Difference between revisions of "Part:BBa K2539500"

Revision as of 12:01, 26 October 2020

PalcA-Regulated GFP Expression Construct

This construct uses the inducible promoter PalcA (BBa_K2092002). PalcA is activated in the presence of both ethanol and a transcription factor, alcR (BBa_K2092001), and was originally isolated from the fungus Aspergillus nidulans (Panozzo et al., 1997). GFP is used as a visible reporter so we can characterize the function of the promoter PalcA.

Construct Design

The PalcA promoter is placed in front of a strong RBS (BBa_B0034), GFP (BBa_E0040), and double terminator (BBa_B0015). This places the expression of GFP under the control of the PalcA promoter.

PCR Check Results

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 2 kb.

References

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.

CPU_CHINA's 2020 Characterization

We constructed a vector in E.coli for Functional Verification. The PalcA promoter is placed in front of a gfp and a T7 terminator. This places the expression gfp under the control of the PalcA promoter.

We used enzyme-labeled instrument for quantitative analysis of gfp expression, to confirm whether the promoter works or not.

Figure 1.GFP Fluorescence Intensity of E. coli cultures with varying amounts of ethanol.

Over 3 hours, the sample with 1% (1000X) ethanol (EtOH) concentration had the biggest increase in fluorescence.

Our results matched the general expected trend (Figure). Addition of 1% (1000X) ethanol (EtOH) concentration seemed to yield the highest fluorescence, and lower amounts of 0.1& and 0.2% ethanol were not significantly different than the controls. Over 0.4% ethanol seemed to kill the bacteria (5 mL of culture), which matches literature thresholds of ethanol tolerance [2]. In our tests, 1% ethanol yielded the greatest amount of GFP but this effect was quite weak . In summary, the ethanol promoter, PalcA, is functional in the presence of a specific concentration of ethanol, but is**quite** inefficient.

Reference

[1] 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.

[2] 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