Team:Manchester/Results2

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 (Eut) bacterial microcompartment of Escherichia coli. Constructs were cloned into the plasmid pSB1C3 and transformed into DH5α E. coli chemically competent cells.

We managed to transform colonies combining EutS and EutMN to give us the construct EutSMN within the plasmid pSB1C3. We then obtained colonies which we believed to contain EutSMNLK, however we were unable to obtain pure plasmid from this construct containing all microcompartment subunits as the transformed colonies became too sickly. We hypothesize that this was due to the expression of EutMN and EutLK being highly toxic to cell and reducing growth rate.

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.

This would ultimately interfere with the real-life implementation of our bacteria in a continuous culture system. In a continuous culture system it is important to balance biomass accumulation with PPK expression and phosphate accumulation. When phosphate levels are low, the cells should invest their resources into growth, rather than phosphate accumulation. To alleviate this problem, we designed and modelled a regulatory operon that aims to regulate the synthesis of microcompartment proteins under different external phosphate levels.