Part:BBa_K2173002
LuxI_LuxR_mrfp1(plux)
LuxI with LVA degradation tag expressed under Plux promoter and a double terminator + LuxR expressed under constitutive promoter J23100 and double terminator + mRFP1 with DAS Degradation tag expressed under PluxL promoter and double terminator Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 879
Illegal NheI site found at 902 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 721
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2514
Illegal AgeI site found at 2626 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1860
Quorum sensing plasmid of the circuit with a reporter
Basic info of LuxI and LuxR
For basic information on LuxI: Part:BBa_C0061
For basic information on LuxR: Part:BBa_C0062
mRFP1
All the basic information about the part is here.
https://parts.igem.org/Part:BBa_E1010
Design
These parts were assembled through 3A assembly. Parts were assembled on psb1C3 which is pMB1 or high copy number plasmid. Through modelling it was inferred that production of AHL should be significantly higher than that of aiiA. So as to eliminate the production factor of luxR, it is constitutively expressed using J23100 promoter.
Characterization
Individual Characterization of k2173000
Fluorescence imaging positive control and K2173001 shows the working of the mRFP1 protein.
We did the fluorescence imaging of the positive control (sfGFP under the control of the lux promoter) and BBa_K2173001 self-amplifying mRFP1 using the Nikon Eclipse T-1 S fluorescence microscope. The positive control showed a high level of fluorescence in both the cases, with similar high fluorescence levels for both mRFP1 and sfGFP, confirming that the mRFP1 protein, as well as the LuxI and LuxR quorum sensing system that we cloned. All images were taken on the Nikon Eclipse Ti-S fluorescence microscope under 10X and 20X magnification.
Characterization in conjugation with K2173000
Fluorimetry
1. Fluorimetry in bulk culture shows oscillatory behavior and characteristic burst
We characterized the circuit using the spectramax M2e spectrofluorometer, using the protocol described in the characterisation section of the project tab. Samples taken every 10 minutes were checked for fluorescence by excitation at 585 nm and emission at 604 nm was recorded. The results were plotted in R in a 3-D plot for the fluorescence at different OD (cell concentration) and time values. We then ran a support vector regression algorithm to find the values of fluorescence at a constant OD value. We chose to plot the fluorescence at the root mean square OD value, in order to best account for and normalize the results for variations in the OD value throughout the experiment.
We were able to capture the oscillatory behavior of the circuit in a time course of ~150 minutes. Plotted against the negative and positive control (figure 1), we see our iDanino circuit showing a sudden burst of fluorescence at a time of around 150 minutes, which is similar to what has been reported by Danino et al (nature, 2011). This shows that our system seems to be in an oscillatory behavior in bulk, indicating the working of the circuit as expected.
Fig.1
2. Characterizing the spike in the oscillations of the iDanino circuit
The spike in the RFU levels of the circuit is apparent from the figure. We further calculated the max/min ratio and max – min values of all the three samples, which is indicative of the gain in amplitude of the values (table 1). We see that the highest value of max/min ratio (relative gain) and max – min (absolute gain) values obtained are for the iDanino circuit, indicating a solid gain in oscillations.
3. Results of the fluorimetry of iDanino circuit is in accordance with the Danino circuit fluorimetry values
We ordered the plasmids pTD103_aiiA and pTD103_LuxI_sfGFP, constructed originally by Tal Danino, from addgene, and ran the same experiment for these plasmids, which we used as a further proof of the working of our circuit (since the pTD plasmids were confirmed to be working). Running the fluorimetry experiment for the pTD plasmid containing cultures (positive control, negative control, and the complete circuit), we plotted fluorescence at the root mean square value of cell concentration after support vector regression based curve fitting. We saw a similar spike in the OD values in this case as we saw for our own iDanino cultures, indicating that our oscillator worked as expected.
Microfluidics and Microscopy
1. Microfluidic chamber construction
We constructed microfluidic chambers of different channel sizes and dimensions, by laser etching on acrylic sheets using the Epilog LASER FUSION M2™ laser etching machine. The chambers were designed on Corel Draw X6 and fed to the machine, which then etched the channel out. Through holes and the outer rectangular cut was also made using the same machine. High resolution microscopy shows the etch marks when focused on the channels (figure 2). These marks were, however, found to not be inhibitory to the imaging of the cells in the channel in any way, and we were able to take images of the cells in a satisfactory manner.
2. Microfluidic testing shows synchronized oscillations in the iDanino circuit, cultured at 37oC
In the microfluidic device, E. coli cells were loaded from the cell port while keeping the media port at sufficiently higher pressure than the waste port below to prevent contamination. Cells were loaded into the cell traps by manually applying pressure pulses to the lines to induce a momentary flow change. The flow was then reversed and allowed for cells to receive fresh media with 0.075% Tween20 which prevented cells from adhering to the main channels and waste ports. The images of the cell trap in the center were taken over different time points (typically images were taken every 7-10 minutes), and the mean fluorescence of each of the images was analyzed and plotted, which shows the oscillatory trend as predicted by Danino et al. (figure 3).
Figure-3 The images were analyzed using ImageJ. Within each image, to test synchronization, we picked up 100 overlapping squares of similar dimensions, and measured the mean fluorescence within each square, and then took the average and variance from all of these squares combined.
i) The values of each individual square was very close to the moving average of all the squares combined.
ii) Variance vs time showed that there variance over time across the cells changed very little indicating that all the cells were fluorescing in synchrony. Null hypothesis stated was that cells were not oscillating in synchrony. High variance would have justified null hypothesis.
3. Image Analysis results confirm synchronization
Image analysis using ImageJ showed a very small value of variance as compared to the mean oscillation value, which confirmed that the oscillations among the cells were synchronized, i.e. all the cells were in the same/similar phases at every time point. The results are shown in the graph 3.(ii)
4. Switching of temperature of the iDanino circuit causes oscillations to stop and toggles to a 0 or 1 state, depending on the reporter type used.
Cultures of the iDanino system were grown at 37oC until it reached a cell concentration (O.D.600) value of ~1, following which the temperature was switched to 30oC. Samples from this culture were taken at different times, and fluorescence microscopy for these samples was done at 10X and 20X magnification. We saw the following trends in the images:
Figure 4 :Part – Bba_K2173002 -If the reporter (mRFP1) was placed downstream of a Pluxλ promoter , when the temperature was brought down to 30oC, the oscillations stopped and an increase in the level of mRFP1 was seen, until a final saturation level was reached. This corresponds to a constant “ON” state in the toggle switch
Hence, these results show that we have successfully designed and tested the iDanino in such a manner that it has a temperature dependent switch from a natural oscillator to a bistable toggle switch.
Modelling
Equations involved in the iDanino circuit
As we change the temperature from 37 degree celsius to 30 degree celsius at time 200 minutes, the lambda repressor starts repressing AiiA, thus bringing its concentration down to zero. Consequently, the concentration of LuxI starts increasing uninhibited by AiiA.
Verification of Part
- We did agarose gel electrophoresis of the undigested plasmid, singly-digested plasmid and the doubly-digested plasmids.
- For further confirmation, we got the part sequenced from Thermo Fisher Scientific.
Gel run of singly-digested plasmid
References:
Danino, Tal, et al. "A synchronized quorum of genetic clocks." Nature463.7279 (2010): 326-330. Waters, C., and Bassler, B., 2005: Quorum sensing: cell-to-cell communication in bacteria.
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