1. Inorganic phosphate ions enter the periplasm through the PhoE phosphorin.
2. Once in the Periplasm phosphate can enter the cytoplasm in one of two ways – non-specific (through Pit) or specific transport. For the latter, the substrate binding protein (PstS) of the Phosphate-specific ABC transporter (Pst) binds the phosphate ion.
3. It then brings the ion in close proximity to the transporter’s transmembrane domains – the permeases PstC and PstA. These two proteins provide the translocation pathway and the phosphate’s uptake is energized by a homodimer of PstB (an ATP-ase protein that hydrolyses ATP).
4. Phosphate ions can then be used for cellular metabolism which includes ATP formation from ADP and phosphate by the F-ATPase protein. ATP is the substrate for the Polyphosphate kinase enzyme (PPK1) which reversibly transfers the γ-phosphate of ATP onto a chain of polyphosphate (PolyP). Amongst many roles polyphosphate acts as energy and phosphate storage molecule.
5. Polyphosphate can be used to generate ATP from ADP and in the case of phosphate shortage, the cell releases orthophosphate residues using the Exopolyphosphatase (PPX) enzyme which cleaves a residue at a time from PolyP’s

Phosphate assay

The aim of the phosphate assay was to create a reliable method of determining the concentration of phosphate both inside of the cell and within the cell growth media. As a starting point, we used an ABCAM colorimetric assay kit (ab65622) containing a phosphate reagent dye composed of malachite green and ammonium molybdate. A chromogenic complex is formed between the dye and the phosphate ion (PO43-), producing an intense absorption at around 650nm. The kit also contains a 10mM phosphate standard solution, which can be diluted to create a series of standards (protocol can be found on our wiki under phosphate assay protocols).

Our first phosphate assay using the ABCAM kit was run using the wild-type BW25113 Escherichia coli as well as the ∆ppk and ∆ppx knockout strains from the Keio collection1. This assay involved experimenting with the quantity of cells under test and the volume of sample used in each well. From our results we found that the most reliable results were gained using 108 cells. We also found that phosphate within the cells can be detected and varied between different strains (Figure 1).

Figure 1: A graph to show results from phosphate assay using ABCAM kit comparing two strains from the KeiO collection ∆ppk and ∆ppx and the parent strain BW25113. Error bars show the standard error of the mean from concentrations calculated from technical repeats. Results were measured using a BMG FLUOstar Omega plate reader at OD650 and concentrations calculated from known phosphate concentration standards.

From these results, we proceeded to run a second assay using two strains of ∆ppx (47A7,48A7) and two strains of ∆ppk (5D10, 6D10), along with the parent strain BW25113. Following sonication of the cell samples, we added 50uL of 1 moldm-3 formic acid to hydrolyse the chains of polyphosphate to orthophosphate. This step was added because we suspected that the ABCAM kit detected phosphate ions but not polyphosphate as the assay was only developed for the detection of orthophosphate; correspondence with ABCAM stated that the assay had not been used before to detect polyphosphate. Volumes of 10uL, 15uL, 20uL and 100uL of sample were added to each well. A BMG FLUOstar Omega plate reader was used to measure the absorbance at 650nm and the phosphate concentration in each well was determined using a series of standards with known phosphate concentration.

However, these results showed that the phosphate concentrations recorded were far lower than before the acid hydrolysis step was incorporated. This is contrary to our hypothesis that the concentration of phosphate would increase as the polyphosphate in the cells was hydrolysed. These results may have been due to the acid addition and resulting decrease in pH affecting the complex formed between the phosphate ions and dye. These results therefore did not confirm our theory that the kit does not detect polyphosphate Therefore in order to effectively test the ability of the kit to detect polyphosphate; we planned to use a glass milk solution as a filter in our samples to separate the polyphosphate from the rest of the lysate.

Preparing the glass milk involved using a fume hood located in the chemistry teaching labs and heating concentrated nitric acid almost to boiling; a 50% water to glass milk slurry was made and stored at room temperature. As well as using glass milk to determine cellular polyphosphate concentration, we planned to use NMR to create a spectrum showing two separate peaks for orthophosphate and polyphosphate which could be used to calculate concentration. We used impure DNAse to digest the DNA and RNA found within our samples. This was because the phosphate backbone in DNA produces a peak in 31P NMR which overlaps with the orthophosphate peak, making the spectrum difficult to analyse. However, from the results shown below in figure 2, the acid hydrolysed the DNA resulting in no overlapping peaks.

Figure 2: An NMR spectrum showing analysis of BW25113 cell lysate with and without DNAse. No polyphosphate peak can be observed, however a clear 31P peak can be seen, indicating the presence of orthophosphate. The results were gained following 18000 runs on a Bruker NMR spectrometer.

Our next assay focussed on altering the polyphosphate assay by finding a way to hydrolyse the polyphosphate chains within our modified cells without affecting the complex formed between the malachite green, ammonium molybdate and inorganic phosphate. Biological repeats of a BW25113 colony was used, each set of three repeats contained a different volume of 1M formic acid, ranging between 0uL and 100uL. The samples were not neutralised before plating. The results can be seen in figure 5 below. This assay showed concentrations similar to those calculated in the previous assay; however the general trend shows that as the volume of acid added increases, the concentration of phosphate recorded decreases. This therefore indicates that the chromogenic complex formed between the phosphate reagent dye and the orthophosphate is affected by the formic acid through the decrease in pH.

Figure 3: Polyphosphate assay following hydrolysis of polyp chains using varying volumes of 1M formic acid. Error bars are calculated using standard error of the mean cellular phosphate concentrations of technical repeats. Results were measured using a BMG FLUOstar Omega plate reader at OD650 and concentrations calculated from known phosphate concentration standards.

A potential solution to this was to add 1M NaOH to neutralise the acid hydrolysed solution before adding the phosphate reagent dye. However, a large volume of alkali was needed to neutralise the solution which created large dilution factors. Figure 6 shows a correlation between increased formic acid volume and decreased phosphate concentration. From the results shown in figure 6, we can see that there is a large variation in the phosphate concentrations; we also found that at small volumes of acid addition higher phosphate concentrations were detected.

Figure 4: Polyphosphate assay using varying volumes of formic acid and neutralising using 1M NaOH. Error bars are calculated using standard error of the mean cellular phosphate concentrations of technical repeats. Results were measured using a BMG FLUOstar Omega plate reader at OD650 and concentrations calculated from known phosphate concentration standards.

Following from this, we ran an assay on three colonies of BW25113 using our pre-prepared glass milk solution. In this assay, we used glass milk to remove the polyphosphate from the cell lysate. We then tested samples that had been subjected to the glass milk as well as samples that hadn’t. Our hypothesis was that if the assay did not detect polyphosphate, we would have seen a significant decrease in the levels of phosphate detected when the glass milk was used. However, if the assay did detect polyp, the phosphate levels would have been roughly the same. From the results presented in the graph below (figure 5), we found no difference between the phosphate concentrations when using or not using glass milk.

Figure 5: phosphate assay using glass milk. Error bars calculated using standard error of the mean concentrations of phosphate in technical repeats of the biological repeats. Results were measured using a BMG FLUOstar Omega plate reader at OD650 and concentrations calculated from known phosphate concentration standards

We placed an order for sodiumhexametaphosphate and polyphosphoric acid. These molecules were both used in literature as controls for polyphosphate. We then modified the acid hydrolysis step by increasing the concentration of NaOH from 1M to 6M to decrease the sample dilution during neutralisation. For this assay, we used the strains BW25113, ∆ppk and ∆ppx . From the results shown in the graph below (figure 8), there is no difference in the cellular phosphate concentrations of ∆ppk , ∆ppx and BW25113 when using different volumes of acid. However, this succeeded in reducing the amount of dilution the samples were subjected to.

Figure 6: Results from phosphate assay using various volumes of 1M formic acid, neutralised with 6M NaOH before plating. Error bars are calculated using standard error of mean phosphate concentrations of technical repeats. Results were measured using a BMG FLUOstar Omega plate reader at OD650 and concentrations calculated from known phosphate concentration standards.

We then decided to join forces with the growth assay team to analyse the phosphate concentration in the growth media of ∆pstA, ∆pstC, ∆pstB, ∆pstS, ∆phoE, ∆ppk, ∆ppx and BW25113 using our phosphate assay kit. This was with the aim of monitoring the change in phosphate concentration in the media following cell growth. LB was tested however an issue arose where the sample was not diluted sufficiently and a precipitant formed. This therefore altered the OD to produce an unreliable reading.

Figure 7: Image of plate showing precipitant due to high phosphate concentrations indicated by arrows.

Figure 8: Results from combined growth and phosphate assay. Error bars calculated using standard error of mean phosphate concentrations in the media of biological repeats. Results were measured using a BMG FLUOstar Omega plate reader at OD650 and concentrations calculated from known phosphate concentration standards.

Following from this, we prepared an additional phosphate assay using BW25113, PPK and PPX using the formic acid hydrolysis using 0uL, 1uL and 5uL of formic acid and neutralising using 6M NaOH. Here we brought all sample volumes to an arbitrary value (200uL) to minimise the effect of varying dilution factors. Using a two way ANOVA, the phosphate concentrations between 5uL of acid added and the concentrations when 1uL and 0uL of acid were added were found to be significant (p < 0.001). The difference between the phosphate concentrations recorded when 0uL and 1uL of formic acid were added were not found to be significant (p > 0.05).

Figure 9: Results from polyphosphate assay. Error bars calculated using standard error of mean cellular phosphate concentrations of technical repeats. Results were measured using a BMG FLUOstar Omega plate reader at OD650 and concentrations calculated from known phosphate concentration standards. *** indicates significant difference. NS indicates no significant difference.

From these results we deduced that there was a significant difference between the volume of acid added and the cellular phosphate concentration. We also drew the conclusion that using 5uL may have hydrolysed a larger volume of polyphosphate than lower volumes of acid and is therefore the most reliable volume to use for further assays.

As part of our collaborations with other iGEM teams from around the world, we performed a phosphate assay on water samples that had kindly been sent to our team. To carry this out we used 200uL per well of each sample and used the dye from the ABCAM kit to analyse the phosphate concentration in each sample. The results from this assay are shown on our wiki.

References

Visualization of polyphosphate granules in E.coli nucleoids using fluorescent microscopy

Introduction

A widely accepted method for in situ polyphosphate (polyP) detection is fluorescence microscopy using a fluorochrome, 4’, 6 – diamino-2-phenylindole (DAPI) at high concentrations ranging from 3 to 50 µg/ ml1. Interaction of polyP with DAPI shifts the emission maxima of the dye from 456 nm emitting blue fluorescence to 526 nm emitting bright yellow- green fluorescence^2,3,4.

Fluorescence microscopy imaging (63x)

Micrographs of immunostained cell coagulates and tissue sections were captured with Nikon Eclipse T300 inverted wide field microscope equipped with a plan apochromat 63x oil immersion objective (NA 1.4) (Nikon, Tokyo, Japan). The images were acquired using Volocity 6.0 software (PerkinElmer, Inc) on the same day using the same exposure time, which allowed a direct comparison between slides.

Confocal microscopy imaging (100x)

Confocal imaging was performed using Zeiss LSM 710 inverted confocal microscope equipped with a plan apochromat 100x oil immersion objective (NA 1.4). The excitation filter was set at 405 nm and emission filter at 526 nm. Image acquisition was performed using Carl Zeiss imaging software ZEN (black edition 2012).

Troubleshooting

The pH of the washing buffer should be 7.0 as the DAPI stain does not react with the polyphosphate granules even at the slight variation of pH. It was also observed that the application of the dye to the specimen at concentrations greater than 25 µg/ ml resulted in only blue light emission making the detection of polyP impossible.

Anticipated results

DAPI-stained polyphosphate granules present in the E.coli nucleoids show bright yellow- green fluorescence at 526 nm upon observation under confocal microscope (Figure 1) whereas the cells devoid of the granules emit a light blue fluorescence at 456 nm.

Figure 1: Detection of polyPgranules in E.coli nucleoids stained with DAPI using fluorescent microscopy. Green signal indicates the presence of polyphosphate granules in the cells of:

• A)Chlorella sp. (example from Mukherjee and Ray, 2015); 63x
• B)Wild type strain BW25113; 100x
• C)Δppk; 100x
• D)Δppx; 100x

References

To be able to tell whether Phil was going to work in environments with variable phosphate concentrations, we devised an assay to measure his growth rates. Using the KEIO collection we were able to determine the effect of each gene we were tampering with. Knocking out phosphate transporters exhibits a growth phenotype, and based on modelling results we expected a change in the growth as phosphate metabolism is interfered with.

Growth assays were based on optical density measurements of cell cultures at 650nm over a set period of time, either 7, 24 or 48 hours. The growth rates and curves of multiple phosphate transporter knockout strains have been compared and analysed to find both a chassis for characterisation of genes, as well as, to experimentally test for a decrease in cell growth.

Initially we decide to test each of our KEIO collection knockouts, these consisted of the interference several genes:

1. ΔpstA - Phosphate ABC transporter permease
2. ΔpstB - Phosphate ABC transporter, ATPase
3. ΔpstC - Phosphate ABC transporter permease
4. ΔpstS - Phosphate ABC transporter periplasmic binding component
5. ΔphoE - Outer membrane phosphoporin protein E
6. Δppk - Polyphosphate kinase
7. Δppx - Exopolyphosphatase
8. Each gene was tested in triplicate in MOPS media (see fine details) containing a variety of phosphate concentrations in order to determine how each phosphate transporter reacts. Before carrying out growth assays with these particular mutants we used colony PCR to verify the knockouts. This showed that the mutations were correct in the bacteria.


Figure 1: Picture showing Colony PCR verification of KEIO collection

Name	Sequence	Estimated Tm °C	Size bp
PhoE for	CCGGCAATATTCATTAAAACTGATACGTC	58	29
Phoe rev	ATTCGCGCGTTAATTAAAATCAGGAAT	58	27
ppk for	CCGTGAATAAAACGGAGTAAAAGTGG	58	26
ppk rev	AGGGTTATTCAGGTTGTTCGAGTGA	59	25
ppx for	AATCACTCGAACAACCTGAATAACCC	58	26
ppx rev	AGTATTAAGCGGCGATTTCTGGTGT	60	25
pstA for	AGCAATATCAACCGTGTTTATTCTTCGC	59	28
pstA rev	CTAAGAATGAGGGGGCACGCTAATG	60	25
pstB for	TGAATCAACCGTAACGACCGGTGAT	61	25
pstB rev	GCACGATGAGGAAAAGATTGCAATG	59	25
pstC for	CCATTAGCGTGCCCCCTCATT	60	21
pstC rev	AAACGCGTTTAACTGAAGAGTAACTTATG	57	29
pstS for	TTTATTAGTACAGCGGCTTACCGCT	59	25
pstS rev	ATGAATCCTCCCAGGAGACATTATG	57	25

Table 1. Primers used for PCR verification

Our first growth experiment was to test two knockouts, Δppx and Δppk , against the with the parent strain BW25113. We did this for 48 hours at concentrations of 1.32mM, 0.1mM and 0 mM phosphate. Overall no real difference was observed in Δppx and Δppk.



Figure 2: Graphs showing a 48 hour growth assay comparing BW25113, ΔPPK and ΔPPX. Cells were grown in MOPS media at different phosphate concentrations of 1.32mM (Figure 1A) 0.1mM (Figure 1B) and 0mM ( Figure 1C). Overall no significant difference observed. Error bars show standard error of the mean. Assay was started at 0.01 OD650 and OD650 was measured using a BMG FLUOstar Omega plate reader.

Our next experiment was to consider the difference in growth of knockouts ΔpstA and ΔpstC compared to that of the parent strain. This assay was performed for only 24h hours as we saw that stationary phase was reached at 12 hours and so it was unnecessary to continue for a longer time period. Also we decided to increase the number of different phosphate concentrations used. We tested the strains at 1.32mM, 0.1mM, 0.05mM, 0.01mM and 0mM phosphate concentration. A significant difference in growth phenotype was observed with ΔpstC as well as ΔpstA showing a reduction in growth at 0.05mM. This corresponds as these knockouts might be candidates to characterise our transport constructs which after transformation in these knockouts should restore the wild type phenotype .



Figure 3: Graphs showing a 24 hour growth assay comparing BW25113, ΔpstA and Δ pstC. Cells were grown in MOPS media with different phosphate concentrations of 1.32mM (Figure 2A) , 1.0mM (Figure 2B), 0.1mM (Figure 2C), 0.05mM (Figure 2D), 0.01mM (Figure 2E) and 0mM (Figure 2F). Error bars show standard error of the mean. Assay was started at 0.01 OD650 and OD650 was measured using a BMG FLUOstar Omega plate reader.

Furthering our tests on the KEIO collection knockouts we performed a growth assay testing ΔpstB and ΔpstS against the parent strain. This assay used the same phosphate concentrations as our previous one and again lasted 24 hours. Overall no real difference was shown.



Figure 4: Graphs showing a 24 hour growth assay comparing BW25113, ΔpstB and Δ pstS. Cells were grown in MOPS media with different phosphate concentrations of 1.32mM (Figure 3A) , 1.0mM (Figure 3B), 0.1mM (Figure 3C), 0.05mM (Figure 3D), 0.01mM (Figure 3E) and 0mM (Figure 3F). Error bars show standard error of the mean . Assay was started at 0.01 OD650 and OD650 was measured using a BMG FLUOstar Omega plate reader.

The final of the KEIO collection knockouts to test was ΔphoE. This was tested against parent strain BW25113 for 24 hours in phosphate concentrations of 1mM, 0.1mM, 0.05mM and 0mM. A slight difference between growth phenotypes was observed, however since PhoE is a phosphoporin which is expressed (REF) under stress a knockout does correspond to a reduction in growth.



Figure 5: Graphs showing a 24 hour growth assay comparing BW25113 with ΔphoE. Cells were grown in MOPS media with different phosphate concentrations of 1mM (Figure 4A) , 0.1mM (Figure 4B), 0.05mM (Figure 4C) and 0mM (Figure 4D). Standard Error Bars. Assay was started at 0.01 OD650 and OD650 was measured using a BMG FLUOstar Omega plate reader.

From the growth assays using the KEIO collection mutants we decided that the best chassis for characterisation of genes would be ΔpstC, since this is the one that showed the greatest change in growth phenotype from the parent strain. Unfortunatly due to unforseen circumstance we were unable to use the plate reader for the remainder of our experminents. Therefore using this data that we collected we applied to a manual version of our growth assay (See Protocols). Due to the limitations of time we have we were unable to use MOPS as the growth was too slow to see a difference. Therefore we switched to LB. We then used this growth assay to focus on characterising our transports, however at the time only Sinorhizobium meliloti pstSCAB could be characterised. Here we tested this construct in triplicate in LB media. The growth assay was performed over 7 hours to capture the growth rate in its exponential phase. Our construct had been put under a promoter induced by IPTG so we decided to test the genes both with and without this due to the stress induced by IPTG. When IPTG is added to the strains there is a significant difference observed between the growth rates (ANOVA, Greenhouse-Geisser: F=12.006; d.f. =1.784; P=0.002). Post hoc LSD test showed a significant difference between the parent strain BW25113 and ΔpstC or ΔpstC+smp however there is no significant difference between ΔpstC and ΔpstC+smpst alone. Similarly when there is no IPTG added to the cells there is a significant difference observed between the growth rate of the strains (ANOVA, Greenhouse-Geisser: F=9.506; d.f.= 1.405 ; P=0.008). Again a post hoc LSD test showed significant difference between BW25113 against ΔpstC or ΔpstC+smpst. When comparing ΔpstC+smpst with IPTG and ΔpstC+smpst without IPTG there is a significant difference in the growth rates being observed (ANOVA Greenhouse-Geisser: F=15.167; d.f.= 1 ; P=0.006).



Figure 6: Graphs showing 7 hour growth assays comparing BW25113, ΔpstC and ΔpstC+smpst. Cell were tested LB without IPTG (Figure 6A) and with IPTG (Figure 6B). Error bars show standard error of the mean. Assay was started at 0.1 OD650



Figure 7. A graph showing a comparison of the growth rates observed in ΔpstC+smpstC with and without IPTG. Assay was started at 0.1 OD650 .

Fine Details

What is MOPS media?

Shortened for "3-(N-morpholino) propanesulfonic acid", it has an almost completely neutral pH and can be used as a replacement media for LB that doesn't have phosphate in it. This allowed us to measure growth at variable concentrations of phosphate by adding the amount of phosphate we desired.

What is the KEIO^[1] collection?

This is biggest collection of knockout strains of Escherichia coli using parent strain BW25113. The knockout strains were created through single-gene deletions of all nonessential genes.




References

1. Baba T1, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol .2006.0008. (Feb).p1-11