Part:BBa_K4491132
Autoregulatory Negative feedback circuit
Negative feedback is a type of circuit design motif that exists in nature to improve system response. The purpose of the experiment is to characterise the autonegative feedback circuit by seeing how it adapts to constant disturbance as a perturbation. Using the mother machine, a microfluidic device, we are able to control the medium the cells are cultured in. We will image the cells with time-lapse microscopy at 1 µM Vanilic acid for 3 hours, making sure that the steady state value is reached. We then perturb the system by switching the culture medium to 10 µM vanillic acid for another 3 hours to see how the system adapts.
Contents
| Autoregulatory negative feedback circuit with VanR | |
|---|---|
| Function | Autoregulatory negative feedback |
| Use in | Bacteria |
| Chassis Tested | E. coli |
| Abstraction Hierarchy | Device |
| Parts of the composite part | p_vanCC, B0032, VanR^AM, DT5 |
| RFC standard | RFC10 & RFC23 & RFC25 & RFC1000 compatible |
| Backbone | pJUMP27-1A |
| Submitted by | Cambridge iGEM 2022 |
Design
We have designed an autonegative feedback loop (nFc) to study the effect of adaptation. An autoregulatory negative feedback refers to a motif that involves a species feeding back to downregulate itself. The feedback species we have chosen is VanR and the promoter we have chosen is P_VanCC, the effect of VanR on P_VanCC at different concentration of Vanillic acid pf inducer is well characterised by Meyer et al., (2019). Essentially, VanR will act on P_VanCC, repressing its production, following a Hill Function dynamics. In order to characterise the amount of vanR, we have created an operon (MegaT (BBa_K4491009) with a RBS, mVenus and Terminator after VanR coding sequence (CDS).
Build
We perform JUMP assembly to clone the transcription unit into pJUMP27-1A using the enzyme BsaI to clone the transcription unit into the main cloning site. For the protocol we used for JUMP assembly, please refer our wiki page.
Characterisation
To test the effect of the negative feedback circuit in response to perturbation, we have decided to conduct the experiment in a mother machine.
The following table shows the strain of cells and culture conditions overnight we have used for the characterisation of the negative feedback circuit. The test strain we have used is called SB7, a strain with mCherry integrated into chromosomal DNA. We have used E. coli SB6, a strain with mVenus integrated into the chromosomal DNA as a positive control and E. coli SB5, a strain with CFP integrated into the chromosomal DNA as a positive control.
As we need to visualise the cells and the mother machine under the microscope (Nikon TI2 Eclipse), we have to have appropriate microscope settings to image the cells. We are using the Nikon TI2 Eclipse microscope with phase contrast, mCherry, YFP and CFP and take images along the trenches.
We have prepared 2 media which was put into the mother machine in the first 3 hours and last 3 hours of the experiment respectively. The first media we have made consists of EZRDM, pluronic and 1μM Vanillic acid. The second media we have made consists of EZRDM, pluronic and 10μM Vanillic acid. The explanation of the choice of the media is explained below.
Explanation
With 1μM vanillic acid added, vanR produced represses on p_vanCC, inhibiting mVenus production. After the circuit reaches its steady state, we introduce the perturbation as 10μM vanillic acid. As this is a negative feedback loop but not robust perfect adaptation, 10μM of vanillic acid should act to inhibit the repression of vanR on p_vanCC so the signal of mVenus is expected to increase. The concentration of vanillic acid chosen for the experiment is based on the characterisation from Meyer et al., (2019). To allow the system to have a large dynamic range, we have chosen concentrations of vanillic acid that lie on the lower end and the higher end of the slope of the Hill curve. As we are interested in a positive perturbation, we started the system off with a vanillic acid concentration of 1μM then use 10μM vanillic acid as a perturbation.
Through looking at the data from the microscope as a quick analysis, we have realised that only the cells in the middle of the field of view survived but those on the side are all dead at the end of the experiment. Moreover, this happens consistently in all the field of views we have captured. We proceed on looking at the time-lapse images and realise that the cells on the periphery of the field of views are slowly dying out as the experiment proceeds. As the experiment requires using high energy photons to detect CFP emission from SB6 strain, we suspect that this effect we saw from such quick-analysis is due to the effect of phototoxicity. As the different field of views are close together and the area that the excitation light shines on is wider than the field of view itself, the cells on the periphery are excited for more than once during every cycle of taking images, leading to their death. As we are interested in comparing the mean intensities of mVenus expression between 0µM and 100µM Vanillic acid, we perform a t-test between the 2 samples and confirm that the 2 means are statistically significantly different from each other.
As the cells in the middle of the field of view are largely unaffected, we looked at the time-lapse images to see whether we see the effect we expect after the addition of vanillic acid. As we are going through the images, we see that the cells increases in intensity of mVenus as expected. However, towards the end of the experiment, we see signs that the cells are “struggling” and getting dimmer, probably also due to the phototoxicity brought about by the UV light that excites CFP.
To show that the fluorescent intensities of cells in the presence of perturbation of vanillic acid, we plotted a time series data of the cells that survived the imaging. However, as it is evident that some cells are dying as the imaging goes on and lost their fluorescent, we have limited the time range we analyse the time series data. The following figure shows the change in intensity of 3 cells as an example to see how the intensity of the cells have increased with the change of medium from 1µM of vanillic acid to 10µM of vanillic acid.
To visualise the effect of vanillic acid on the fluorescent intensity, the distribution of fold change of fluorescent intensites of all the cells is plotted below. The fold change is calculated by the ratio of the intensity of the end point and the intensity of the starting point. Apart from the fold change, the halfway point, the time point where half of the maximum intensity is reached, is also being plotted out as a distribution to look at the response time of the circuit.
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