Difference between revisions of "Team:Manchester/Results"
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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 construct had not been induced,+ indicates construct had been induced. Red arrows indicate predicted size of bands, also shown in table 1. | <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 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. | <p><font color="#111"><center></h3><b>Table 1.</b>Predicted sizes of Eut proteins and the associated tags. | ||
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Revision as of 15:28, 31 October 2017
Results
PPK
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Eut Bacterial Microcompartment Expression
Previous iGem teams have found that microcompartments have proved difficult to work with. 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.
Localization Tag
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Localisation Tag Characterization using Microscopy
We aimed to confirm our constructs were working via separate means so we designed all of the tag-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 tag throughout the cell, whether the tag localised in the presence of Eut and to see the level of expression based on fluorescence level.
As expected, without the expression of any Eut subunits, the tag showed a homogeneous distribution throughout the cells with no localization. The difference in fluorescence was most pronounced between low promoter and the other two as shown (in another figure? There’s a graph showing medium and high expression levels as very similar).
As medium promoter and high promoter had a similar level of expression, 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 localization.
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 with the low promoter as successful construction of High+EutSMNLK was not possible before we ran out of time. 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.
After successful expression of Low+EutSMNLK we decided to transform EutLK-Low-PduD-mCherry-PPK and EutSMN together. We reasoned that this SMNLK+PPK construct would allow us to determine whether it actually worked via DAPI staining. The polyphosphate chains produced by PPK can be stained using DAPI 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.
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.
