Part:BBa_K3306002

K+ responsive KdpF promoter with GFP
G-mutant KdpF-promoter upstream of GFP

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. pKdpF is the promoter for KdpFABC operon and the design based is on the iGEM HKUST-Rice 2015 part BBa_K1682004. We have assembled KdpF promoter upstream of GFP protein to check the expression levels and the activity of the promoter in the presence of different concentrations of potassium present in serum.

Biology of KdpFABC operon

Potassium is one of the vital elements of a living organism. It is required for key cellular activities in prokaryotes as well as in eukaryotes. To survive, bacteria are forced to monitor their environment constantly and to adapt to changing conditions immediately. Therefore, bacteria have established special signal transduction systems to execute adaptive responses to changing environmental conditions. The bacterial species have three channels like Trk/Ktr/HKT, Kup/HAK/KT and Kdp to uptake monovalent potassium ion. Among the three, KdpFABC operon works under an inducible promoter and the only transport system whose expression is regulated and induced by environmental potassium level and when the other transport system like Trk and Kup are nonfunctional.

Fig 1. KdpFABC operon interaction schematics (Source:https://med.nyu.edu/skirball-lab/stokeslab/kdp.html)

Kdp-ATPase is a complex consisting of four subunits KdpFABC operon which encodes proteins responsible for stabilizing the complex and for binding and translocation of potassium by KdpF and KdpA proteins respectively. KdPB and Kdp C are associated with ATP hydrolysis. Kdp-ATPase system even consists of two-component response regulatory KdpDE which comprises of KdpD and KdpE proteins where KdpD is a histidine kinase and KdpE is a response regulator. Potassium concentration, ionic strength and ATP content stimulate KdpD to undergo auto-phosphorylation, with its phosphoryl group transferred to the transcriptional regulator KdpE. The phosphorylated KdpE (KdpE~P) binds to a 23-bp T-rich sequence in the promoter of kdpFAB and activates the expression of the transporter components KdpFABC.

Construction of composite part

The G mutated KdpF promoter was assembled with GFP reporter (BBa_E0040) via RFC10 standard. We got potassium promoter with modified EcoRI sites synthesized from DeNovo Tech India, which was used as template to amplify the promoter fragment.

Figure 1. Construction of KdpF promoter in GFP plasmid. Agarose gel electrophoresis of DNA fragments obtained from PCR amplification using Kdp-F and Kdp-R primers 1. Potassium promoter 2. Colony PCR of BBa_K3306002 recombinant plasmid 3. Colony PCR of BBa_E0040. M: 100 bp DNA ladder.

Our PCR amplified promoter was cloned upstream of GFP derived from Aequeora Victoria [BBa_E0040]to construct composite part. The resultant plasmid was then transformed in E. coli JM109 competent cells. To check the correct insertion of the fragment, few colonies were sub-cultured and used to extracted plasmids. Colony PCR of the isolated recombinant plasmid was performed using KdpF promoter-specific forward and reverse primers gave expected band of 78 bp confirms that potassium promoter is successfully integrated into BBa_E0040 (Figure 1, Lane 2, 3)

Characterization

Qualitative data

To establish whether reporter strains could successfully detect the presence of potassium ions, we did an initial qualitative fluorescence microscopy test with the reporter strain incubated with 0.5mM potassium. Figure 2 shows fluorescence images from this test, where it can be seen that incubation of our reporter strain with potassium leads to increase GFP expression (Figure 2B) compared to basal GFP expression levels in cells grown in absence of potassium.

Fig 2. Fluorescence Microscopy. A. Control - not induced; B. Test induced - induction with potassium; C. Negative control

Quantative data

Fluorescence measurement in potassium standard solutions

To determine the dynamic range in which KdpF promoter works, different concentrations of KCl was made to induce GFP expression, the reporter strain was further analyzed with a dose-dependence study. Transformants with GFP alone (BBa_E0040) and GFP with potassium promoter (K+ BBa_E0040) were grown on LB media. The OD600 of the cells was measured and equalized to 0.1 OD to ensure comparability. The cells were diluted and the relative concentration of GFP was recorded in Tecan Infinite m200 fluorescence spectrophotometer at 600nm. The graph in Figure 3 clearly indicates that at different concentrations of potassium, there is also an increase in the expression of GFP, but it is not linear in nature.

Figure 3. Changes in fluorescence with varying potassium ion concentration.

Fluorescence measurement in serum

From the implementation point of view, we wanted to evaluate the performance of KdpF promoter in presence of blood/serum as a source of potassium ions to simulate the conditions that PEred would be exposed to in the future. This is achieved by growing our transformants in different K+ concentrations which are obtained from diluting serum in LB. Blood serum was collected and potassium ion concentration in serum was analyzed using Microlyte electrolyte analyzer.

Electrolyte	Concentration
K+	4.3 mM
Na+	14.3 mM
Cl-	110 mM

Figure 4. Emission of reporter strain with BBa_K3306002 in presence of serum.

Figure 4. suggests that the potassium promoter starts expressing GFP at different concentrations wherein elevated levels of expression were found at the concentration of 1.7mM. But this was not significantly high compared to remaining potassium concentrations. This clearly indicates that the induction of GFP expression by KdpF promoter remained insensitive to the variation in potassium concentration in serum. We can hypothesize that due to the high osmolarity of serum, the expression of Kdp pathway becomes independent of potassium in the environment. This hypothesis would have to be validated with further experiments.

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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 722