Difference between revisions of "Team:Manchester/Results"
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<p><font color="#111">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.<br> | <p><font color="#111">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.<br> | ||
| + | <br> | ||
| + | Biobricks used in the following section are as follows:<br> | ||
| + | <a href="http://parts.igem.org/Part:BBa_K2213006"target="_blank"><b>Low promoter</b></a><br> | ||
| + | <a href="http://parts.igem.org/Part:BBa_K2213007"target="_blank"><b>Medium promoter</b></a><br> | ||
| + | <a href="http://parts.igem.org/Part:BBa_K2213008"target="_blank"><b>High promoter</b></a><br> | ||
| + | <a href="http://parts.igem.org/Part:BBa_K2213000"target="_blank"><b>Eut S</b></a><br> | ||
| + | <a href="http://parts.igem.org/Part:BBa_K2213012"_blank"><b>Eut SMN</b></a><br> | ||
| + | <a href="http://parts.igem.org/Part:BBa_K2213013"_blank"><b>EutLK+Low+PPK</b></a><br> | ||
<br> | <br> | ||
<center> | <center> | ||
| Line 167: | Line 175: | ||
</center> | </center> | ||
<br> | <br> | ||
| − | <p><font color="#111">As expected, without the expression of any Eut subunits, the | + | <p><font color="#111">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:<br> |
| + | <br> | ||
| + | <center> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/bd/TagExpression800p.jpeg" width="800" height="317"> | ||
| + | </center> | ||
<br> | <br> | ||
| − | As medium promoter and high promoter | + | 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.<br> |
<br> | <br> | ||
| − | 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 | + | 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> |
<br> | <br> | ||
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.<br> | 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.<br> | ||
<br> | <br> | ||
| − | SMNLK was visualized | + | 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.<br> |
<br> | <br> | ||
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.<br> | 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.<br> | ||
| Line 186: | Line 198: | ||
</center> | </center> | ||
<br> | <br> | ||
| − | <p><font color="#111"> | + | <p><font color="#111">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 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.<br> |
<br> | <br> | ||
| + | By modifying the protocol found <a href="https://www.nature.com/protocolexchange/protocols/4075#/main"target="_blank"><b>here</b></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>). | ||
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> | 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> | ||
<br> | <br> | ||
Revision as of 16:15, 31 October 2017
Results
PPK
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. Vestibulum faucibus augue a pretium interdum. Phasellus eros felis, tincidunt eget odio a, imperdiet consequat ipsum. Duis bibendum mattis efficitur. Etiam auctor id elit at fringilla. Donec pretium, ex id pellentesque varius, risus lacus pellentesque tortor, non tincidunt eros libero vel mi. Morbi viverra tempor mattis. Curabitur mattis sed dolor in aliquet. Maecenas nec metus at nibh gravida commodo id vel dolor. Morbi commodo cursus lacus, et ullamcorper nibh lacinia in. Mauris lectus enim, sodales vel dolor eget, volutpat volutpat purus. Quisque vitae odio pellentesque, pulvinar lorem eu, congue ligula. Morbi magna sem, pharetra sed sagittis quis, aliquam sit amet purus. Aliquam eget est ut ligula facilisis gravida ut ut ante. Nullam quis venenatis nulla. Nam facilisis ex in ligula consequat posuere. Pellentesque id arcu mattis, gravida elit a, hendrerit ante. Integer mollis justo sed vestibulum vehicula.
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.
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.)
Localization Tag
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. Vestibulum faucibus augue a pretium interdum. Phasellus eros felis, tincidunt eget odio a, imperdiet consequat ipsum. Duis bibendum mattis efficitur. Etiam auctor id elit at fringilla. Donec pretium, ex id pellentesque varius, risus lacus pellentesque tortor, non tincidunt eros libero vel mi. Morbi viverra tempor mattis. Curabitur mattis sed dolor in aliquet. Maecenas nec metus at nibh gravida commodo id vel dolor. Morbi commodo cursus lacus, et ullamcorper nibh lacinia in. Mauris lectus enim, sodales vel dolor eget, volutpat volutpat purus. Quisque vitae odio pellentesque, pulvinar lorem eu, congue ligula. Morbi magna sem, pharetra sed sagittis quis, aliquam sit amet purus. Aliquam eget est ut ligula facilisis gravida ut ut ante. Nullam quis venenatis nulla. Nam facilisis ex in ligula consequat posuere. Pellentesque id arcu mattis, gravida elit a, hendrerit ante. Integer mollis justo sed vestibulum vehicula.
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.
Biobricks used in the following section are as follows:
Low promoter
Medium promoter
High promoter
Eut S
Eut SMN
EutLK+Low+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 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.
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.
