Line 154: | Line 154: | ||
<br><br>The results indicate that the species within this module reach steady states at time points unique to that species. | <br><br>The results indicate that the species within this module reach steady states at time points unique to that species. | ||
<center> | <center> | ||
− | <br> <img src='https://static.igem.org/mediawiki/2016/d/d2/T--Imperial_College--C4.png'> <p><br> <b> Figure 1:</b> Production of C4 AHLs and C14 AHLs against time <br><br></p> | + | <br> <img src='https://static.igem.org/mediawiki/2016/d/d2/T--Imperial_College--C4.png'> <p><br> <b> Figure 1: </b> Production of C4 AHLs and C14 AHLs against time <br><br></p> |
</center> | </center> | ||
<center> | <center> | ||
− | <br> <img src=' | + | <br> <img src='https://static.igem.org/mediawiki/2016/8/8b/T--Imperial_College--C4RvsC14R.png'> <p><br> <b> Figure 2: </b> Production of C4R and C14R regulatory proteins against time <br><br></p> |
</center> | </center> | ||
<center> | <center> | ||
− | <br> <img src='https://static.igem.org/mediawiki/2016/f/fe/T--Imperial_College--C4ComplexvsC14Complex.png'> <p><br> <b> Figure 3:</b>C4:C4R and C14:C14R complex formation against time<br><br></p> | + | <br> <img src='https://static.igem.org/mediawiki/2016/f/fe/T--Imperial_College--C4ComplexvsC14Complex.png'> <p><br> <b> Figure 3: </b>C4:C4R and C14:C14R complex formation against time<br><br></p> |
</center> | </center> | ||
</p> | </p> | ||
Line 187: | Line 187: | ||
<p><br>Using STAR (Short Transcription Activating RNA) technology, we were able to develop a novel method of comparing the sizes of two populations from their quorum signal concentrations. | <p><br>Using STAR (Short Transcription Activating RNA) technology, we were able to develop a novel method of comparing the sizes of two populations from their quorum signal concentrations. | ||
− | <br><br>We used RNAstruct developed by | + | <br><br>We used RNAstruct developed by Matthews Lab to help aid the development of the ANTISTAR. This software allowed us to determine the secondary structure and free energy to optimize the way in which our ANTISTAR sequence was designed. This was done so that our ANTISTAR sequence would have as high an affinity to the STAR sequence as was possible. |
<center> | <center> | ||
Line 223: | Line 223: | ||
\[STAR\rightarrow\varnothing\] | \[STAR\rightarrow\varnothing\] | ||
− | \[antiSTAR | + | \[antiSTAR\rightarrow\varnothing\] |
− | \[STAR_{complex | + | \[STAR_{complex}\rightarrow\varnothing\] |
− | <p><br><br>Simulation Results:<br> | + | <p><br><br><specialh3>Simulation Results:<br></specialh3> |
+ | <p> | ||
Induced production of STAR and ANTISTAR was shown to be much higher than their basal production. | Induced production of STAR and ANTISTAR was shown to be much higher than their basal production. | ||
+ | </p> | ||
<center> | <center> |
Revision as of 00:38, 20 October 2016
<!DOCTYPE html>
The first stage of our modelling process was to construct a single cell in silico model of our circuit. Our model was built using mass action kinetics in Simbiology (Matlab toolbox) and built up reaction by reaction.
We first separated the models into 3 modules: Quorum Communication, STAR-antiSTAR Comparator and Growth Regulation.
We constructed the four quorum systems that we considered viable choices for our system (cin, rhl, lux and las) to allow us to directly compare the expected behaviour and plan our growth module experiments accordingly. We designed the overall model for the Rhl and Cin systems (Chen et al., 2015) as they have been previously shown to operate with minimal crosstalk.
We used numbers obtained from Chen et al for C4 and C14 production. This is a high level production term that ignores parts of the central dogma. We made this assumption due to the limited data on the enzymatic kinetics of the autoinducer synthases.
The Rhl system consists of an autoinducer synthase RhlI (that produces C4 AHL) and a transcriptional regulator RhlR that dimerises to activate a pRhl promoter which activates STAR transcription.
The Cin system consists of an autoinducer synthase CinI (that produces C14 AHL) and a transcriptional regulator CinR that dimerizes to activate a pCin promoter which induces Anti-STAR production.
Communication module:
The results indicate that the species within this module reach steady states at time points unique to that species.
Figure 1: Production of C4 AHLs and C14 AHLs against time
Figure 2: Production of C4R and C14R regulatory proteins against time
Figure 3: C4:C4R and C14:C14R complex formation against time
Using STAR (Short Transcription Activating RNA) technology, we were able to develop a novel method of comparing the sizes of two populations from their quorum signal concentrations.
We used RNAstruct developed by Matthews Lab to help aid the development of the ANTISTAR. This software allowed us to determine the secondary structure and free energy to optimize the way in which our ANTISTAR sequence was designed. This was done so that our ANTISTAR sequence would have as high an affinity to the STAR sequence as was possible.
Figure 4:Secondary structure of our ANTISTAR sequence
To calculate the kinetics of the RNA interactions that occur within this module we adapted a method developed by Eric Winfree known as DNA strand displacement kinetics (Zhang and Winfree, 2009).
Comparator module:
Induced production of STAR and ANTISTAR was shown to be much higher than their basal production.
Figure 5:Basal vs Induced STAR production
Figure 6:Basal vs induced AntiSTAR production
Figure 7:STAR:AntiSTAR Complex formation
Figure 8:STAR:STAR Target Complex formation
TEXT GOES HERE
Works Cited
Chen, Y., Kim, J., Hirning, A., Josi, K. and Bennett, M. (2015). Emergent genetic oscillations in a synthetic microbial consortium. Science, 349(6251), pp.986-989.
Zhang, D. and Winfree, E. (2009). Control of DNA Strand Displacement Kinetics Using Toehold Exchange. J. Am. Chem. Soc., 131(47), pp.17303-17314.