Difference between revisions of "Team:IIT Delhi/Oscillations"

 
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       <a href="/Team:IIT_Delhi/Circuit_Design">Circuit design and construction</a>
 
       <a href="/Team:IIT_Delhi/Circuit_Design">Circuit design and construction</a>
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       <a href="/Team:IIT_Delhi/Microfluidics">Microfluidics and Fluroscence</a>
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       <a href="/Team:IIT_Delhi/Microfluidics">Microfluidics and Fluorescence</a>
 
       <a href="/Team:IIT_Delhi/Photobleaching">Photobleaching</a>
 
       <a href="/Team:IIT_Delhi/Photobleaching">Photobleaching</a>
 
       <a href="/Team:IIT_Delhi/Promoter">Promoter strength</a>
 
       <a href="/Team:IIT_Delhi/Promoter">Promoter strength</a>
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             <h2 class="h2font">Characterization of Promoter Strengths</h2>
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             <h2 class="h2font">Oscillations: Sinusoids and Squares</h2>
  
 
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<h2 id="pfont">Before going on to the oscillations, we wanted to characterize the promoter strengths of our newly submitted promoters, since a proper characterization for these parts did not exist. Therefore, we set up experiments in a plate reader, for the 5 promoters used in the Murray et al 5n1 system, of which 2 were also a part of the square wave generating oscillator. <br>
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<h2 id="pfont">Having constructed our square wave circuit via cloning and synthesis, and having obtained Murray et al’s 5n1 plasmid (from addgene), we then tested out our system in microfluidic chambers, and obtained time lapse images, placed under a fluorescence microscope.<br><br>
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Parts containing the fluorescent GFP reporter (BBa_E0040) under the 5 repressible promoters (pLac, pTet, pCI, pPhlF, pSrpR) were cloned and created, and grown overnight in LB. The cultures were then diluted 1:50 and 1:200 in LB, and put into a plate reader, with 4 replicates. Growth was observed via absorbance at 600 nm, and fluorescence was observed by excitation at 485 nm and emission at 511 nm.
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Cells containing the square wave circuit were first grown overnight in LB medium containing 100 ug/ml of antibiotic (ampicillin). The next day, the culture was diluted 1:100 in LB containing the appropriate concentration of ampicillin, and loaded into the microfluidic chamber (refer to microfluidic section for design specifications of the chamber). Cells caught at one end of the T junction were imaged, and a single cell among them was isolated. Flow rate ensured that as the cell divided, the daughter cells flowed out along with the fresh media flowing past the straight part of the channel, without taking the cells in the perpendicular part of the channel along with it.<br><br>
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Cells were also diluted, and induced by adding a minimal concentration of anhydrotetracycline (aTc < 0.1 nM). This barely induced the system, and set the oscillations into motion. The cells were loaded into 96 well plates with 4 replicates for each part, and the time evolution of GFP was seen. The data showed a good correlation with a square wave of the same time period imposed on it (shown in red in the graph). <br><br>
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We see in Fig. 1 that the decreasing order of strengths for the five repressible promoters is pLac, pcI, pPhlF, pTet and pSrpR. This is also evident in Fig. 2 when all the 5 promoters settle to a steady state. Fig. 3 depicts the protein production rates for the promoters.  pLac has the largest production rate reaffirming its highest strength. However, despite dominating completely in terms of protein production rate, we see in Figs. 2 and 4 that in the initial transient phase, pLac has lower relative strength compared to pTet and pcI. This is due to the highest dilution rate for pLac in this regime, which can be seen in Figs. 5 and 6. Similar arguments can be made about pcI.
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<img src = "https://static.igem.org/mediawiki/2017/1/19/T--IIT_Delhi--Results_Oscillations_Picture-3.png" style='border:3px solid #000000' width =95%><br>  
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<h6>Figure – Plate reader data showing square wave oscillations when a culture of the square wave generator was induced using a minimal concentration of aTc. One single time period of the square wave was captured.</h6>
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<h6>Fig 1. Relative Strength for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The decreasing order of relative strength is pLac>pcI>pPhlF>pTet>pSrpR. Rlative strengths have been averaged over 4 trials. </h6>
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<h2 id = "pfont"> The oscillations obtained in the microfluidic chamber are shown below – <br><br>
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<img src = "https://static.igem.org/mediawiki/2017/3/38/T--IIT_Delhi--Promoter_Strength_pic-2.png" style='border:3px solid #000000'>
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<h6>Fig 2. Relative Strength over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.</h6>
 
 
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Finally, the system was operated at various temperatures, to study the temperature robustness. No noticeable change in the qualitative nature of the oscillations was seen. Also, growth curves at three different temperatures were plotted, and not much difference in the growth rates was seen. <br><br>
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<h6>Fig 3. Protein production rate over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.</h6>
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<h6>Fig 4. Relative Strength versus OD over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.</h6>
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<h6>Fig 5. Dilution rate over time for the five repressible promoters, including the novel promoterspPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.</h6>
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<h6>Fig 6. OD over time for the five repressible promoters, including the novel promoters pPhlF and pSrpR, used in the construction and analysis of 5n1. The plots have been averaged over 4 trials.</h6>
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As shown in Fig. 2, pLac and pcI have three distinct regions of behavior in terms of relative strength over time. In the initial transient phase, dilution rate is higher than the protein production rate, thus we see that relative strength per OD decreases over time. The middle corresponds to overcompensation phase, where protein production rate surpasses the dilution rate. Finally, the relative strength settles a steady state value, entering an exact compensation between protein production and dilution rates. Contrary to pLac and pcI, the other promoters don’t exhibit the overcompensation phase.
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Latest revision as of 22:19, 1 November 2017

iGEM IIT Delhi

Overview Squarewave Generator Interlab
Circuit design and construction Microfluidics and Fluorescence Photobleaching Promoter strength Oscillations
Basic Parts Composite Parts Improved Parts Part Collection
Overview Writing a Model Deterministic Model Stochastic Model Bifurcation and Squareness Resource sharing
Human Practices Integrated Practices Public Engagement Collaborations Safety
Overview GMM Legislation iGEM Berlin Mentoring IISER Mohali iGEM Glasgow
Lab Records Protocols Recipes Medal Criteria
The Team Attributions

Oscillations: Sinusoids and Squares

                                                                                                                                                                                                                 

Having constructed our square wave circuit via cloning and synthesis, and having obtained Murray et al’s 5n1 plasmid (from addgene), we then tested out our system in microfluidic chambers, and obtained time lapse images, placed under a fluorescence microscope.

Cells containing the square wave circuit were first grown overnight in LB medium containing 100 ug/ml of antibiotic (ampicillin). The next day, the culture was diluted 1:100 in LB containing the appropriate concentration of ampicillin, and loaded into the microfluidic chamber (refer to microfluidic section for design specifications of the chamber). Cells caught at one end of the T junction were imaged, and a single cell among them was isolated. Flow rate ensured that as the cell divided, the daughter cells flowed out along with the fresh media flowing past the straight part of the channel, without taking the cells in the perpendicular part of the channel along with it.

Cells were also diluted, and induced by adding a minimal concentration of anhydrotetracycline (aTc < 0.1 nM). This barely induced the system, and set the oscillations into motion. The cells were loaded into 96 well plates with 4 replicates for each part, and the time evolution of GFP was seen. The data showed a good correlation with a square wave of the same time period imposed on it (shown in red in the graph).


Figure – Plate reader data showing square wave oscillations when a culture of the square wave generator was induced using a minimal concentration of aTc. One single time period of the square wave was captured.

The oscillations obtained in the microfluidic chamber are shown below –



Finally, the system was operated at various temperatures, to study the temperature robustness. No noticeable change in the qualitative nature of the oscillations was seen. Also, growth curves at three different temperatures were plotted, and not much difference in the growth rates was seen.



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