Team:Manchester/Results

Accumulation of poly-phosphate by poly-phosphate kinases (PPKs) is an important mechanism of phosphate uptake for enhanced biological phosphate removal (EBPR) in water treatment plants (Mino, van Loosdrecht and Heijnen, 1998). Degradation of polyphosphate in cells limits its accumulation (Akiyama, Crooke and Kornberg, 1992).

Encapsulation of PPKs within bacterial microcompartments has been shown to be a viable approach to enhancing accumulation of phosphate in Escherichia coli (Liang et al., 2017). This was achieved by co-expression of a recombinant Citrobacter freundii 1,2-propanediol utilisation (Pdu) microcompartment operon with E. coli PPK that had been fused with an N-terminal sequence that had been shown to direct heterologously expressed proteins towards the interior of the microcompatment. We wanted to build on this work by identifying a polyphosphate kinase with a higher turnover rate than the E. coli PPK, and directing it towards the a heterologously expressed bacterial microcompartment. We used the Braunschweig Enzyme Database (BRENDA) to compare specific activities of polyphosphate kinases and identified the class II polyphosphate kinase from Corynebacterium glutamicum (cgPPK) as a suitable candidate, having a 30 fold greater turnover rate than that of the E. coli PPK ( Figure 1; Lindner et al., 2007; Kornberg and Simms, 1956).

Figure 1. Comparison of specific activity between the E. coli class I PPK and the Corynebacterium glutamicum class II PPK

We used the SwissModel (Schwede, 2003) server to generate a homology model of the structure of the cgPPK enzyme based on its amino acid sequence. SwissModel identified an existing crystal structure of class II polyphosphate kinase from Sinorhizobium meliloti (Figure 2).

Figure 2: cgPPK2 model Homology model of cgPPK generated using SwissProt (AS Rose et al., 2016; 2015)

We believed that a fused PduD(1-20) sequence would be sufficient to direct recombinant cgPPK towards the interior of the Ethanolamine utilisation (Eut) bacterial microcompartment, owing to a hydrophobic motif that it shares with the N-terminal sequences of EutE and EutC, which are targeted to the Eut microcompartment in Salmonella enterica (Jakobson et al., 2015). We designed three constructs that would allow us to study whether the PduD(1-20) sequence could localise cgPPK to the microcompartment (Figure 3). Two of the constructs (PduD(1-20)_mCherry_cgPPK and PduD(1-20)_cgPPK_mCherry) contained an mCherry fusion that would allow us to determine subcellular localisation of the enzyme via microscopy. All three constructs contained a C-terminal hexa-histidine sequence that would allow purification with immobilised nickel-affinity media.

Figure 3. Architecture of the constructs based on the class II polyphosphate kinase from Corynebacterium glutamicum (cgPPK) (PduD(1-20)_mCherry_cgPPK; PduD(1-20)_cgPPK_mCherry;cgPPK.)

The constructs shown above were cloned into the pNIC28-bsa4 vector downstream of a T7 promoter for overexpression. The vectors were transformed into BL21 (DE3) E. colicells and cultured under similar conditions to those used by Lindner et al. (2007) to express recombinant cgPPK: The liquid cultures were grown at 37 ˚C and 180 rpm until the OD600 reached 0.5 – 0.6, at which point Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. The cultures were grown for a further 4 hours at 37 ˚C and 180 rpm before harvest by centrifugation at 5,000 rpm for 10 minutes. The cell pellets were frozen at -20 ˚C for later purification.

Figure 4. SDS-PAGE analysis of fractions from the Ni-NTA agarose purification of the cgPPK constructs. Ladder: Precision Plus Protein™ (Biorad). Black arrows indicate where a band with a molecular mass corresponding to that of the construct should be.

All three constructs were purified using Ni-NTA agarose (QIAgen) according to our agarose purification protocol. The SDS-PAGE analysis showed that the elution fraction of the PduD(1-20)_cgPPK_mCherry construct gave a band of around 30 kDa rather than the expected 66 kDa. The bright pink colour of the concentrated elution fraction suggested that the mCherry unit was intact. This suggested that the PduD(1-20)_cgPPK region of the construct was cleaved before or during the purification. It wasn’t clear from our results why this construct was unstable, but we reasoned that it was too unstable for our use, so no further attempts to express and purify it were made.

The cgPPK construct was purified to near-homogeneity (Figure 4) to a calculated final concentration of 0.89 mg/ml. The presence of kinase activity of the construct was confirmed by data generated using the Promega ADP Glo™ kinase assay kit (Figure 5).

Figure 5. Qualitative kinase activity assessment of the cgPPK and PduD(1-20)_mCherry_cgPPK constructs, assessed using the Promega ADP Glo (TM) Kinase assay

The PduD(1-20)_mCherry_cgPPK co-purified with a ~70 kDa protein in roughly equimolar quantities. The co-purified protein may be the Gro EL heat shock protein (Thain et al., 1996), which may be the result of mis-folding. The PduD(1-20)_mCherry_cgPPK construct retained a bright pink colour and kinase activity (Figure 5), which suggests that both the mCherry and the cgPPK domains folded correctly. It’s possible that the putative heat shock protein bound to the N-terminal PduD(1-20) targeting peptide as a result of its hydrophobicity.

Fluoresence microscopy imaged of 4',6-diamidino-2-phenylindole (DAPI)-stained cells expressing all five Eut microcompartment structural genes and PduD(1-20)_mCherry_cgPPK suggested that the construct localised within the bacterial microcompartment and synthesized polyphosphate.

Expression Optimization
Expression of the pNIC28-bsa4:PduD(1-20)_mCherry_cgPPK construct was optimised using a Design of Experiments-based method. mCherry fluorescence in raw culture would be measured by a plate reader as a proxy for proxy for protein yield. JMP software from SAS was used to design an initial round of 20 experiments that would screen the following factors for their effect on protein yield:

OD600 at induction (0.2 - 0.8)
[IPTG] at induction (0.1 – 1 mM)
Growth temperature after induction (20 – 37°C)
Growth period after induction (4 – 24 hours)

The initial design took into account potential interactions between the factors, but did not include mid-points between the extremes of each factor. All cultures were grown from a clonal glycerol stock in Luria-Bertani (LB) broth supplemented with 100 µg/ml kanamycin. The initial growth phase prior to induction was always carried out in a single flask at 37°C and 180 rpm. At the point of induction, the flask was split into smaller shake flasks which met the specified growing conditions and the resulting mCherry fluorescence quantified (Figure 6).

Figure 6. Relative mCherry fluorescence from raw liquid culture expressing the PduD(1-20)_mCherry_cgPPK construct under different conditions.

Background

Using JMP, we fit a linear model to our data using standard least squares regression analysis (R²=0.99, Figure 7). The effects summary outlined the significant individual factors and the two-way interactions between the factors that affected yield (Figure 8).

Figure 7. Predictive vs. actual plot of the linear model generated from the experimental data (R²=0.99)
Figure 8. Effect summary of input factors and their interactions on yield of PduD(1-20)_mCherry_cgPPK

Figure 8. The interaction profile of the input factors, maximised for yield. OD600 at induction consistently correlated with higher yields. The profiler also suggests that optimal conditions for expression of PduD(1-20)_mCherry_cgPPK lie close to 20°C and a post-induction growth period of 24 hours or more.

The model suggested that OD600 at the time of induction had consistent positive correlation with yield, so we decided that for future experiments we would fix this value to 0.8, which allowed us to look at other factors with greater resolution. The model also suggested that the optimal conditions for PduD(1-20)_mCherry_cgPPK lay outside of our initial parameters. We designed a second round of 20 experiments that would explore the effects of our input factors slightly closer to the predicted optimal values. The second round of experiments resulted in yields that were beyond the detection capability of the plate reader using the gain settings that were used for the first round (Figure 8), so the gain had to be lowered. This meant that the results from the two rounds were no longer comparable and could not be modelled together. However we could be sure that the yields of the second round exceeded those of the first (Figure 9).

Figure 9. mCherry fluorescence measurements from the 24 hour time points from round 2 of optimisation (Pink) plotted alongside the mCherry fluorescence measurements from the round one experiments, which used the same gain setting. The readings from the second round have saturated the detector, so we had no choice but to change the gain settings on the plate reader. This meant that we could no longer directly compare data from round 1 and round 2.

Figure 8. mCherry fluorescence of raw culture from round 2 of expression optimisation. Fluorescence intensity for all round 2 experiments are greater than those of round 1.

Once again, we fit a model to the data (R² =0.80, Figure 9) which predicted that post-induction growth period and post-induction incubation temperature had the most significant effects on mCherry fluorescence (P < 0.05 in both cases); variation in IPTG concentration within the experimental range (1-10 mM) was not found to have a particularly significant effect on mCherry fluorescence (P = 0.15). Figure 10 shows the effect of post-induction growth period and post-induction incubation temperature on mCherry fluorescence.

Figure 9. Actual vs. predictive plot generated using the model fitted to the second round of expression optimisation data

Figure 10. Effects summary of the second round of expression optimisation. Post induction temperature and the harvest time had the most significant effect on yield within the parameters tested

The model predicted that within the limits of the tested parameters, optimal expression of PduD(1-20)_mCherry_cgPPK could be achieved by inducing the culture to a final concentration of 1mM IPTG when the OD600 reached 0.8, followed by a 48 hour growth period at 24°C at 180 rpm.

A culture of BL21 (DE3) cells harbouring pNIC28-bsa4:PduD(1-20)_mCherry_cgPPK was grown under the predicted optimal conditions for mCherry yield per volume of culture. On attempt to purify the recombinant protein according to our agarose purification protocol, we found that after repeated attempts to disrupt the cells by sonication the bright pink recombinant protein was insoluble and could not be separated from the cell debris under non-denaturing conditions. (Figure 11).

Figure 11. SDS-PAGE analysis of the purification stages of PduD(1-20)_mCherry_cgPPK expressed under predicted optimal conditions. A large amount of the protein of the expected weight (66 kDa) appears to have been retained in the insoluble (insol) stage, with a roughly equimolar 70 kDa band that may correspond to a heat shock protein.

From this we concluded that mCherry fluorescence in raw culture was not a suitable proxy for soluble protein yield because it did not account for protein aggregation in vivo. In retrospect, a more useful, but more labour intensive end point would have been mCherry fluorescence in the supernatant following cell disruption and removal of cell debris by centrifugation.

Eut Bacterial Microcompartment Expression

Previous iGem teams have found that microcompartments have proved difficult to work with (Dundee 2011, Hong Kong 2013, CU-Boulder 2016). Because of this, we thought it would be beneficial to work on optimising the formation of micro-compartments. These three constructs (EutS, EutMN and EutLK) were each combined with an independent inducible promoter (see figure 1) to enable variable synthesis of micro-compartment proteins and allow us to optimise micro-compartment formation with varying induction levels. We designed a collection of experiments, varying in complexity in order to prove that microcompartment formation was induced by our promoters. We also wanted to understand if microcompartment protein synthesis induced stress within our chassis and affected growth.

Figure 1. Architecture of EutS, EutMN, and EutLK constructs based on the ethanoalamine utilisation bacterial microcompartment of Escherichia coli. Constructs were cloned into the plasmid pSB1C3 and transformed into DH5α E. coli chemically competent cells.

We induced our constructs with their respective reagents for 4 hours and 20 hours before collecting soluble and insoluble proteins, see our induction protocol here. These samples were then run on a 12% Tris-Glycine SDS-Page gel. Unfortunately, we were unable to see any bands of increased intensity, see figure 2. and the corresponding table 1. for the bands we were expecting.

Figure 2. 12% Tris-glycine SDS page gels of soluble and insoluble Eut S, MN, SMN and LK construct proteins. - indicates construct had not been induced,+ indicates construct had been induced. Red arrows indicate predicted size of bands, also shown in table 1.

Table 1.Predicted sizes of Eut proteins and the associated tags.

Due to lack of results from our SDS-Page analysis we decided to specifically target the HIS and FLAG tags associated with our Eut proteins by performing a Western blot (see figure 3.). See our Western blot protocol here.

Figure 3. Western blot analysis of EutS, EutM, and EutN protein production from cultures transformed with MN (BBa_K2213001) and SMN (BBa_K2213012) constructs. Nitrocellulose membranes A and B, blotted using mouse anti-His mAb (clone HIS-1, sigma) and mouse anti-FLAG mAb (clone M2, Sigma), respectively. Goat IRDye 800CW-conjugated anti-mouse igG pAb (Abcam) used on both A and B. Induced and non-induced culture protein lysates indicated by + and - respectively. Bands of interest indicated by black arrows.

Here we observe 2 bands at approximately 70kD, a product of the EutMN construct induced with tetracycline at a concentration of 0.1 μM and EutSMN induced with both tetracycline at a concentration of 0.1 μM and IPTG at a concentration of 250 μM. The size of this band, its occurrence in conjunction with EutM and the absence of a band in conjunction with the anti-FLAG antibody has led us to hypothesize that this band corresponds to a dimer of GFP-EutM.

Following our findings from the Western blot, we focused our induction trials on GFP fluorescence. This allowed us to determine if the expression of EutM had been successful. There was a significant increase in fluorescence at both the 4 and 20-hour time point (p = 0.0016 and p = 0.0054 respectively), produced by cells containing the EutMN construct under inducing conditions. Similarly, there was a significant increase in fluorescence produced by cells containing the EutSMN construct at both the 4 and 20-hour time points (p = 0.002 and p = 0.0007 respectively). This confirmed that the TetR promoter was working as expected, controlling the induction of the EutMN construct (see figures 4 and 5).

Figure 4. Average OD corrected fluorescence (Ex. λ 470-15 / Em. 515 – 20 nM) measurements of EutS, EutSM, EutSMN and EutLK constructs, non-induced and induced taken after 4 hours. Error bars show the SEM.

Figure 5. Average OD corrected fluorescence (Ex. λ 470-15 / Em. 515 – 20 nM) measurements of EutS, EutSM, EutSMN and EutLK constructs, non-induced and induced taken after 20 hours. Error bars show the SEM.

Throughout the GFP induction trial we also recorded optical density measurements at 600nM for each of our constructs. OD readings were taken at 0 hours, 4 hours and at 20 hours (see figure 6). We observed that between 4 and 20 hours, the OD of cultures containing the constructs EutMN, EutSMN and EutLK were reduced by 75%, 81% and 67% respectively. In contrast to this, the OD of the EutS culture continued to rise and had increased by 45% when the final reading was taken at 20 hours. This suggests that the production of microcompartment subunits EutM, EutN, EutL and EutK are toxic to the cell, however, the production of EutS may be less toxic. This may be due to less strain being put on the cell due to the expression of a single microcompartment subunit, rather than multiple subunits being expressed simultaneously. Overall this data indicates that the expression of complete microcompartments is likely to be toxic to the cell and should be highly regulated.

Figure 6. Average optical density at 600 nM of Eut S, EutMN, EutSMN constructs induced and non-induced. Measurements were taken at 0 hours, 4 hours and 20 hours.

Anderson Promoter Characterisation

We aimed to confirm the function of the different PduD-Anderson promoter constructs by expressing them with mCherry attached so that they could be visualised via fluorescent microscopy. We visualised the different strength tag-promoter constructs, using mCherry fluorescence to check the expression level of each construct.

Biobricks used are as follows:
BBa_K2213006: LowPromoter_PduD(1-20)_mCherry (Low)
BBa_K2213007: MediumPromoter_PduD(1-20)_mCherry (Medium)
BBa_K2213008: HighPromoter_PduD(1-20)_mCherry (High)

Figure 1. Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.

As expected, the level of fluorescence increases with each strength promoter. The difference in fluorescence was most pronounced between low promoter and the other two. It is important to note that the expression levels of the medium and high strength promoters are very similar. This result was unexpected and should be taken into consideration when choosing a suitable promoter strength.

The different tag expression levels under each promoter was further investigated via fluorescence microscopy.

Figure 2. Fluorescence microscopy images of Low, Medium and High strength Anderson promoter-PduD construct associated mCherry (OD600: 0.2) expressed in the absence of Eut.

As shown, there is a gradient of fluorescence, substantiating the previous results. These results demonstrate proper functioning of the low promoter and highlight the irregular expression levels between the medium and high promoters, suggesting one or both are not functioning as intended.

Localisation Tag Characterisation using Microscopy

We aimed to confirm that our Eut microcompartments and pduD localisation tag were compatible. We visualised the tag-promoter constructs via fluorescence microscopy using mCherry to check the distribution of the tag throughout the cell and investigated whether the tag localised in the presence of Eut subunits.
(As medium promoter and high promoter had similar levels of expression, we decided to just use low and high in combination with Eut subunits for characterisation).

Biobricks used are as follows:
BBa_K2213006: LowPromoter_PduD(1-20)_mCherry (Low)
BBa_K2213007: MediumPromoter_PduD(1-20)_mCherry (Medium)
BBa_K2213008: HighPromoter_PduD(1-20)_mCherry (High)
BBa_K2213000: LacUV5_EutS (EutS)
BBa_K2213012: LacUV5_EutS_TetR_EutMN (EutSMN)
BBa_K2213013: araB_eutLK_LowPromoter_PduD(1-20)_mCherry_cgPPK2 (EutLK-Low-PduD-mCherry-PPK)

Figure 3. Fluorescence microscopy images of Low and High strength Anderson promoter-PduD construct associated mCherry expressed alone and co-expressed with Eut subunits.

Levels of fluorescence from Low+EutS were too low to properly visualize. High+EutS showed slightly heterogeneous distribution of fluorescence, the fluorescence was slightly granular with some brighter areas and some darker areas but no well-defined localisation.

Low+EutSMN images were very dim but visualization was possible. The fluorescence was granular but mainly homogeneous throughout the cell. High+EutSMN showed quite well defined localisation in a number of cells.

EutSMNLK was visualized by combining EutLK-Low-PduD-mCherry-PPK and EutSMN. Fluorescence from Low+EutSMNLK showed quite well-defined localisation with a number of cells showing relatively round accumulations of fluorescent tag, suggesting proper BMC formation.

(It is important to note that the majority of the accumulations seen occur at or near the end of the cells. This could be indicative of protein aggregates and not proper BMC formation (Bednarska et al., 2013)).

An added benefit of our SMNLK+PPK construct is that it would allow us to determine whether it actually worked via DAPI staining. The polyphosphate chains produced by PPK can be DAPI stained and visualised by setting the excitation filter at 370nm and emission filter at 526nm, and as such both mCherry and DAPI could be visualised in the same cells. This would allow us to see any co-localisation of the two signals, demonstrating successful Eut subunit expression, successful tag localisation and successful PPK activity.

By modifying the protocol found here, we were able to DAPI stain our cells (our modified protocol can be found here). A control DAPI stain was performed along side Medium promoter tag expression to inspect the DAPI distribution in the absence of polyphosphate and whether the staining procedure interfered with mCherry distribution.

Figure 4. Visible light, mCherry and DAPI signals from DAPI stained E. coli expressing Medium strength Anderson promoter-PduD construct.

As shown, the distribution of both mCherry and DAPI were homogeneous with no obvious clumping or accumulation. This suggested that if our construct was working properly, we would see an accumulation of fluorescent signal for both mCherry and DAPI in the same place within the cell.

So in line with this, we DAPI stained a 24h induction of Low+EutSMNLK+PPK:

Figure 5. Fluorescence microscopy images of promoter-PduD associated mCherry and DAPI stained polyphosphate.

This heterogeneous distribution of DAPI indicates successful dying of polyphosphate and confirms the activity of our PPK along with its successful localisation into our bacterial microcompartment. These findings demonstrate the proof-of-concept functionality of our Phosphostore system.

References

Bednarska, N., Schymkowitz, J., Rousseau, F. and Van Eldere, J. (2013). Protein aggregation in bacteria: the thin boundary between functionality and toxicity. Microbiology, 159(Pt_9), pp.1795-1806.

AS Rose, AR Bradley, Y Valasatava, JM Duarte, A Prlić and PW Rose. Web-based molecular graphics for large complexes. ACM Proceedings of the 21st International Conference on Web3D Technology (Web3D '16): 185-186, 2016. doi:10.1145/2945292.2945324

AS Rose and PW Hildebrand. NGL Viewer: a web application for molecular visualization. Nucl Acids Res (1 July 2015) 43 (W1): W576-W579 first published online April 29, 2015. doi:10.1093/nar/gkv402

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Email: manchesterigem17@gmail.com