Difference between revisions of "Team:Manchester/Results"

Background
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).
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. 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 turnover rates 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). 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 localize cgPPK to the microcompartment. 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.

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, 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.

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.

Figure 1. 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.