Difference between revisions of "Team:Imperial College/Proof"

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<p><b>Figure 1:</b> Picture of the different colours obtained by manually mixing different ratios of colored cells.</p>
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<p><b>Figure 1:</b> </p> Picture of the different colours obtained by manually mixing different ratios of colored cells.
 
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<br><br>Our data revealed that cells expressing different chromoproteins tended to have different growth rates, despite the fact that they all were similar in size. This aligns with the theory that co-cultures tend to fail due to one population’s growth rate exceeding that of the other. This affirms the need for a genetic circuit to stabilise co-cultures.  
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<p><br><br>Our data revealed that cells expressing different chromoproteins tended to have different growth rates, despite the fact that they all were similar in size. This aligns with the theory that co-cultures tend to fail due to one population’s growth rate exceeding that of the other. This affirms the need for a genetic circuit to stabilise co-cultures.
  
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The cells transformed with the efoRed+GP2 construct showed a decrease in growth rate when induced with arabinose, suggesting that our circuit can be a suitable system for controlling the growth of colored cells.
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The cells transformed with the efoRed+GP2 construct showed a decrease in growth rate when induced with arabinose, suggesting that our circuit can be a suitable system for controlling the growth of colored cells.</p>
 
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Revision as of 20:49, 19 October 2016

Colour proof

In order to demonstrate one of the many possible applications of our ratio control circuit, we decided to use chromoproteins as visual proof of concept. We were able to create a multitude of colours by mixing different ratios of E. coli cultures expressing the chromoproteins. We hope that this provided a glimpse of the power of co-culture in the production of composite biomaterials.
Key Achievements

Built constructs for expressing 7 different chromoproteins
Produced a construct linking GP2 expression to chromoprotein expression
Showed that Gp2 can be used to control the growth of cells and consequently the production of proteins

Overview

Chromoproteins are brightly coloured when expressed, and the colour is clearly visible to the naked eye, unlike fluorescent proteins. We want to use these proteins to produce coloured cells to demonstrate how co-cultures can be used to create varying shades of colour, providing a visual reference for the ratio of two cells. Chromoproteins are of great interest to the synthetic biology community for used as reporters and also as a new form of non-toxic biological pigments. Colours provide a simple way of demonstrating how different ratio and composition of materials can generate an array of products with different properties.

Our Approach

We hoped to work with multiple chromoproteins for our demonstration, so we selected 7 gene sequences from the 2016 iGEM distribution kit. These are: spisPink, amajLime, amilGFP, fwYellow, eforRed, gfasPurple and cjBlue. We assembled these coding sequences with an RBS part with a built-in Anderson promoter and a terminator. We next transformed these constructs into Top10 cells for characterization.

Experimental Design

We first aimed to simply demonstrate that multiple colors could be produced by mixing coloured cells. We then attempted to recreate these colors by growing cells in co-culture, as color production by co-culture is more efficient and more scalable.

We measured the growth rate of cells producing different chromoproteins by using a plate reader to measure the optical density of isolated populations over 10 hours In order to determine if different types of chromoprotein expression were influencing the growth rate differently.

We want to show that growth control can be used to produce a stable co-culture that can maintain its ratio overtime. Therefore we combined the arabinose-inducible GP2 construct with a construct for the chromoprotein eforRed. We then induced the cells with arabinose to observe the effect of GP2 on the population stability of the colored cells.



Results

In our cell mixing experiment, we were able to produce over 70 different colors from the 7 base chromoproteins, indicating that a wide range of biological colours can potentially be produced by chromoprotein mixing.


Figure 1:

Picture of the different colours obtained by manually mixing different ratios of colored cells.



Our data revealed that cells expressing different chromoproteins tended to have different growth rates, despite the fact that they all were similar in size. This aligns with the theory that co-cultures tend to fail due to one population’s growth rate exceeding that of the other. This affirms the need for a genetic circuit to stabilise co-cultures. The cells transformed with the efoRed+GP2 construct showed a decrease in growth rate when induced with arabinose, suggesting that our circuit can be a suitable system for controlling the growth of colored cells.



Growth control


Figure 2: Plot of Growth rate versus time for Escherichia coli Top 10 cells expressing various color constructs.



We picked a chromoprotein that does not effect growth as much as the others and ligated in our arabinose-inducible GP2 construct. This GP2 construct allowed us to control the growth of the coloured cells and provide a way to maintain a stable co-culture.


Figure 3: Plot of Growth rate versus time for the eforRed with growth control gp2 construct Escherichia coli Top 10 cells.