Difference between revisions of "Team:Manchester/Results"

 
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    <h4>Polyphosphate Kinase (PPK)</h4>
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<p><font color="#111">Background</p>
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<p><b>Overview:</b></p>
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<pBackground>
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Accumulation of poly-phosphate by poly-phosphate kinases (PPKs) is an important mechanism of phosphate uptake from in enhanced biological phosphate removal (EBPR) plants (Mino, van Loosdrecht and Heijnen, 1998). Degradation of polyphosphate in cells limits its accumulation (Akiyama, Crooke and Kornberg, 1992).
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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 utilization (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.
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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 as a suitable candidate, having a 30 fold greater turnover rate than that of the E. coli PPK (Lindner et al., 2007; Kornberg and Simms, 1956).
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<p style="margin-left: 40px">1. <a href="https://2017.igem.org/Team:Manchester/Results1">Polyphosphate kinase</a> purification and optimization.</p>
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<p style="margin-left: 40px">2. <a href="https://2017.igem.org/Team:Manchester/Results2">Eut microcompartment</a> expression.</p>
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<p style="margin-left: 40px">3. <a href="https://2017.igem.org/Team:Manchester/Results3">Localization Tag</a> characterization.</p>
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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).  
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<p style="margin-left: 40px">4. <a href="https://2017.igem.org/Team:Manchester/Results4">Anderson Promoters</a> characterization.</p>
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We designed three constructs that would allow us to study whether the PduD(1-20) sequence could localize cgPPK to the microcompartment (Figure x). Two of the constructs (IDs) contained an mCherry fusion that would allow us to determine subcellular localization of the enzyme via microscopy. All three constructs contained a C-terminal hexa-histidine sequence that would allow purification.
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<p style="margin-left: 40px">5. <a href="https://2017.igem.org/Team:Manchester/Results5">References</a></p>
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The constructs were cloned into the pNIC28-bsa4 vector downstream of a T7 promoter for overexpression. The vectors were transformed into BL21 (DE3) cells 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 0.5mM. 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.
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All three constructs were purified using Ni-NTA agarose (QIAgen) according to our protocol (Protocol).
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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.
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The cgPPK construct was purified to homogeneity (Figure x) 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 x).  
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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 x), 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.
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PPK Activity - Luminesence.png
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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 localized within the bacterial microcompartment and synthesized polyphosphate.
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<a class="floatleft1 project projectlink" href= " https://2017.igem.org/Team:Manchester/Results1 " ><img src="https://static.igem.org/mediawiki/2017/7/7c/T--Manchester--Next_Arrow17.jpg"></a>
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    <h4><font color="#111">Eut Bacterial Microcompartment Expression</font></h4>
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<a class="floatright1 project projectlink" href= " https://2017.igem.org/Team:Manchester/Description1 " ><img src="https://static.igem.org/mediawiki/2017/0/0a/T--Manchester--BACKARROW17MAN.jpg"></a>
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<p><font color="#111">Previous iGem teams have found that microcompartments have proved difficult to work with (<a href="https://2011.igem.org/Team:Dundee"target="_blank"><b>Dundee 2011</b></a>, <a href="https://2013.igem.org/Team:Hong_Kong_HKU"target="_blank"><b>Hong Kong 2013</b></a>, <a href="https://2016.igem.org/Team:CU-Boulder"target="_blank"><b>CU-Boulder 2016</b></a>). 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, 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.
 
 
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We induced our constructs with their respective reagents for 4 hours and 20 hours before collecting soluble and insoluble proteins. 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 1. and the corresponding table 1. for the bands we were expecting.   
 
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<img src= "https://static.igem.org/mediawiki/2017/8/88/Sds_page_eut_no_leg.jpeg" width="800" height="317">
 
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<p><font color="#111"><center></h3><b>Figure 1.</b> 12% Tris-glycine SDS page gels of soluble and insoluble Eut S, MN, SMN and LK construct proteins. - indicates
 
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construct had not been induced,+ indicates construct had been induced. Red arrows indicate predicted size of bands, also shown in table 1.
 
 
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<p><font color="#111"><center></h3><b>Table 1.</b>Predicted sizes of Eut proteins and the associated tags.
 
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<p><font color="#111">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 2.)
 
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<img src="https://static.igem.org/mediawiki/2017/9/9c/Manchesterwesternblotseutfinalsize.png" width="800" height="317">
 
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<p><font color="#111"><center></h3><b>Figure 2.</b> <b>Western blot analysis of EutS, EutM, and EutN protein production </b>from cultures transformed with MN (<a href="http://parts.igem.org/Part:BBa_K2213001"target="_blank"><b>BBa_K2213001</b></a>) and SMN (<a href="http://parts.igem.org/Part:BBa_K2213012"target="_blank"><b>BBa_K2213012</b></a>) 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.
 
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<p><font color="#111">Here we observed a band corresponding to EutMN 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 (approximately 70 kD), 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 is due to the presence of GFP in a dimeric form.</p>
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<p>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 3 and 4).</p>
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<p><font color="#111"><center></h3><b>Figure 3.</b> 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.</center></center>
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<p><font color="#111"><center></h3><b>Figure 4.</b> 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.</center></center>
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<p>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 5). We observed that between 4 and 20 hours, the OD of cultures containing the constructs EutMN, EutSMN and EutLK were reduced by 75.53%, 81.77% and 67.93% respectively. In contrast to this, the OD of the EutS culture continued to rise and had increased by 45.28% 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. 
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<p><font color="#111"><center></h3><b>Figure 5.</b> 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.
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<p><font color="#111">Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed condimentum turpis ut aliquam pellentesque. Quisque consectetur quam lorem, ut varius elit mollis euismod. Fusce gravida orci metus, nec cursus dui pulvinar quis. Vivamus accumsan magna tortor, quis egestas magna faucibus in. Morbi enim sem, sollicitudin viverra viverra et, tempor sit amet justo. Quisque malesuada magna eu lorem egestas, quis molestie lectus sollicitudin. Donec varius magna vel eros faucibus, vel cursus eros condimentum. Donec justo lacus, tempus sed vestibulum quis, semper vitae nulla. Nam vel accumsan mi. Cras molestie, risus in hendrerit laoreet, sapien mauris ultricies enim, in fermentum ligula nibh a tellus. In vitae sollicitudin nibh. Praesent convallis consectetur vulputate.
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    <h4><font color="#111">Localisation Tag Characterisation using Microscopy</font></h4>
 
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<p><font color="#111">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.<br>
 
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<a href="http://parts.igem.org/Part:BBa_K2213006"target="_blank"><b>BBa_K2213006: LowPromoter_PduD(1-20)_mCherry</b></a> (Low)<br>
 
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<a href="http://parts.igem.org/Part:BBa_K2213007"target="_blank"><b>BBa_K2213007: MediumPromoter_PduD(1-20)_mCherry</b></a> (Medium)<br>
 
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<a href="http://parts.igem.org/Part:BBa_K2213008"target="_blank"><b>BBa_K2213008: HighPromoter_PduD(1-20)_mCherry</b></a> (High)<br>
 
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<p><font color="#111">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.<br>
 
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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 localization in a number of cells.<br>
 
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EutSMNLK was visualized by combining EutLK-Low-PduD-mCherry-PPK and EutSMN. Fluorescence from Low+EutSMNLK showed quite well-defined localization with a number of cells showing relatively round accumulations of fluorescent tag, suggesting proper BMC formation.<br>
 
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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.<br>
 
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>here</a>, we were able to DAPI stain our cells (our modified protocol can be found on <a href="https://2017.igem.org/Team:Manchester/Experiments"target="_blank"><b>the protocols page</b></a>).
 
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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.<br>
 
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<p><font color="#111">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.<br>
 
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<p><font color="#111">As shown, there is a clear heterogeneous distribution of fluorescence within the cells for both signals and they are approximately in the same areas, suggesting . 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.<br>
 
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Latest revision as of 03:46, 2 November 2017

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Results


Overview:

1. Polyphosphate kinase purification and optimization.

2. Eut microcompartment expression.

3. Localization Tag characterization.

4. Anderson Promoters characterization.

5. References