergillus nidulans (Panozzo et al., 1997). AlcR is a gene found in Aspergillus nidulans which encodes for a regulatory protein (Panozzo et al., 1997). AlcR can bind to the alcA and aldA promoters (PalcA and PaldA, respectively). In the presence both AlcR and ethanol or threonine, genes downstream of these promoters are expressed (Felenbok et al., 1988).
GFP is used as a visible reporter so we can characterize the function of the promoter PalcA.
Construct Design
This construct (BBa_K2539550) constitutively expresses alcR, a regulatory protein necessary for the activation of PalcA. We use a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, the alcR sequence (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription. Behind this, the PalcA promoter (BBa_K2092002) is placed in front of a strong RBS (BBa_B0034), GFP (BBa_E0040), and a double terminator (BBa_B0015).
Together, this construct expresses alcR, which can activate the PalcA promoter and express GFP if ethanol is present.
PCR Gel Check
The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
PCR check for BBa_K2539550 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 4.8 kb.
Characterization
We tested E. coli carrying Pconst_alcR+PalcA_GFP (BBa_K2539550) using different concentrations of ethanol. Bacterial cultures with different plasmids were grown overnight. In addition to Pconst_alcR+PalcA_GFP (BBa_K2539550), cultures of Pconst_alcR (BBa_K2539300), PalcA_GFP (BBa_K2539500), and a positive control expressing GFP were also prepared. An initial fluorescence reading was taken using a 96 well plate reader.
When alcR is constitutively expressed and ethanol is present, we hypothesized that if PalcA were functional, then we should see the Pconst_alcR+PalcA_GFP (BBa_K2539550) cultures glow green. To characterize the promoter, we added varying amounts of ethanol into cultures of Pconst_alcR+PalcA_GFP, sealed the tubes to prevent evaporation, and left them in a shaking incubator for 24 hours. Fluorescence absorbance measurements were taken before the addition of ethanol and after 24 hours with ethanol.
Relative fluorescence of E. coli cultures carrying Pconst_alcR+PalcA_GFP (BBa_K2539550) with varying amounts of ethanol. Over 24 hours, the sample with 0.3 g ethanol (EtOH) had the biggest increase in fluorescence. Pconst refers to the constitutive promoter we used (BBa_K880005). Lysogeny broth (LB), Pconst_alcR, and PalcA_GFP were used as negative controls for this experiment.
Our results matched the general expected trend. Addition of 0.3 g of ethanol to Pconst_alcR+PalcA_GFP (BBa_K2539550) seemed to yield the highest fluorescence, and lower amounts of 0.01 g and 0.1 g ethanol were not significantly different than the controls (LB only, Pconst_alcR, and PalcA_GFP). Over 0.5 g of ethanol seemed to kill the bacteria (3 mL of culture), which matches literature thresholds of ethanol tolerance (Chong et al, 2013). In our tests, 0.3 g of ethanol yielded the greatest amount of GFP; however, this effect was extremely weak and the measured fluorescence was about 500 times lower than the positive control (constitutively expressed GFP; not shown here). In summary, the ethanol promoter, PalcA, is functional in the presence of alcR and a specific concentration of ethanol, but is very inefficient.
References
Chong H, Huang L, Yeow J, Wang I, Zhang H, Song H, Jiang R. (2013). Improving Ethanol Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP). PLoS ONE, 8(2), e57628.
Felenbok B, Sequeval D, Mathieu M, Sibley S, Gwynne DI, Davies RW. (1988). The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR. Gene, 73(2), 385–396.
Panozzo C, Capuano V, Fillinger S, Felenbok B. (1997). The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. J Biol Chem. 5;272(36):22859-65.
