Revision as of 19:04, 13 September 2015

G-mutant P_kdpF with native RBS- potassium responsive promoter

Biology of P_kdpF

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

The 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).

Construct for characterization

Fig 2. K+ sensing construct with reporter.

To make a potassium-sensing device, we obtained the promoter, PkdpF, 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 PkdpF 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.

RFU measurement of 4 mutants

Fig 3&4. Activity of PkdpF in E. coli DH10B in different K+ concentrations Fluorescence/absorbance versus [K+] plot is shown on the left while the GFP synthesis rate versus [K+] plot is on the right. A, T(wild type), C, and G represent A mutant, wild type promoter, C mutant and G mutant respectively. 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 are presented in SEM.

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 PkdpF 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 PkdpF variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K+.

Promoter function measurement in different strain

Fig5. Comparison between the activities of PkdpF[-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. Activity of PkdpF in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K+], the activity of PkdpF in TK2240 strain exceeded that in the DH10B strain. Since TK2240 is defective in its Trk and Kup systems, it can only rely on the Kdp system for K+ uptake, thereby explaining the higher promoter activity observed beyond 0.05 mM [K+].

RPU measurement

Fig6. Relative promoter unit (RPU) of PkdpF[-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 0.85% saline 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 bar are presented in SEM.

In order to assemble a device that can be widely used by iGEM community, we characterized PkdpF 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 fold change in RPU from 0 mM to 0.4 mM [K+]. As expected, PkdpF is turned off at high [K+] due to inhibition of KdpD kinase activity by K+.