Team:Manchester/Results

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.

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.)

Figure 2. 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 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.

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).

Figure 3. 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 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 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 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.

Figure 5. 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.

Localization Tag

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Localisation Tag Characterization using Microscopy

We aimed to confirm that our Eut microcompartments and pduD localisation tag were compatible. This was enabled by expressing the pduD-Anderson promoter constructs with mCherry attached so that they could be visualised via fluorescent microscopy. We visualised the tag-promoter constructs using mCherry to check the distribution of the fluorescent reporter throughout the cell. Using fluorescence microscopy we investigated whether the tag localised in the presence of Eut and to see the level of expression based on fluorescence level.

Biobricks used in the following section 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)

As expected, without the expression of any Eut subunits, the tags showed a homogeneous distribution throughout the cells with no localisation. The difference in fluorescence was most pronounced between low promoter and the other two, this matches previous results of tag expression as shown in the figure below:

As medium promoter and high promoter have similar levels of expression, we decided it wouldn't be useful to use medium in further characterization, so only low and high were combined with Eut subunits for visualization.

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 heterogeneous throughout the cell. High+EutSMN showed quite well defined localization in a number of cells.

SMNLK 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.

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.

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 on the protocols page). 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.

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:

As shown, there is a heterogeneous distribution of fluorescence within the cells for both signals and they are approximately in the same areas. This heterogeneous distribution of DAPI indicates the presence of polyphosphate and proves the activity of our PPK along with its successful localization into our BMC. The localisation can be determined using the physical location of both fluorescence signals within the cell.