Difference between revisions of "Part:BBa K1682009"

Latest revision as of 04:33, 19 September 2015

P_kdpF[-15, T>G] - GFP generator

G-mutant P_kdpF - E0240

Biology of P_kdpF

Fig 1. The Kdp K⁺ uptake system in E. coli and the potassium biosensor design.

Potassium ion uptake in E. coli is regulated by several systems under different conditions. The potassium ion transporters, Trk and Kup are constitutively expressed (Epstein & Kim, 1971) while KdpFABC, another transporter is activated under low [K⁺] conditions (Laimins et al., 1981).

The kdpFABC operon is controlled by the KdpDE two-component system (TCS) which consists of KdpD, a membrane-bound sensor kinase, and KdpE, a cytoplasmic response regulator (Polarek, 1992; Walderhaug, 1992). KdpD is stimulated by both intracellular and extracellular K⁺ (Jung, 2000; Jung, 2001; Roe, 2000; Yan, 2011a; Laermann, 2013). KdpD phosphorylates KdpE upon low potassium concentration (Voelkner, 1993; Puppe, 1996; Jung, 1997a; Jung, 2000). Under an increase in [K⁺], KdpD phosphatase activity will be enhanced, causing a decrease in phospho-KdpE and kdpFABC expression. Phosphorylated KdpE turns on the expression of kdpFABC (Zhang, 2014a; Laermann, 2013).

We adopt the promoter P_kdpF from kdpFABC operon with -28 position of transcription start site relative to start the first gene, kdpF. The -10 and -35 box elements have been mapped which are TACCCT and TTGCGA respectively (Sugiura et al., 1992). The transcription factor, phosphorylated KdpE, binds to the P_kdpF at binding site located from -71 to -55 site with reference to the transcription start site (Sugiura et al., 1992; Narayanan et al., 2012).

Construction

Fig 2. K+ sensing construct with reporter.

To make a potassium-sensing device, we obtained the promoter, P_kdpF, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different potassium level can be detected and characterized.

Removal of EcoRI illegal site

To make P_kdpF compatible with RFC10 standard and, as so, readily accessible to iGEM community, we designed variants of it with the EcoRI site removed. We mutated the thymine at -15 position to guanine, cytosine and adenine. The wild type promoter and the 3 variants are expected to be different in activity because of the difference in binding energy between the promoter and RNA polymerase (Brewster, 2012). Therefore, we characterized all of them to compare their strengths by relative fluorescence intensity, so as to obtain comprehensive knowledge in the activity and working range of the four promoters. For convenience, we denote them as A mutant, G mutant and C mutant respectively in the following context.

Characterization

RPU measurement

Fig 3. Relative promoter unit (RPU) of P_kdpF[-15,T>G] at different concentration of K⁺. Cells were pre-cultured in K115 medium overnight at 37°C. The cells were washed 3 times in 0.8% NaCl solution and then sub-cultured in medium with specific [K⁺]. Cells were fixed when OD₆₀₀ = 0.4. The measurement was carried out using fluorescence-activated cell sorting. Error bars are presented in SEM.

In order to assemble a device that can be widely used by iGEM community, we characterized P_kdpF by relative promoter unit (RPU) measurement. Additionally, relative fluorescence unit (RFU) measurement has also been done to compare the activities between wild type promoters and 3 variants. At the lowest [K⁺], the strength of the G mutant promoter was found to be approximately 0.5 RPU. RPU of the promoter decreases as [K⁺] increases. At 0.025 mM [K⁺], RPU values was found to be 0.13. There was about 3.8 times change in RPU from 0 mM to 0.4 mM [K⁺]. As expected, P_kdpF is turned off at high [K⁺] due to inhibition of KdpD kinase activity by K⁺.

RFU measurement of 3 mutants

Fig 4. Activity of P_kdpF in E. coli DH10B in different K⁺ concentrations A) Positions of base substitutions to standardize P_kdpF into RFC10 format. B) Single time point transfer curve for P_kdpF variants along a gradient of [K+]. C) Relative GFP synthesis rate calculated from 3 measurement time points. Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD₆₀₀ = 0.4. 2 other measurements were taken every 15 mins afterwards for GFP synthesis rate. Error bar present SEM from 3 biological replicates.

Both C and G mutants expressed higher fluorescence compared to the wild type and the A mutant. As the [K⁺] went up, the activity of P_kdpF decreased. The expression levels of both the C and G mutant promoters were significantly higher than the wild type promoter, while the A mutant was always the lowest. At 0 mM [K⁺], both the C and G mutants expressed fluorescence which was about 1.7 times higher than the A mutant and wild type promoter. At 0.2 mM [K⁺], expression of both C and G mutants were 1.7 times higher than the wild type promoter, and 3 times higher compared to the A mutant. This might be caused by the difference in binding affinity between RNA polymerase and P_kdpF variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K⁺.

Promoter function measurement in different strain

Fig 5. Comparison between the activities of P_kdpF[-15, T>G] in DH10B and TK2240 strain. Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD₆₀₀ = 0.4. Error bars are represented as SEM.

At [K⁺] lower than 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than that in TK2240 strain. The activity of P_kdpF in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K⁺], the activity of P_kdpF in TK2240 strain exceeded that in the DH10B strain. We are uncertain about what causes the discrepancies in the comparisons.