Difference between revisions of "Part:BBa K2092002"

Latest revision as of 06:46, 11 October 2018

PalcA, improved alcR inducible promoter from A. nidulans

The alcA promoter, PalcA, is originally found in Aspergillus nidulans as a part of the ethanol regulon. PalcA requires its positive transcriptional regulator AlcR to regulate the expression of gene alcA. Gene alcA encodes for alcohol dehydrogenase I (ADHI) which facilitates the interconversion between alcohols and carbonyls.

This part is an improved BioBrick previously characterised by 2011 iGEM DTU-Denmark (BBa_K678001). Sequencing results showed that two Prefix/Suffix restriction sites XbaI and SpeI were absent in the original part. Hence the two missing restriction sites were added using PCR.

Usage and Biology

PalcA is one of the strongest inducible promoters in Aspergillus nidulans commonly used to overexpress proteins [1]. It has been shown that PalcA promoter is also functional in monocotyledonous plant sugar cane [2] and Escherichia coli [3]. Its transcriptional activation is dependent on the binding of its positive transcriptional regulator AlcR with various substrates that employ a hydroxyl group, for example ethanol and threonine. The native PalcA consists of three AlcR binding sites abc. The number and position of the AlcR binding sites on the PalcA are crucial in determining its transcriptional activation strength. It has also been shown that each AlcR target in the PalcA contributes differently to the activation of the downstream protein expression [1].

Characterization and Improving Function (New Data, 2018)

Team: TAS_Taipei 2018

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. Our experiments are results are documented below.

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 [4]. 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). In summary, the ethanol promoter, PalcA, is functional in the presence of alcR and a specific concentration of ethanol, but is very inefficient.

References

[1] Panozzo, C., Capuano, V., Fillinger, S. and 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. Journal of Biological Chemistry, 272(36), 22859–22865.

[2] Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443–454.

[3] Hemmati, H. and Basu, C. (2015). Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein. Biotechnology & Biotechnological Equipment, 29(6), 1043–1052.

[4] 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.

Sequence and Features