Part:BBa_K2173000

aiiA with degradation tag expressed under Plux-lambda hybrid promoter and a double terminator + mRFP

aiiA with degradation tag expressed under Plux-lambda hybrid promoter and a double terminator + mRFP1 with degradation tag under Plux-lambda promoter and a double terminator + heat sensitive C1-lambda under constitutive promoter and a double terminator

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2011
  Illegal NheI site found at 2034
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 196
  Illegal AgeI site found at 1708
  Illegal AgeI site found at 1820
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1054
  

Part K2173000

aiiA with degradation tag expressed under Plux-lambda hybrid promoter and a double terminator + mRFP1 with degradation tag under Plux-lambda promoter and a double terminator + heat sensitive C1-lambda under constitutive promoter and a double terminator.

aiiA

autoinducer inactivation enzyme from Bacillus; hydrolyzes acetyl homoserine lactone(AHL)

Coding region for the autoinducer inactivation enzyme A(aiiA) LVA tagged(for untagged version see Part:BBa_C0160). The gene was originally isolated from Bacillus sp. 240B1 and it encodes an enzyme that catalyzes the degradation of N-acyl-homoserine lactones (AHLs)--quorum sensing autoinducers.

Protein structure of aiiA (Source:RCSB-PDB)

Usage and Biology

The aiiA gene has 750 nucleotides and encodes a protein of 250aa with a predicted molecular mass of 28,036Da and an isoelectric point of 4.7. The protein has no hydrophobic signal pepetide at the N-terminus and therefore it is believe that it is not secreted. This is supported by the observation that when aiiA is expressed in E.coli DH5alpha or Bacillus 240B1 cells no autoinducer inactivation is detected in the supernatants of these cultures. The AiiA protein has no significant overall homology with other known proteins, but based on the presence of two well-conserved motifs it is believed that it is a metalloenzyme.

Figure : Time course of AI inactivation by purified AiiA protein. The purified AiiA protein was diluted to a concentration of 50 ng/μl with 1/15 M phosphate buffer (pH 8.0). Equal volume of diluted AiiA protein and 40 μM OHHL (⧫), ODHL (■), or OOHL (▴) was added in a 1.5-ml Eppendorf centrifuge tube. The reaction was conducted at 28°C. The same concentration of the denatured AiiA (boiling for 5 min) and OHHL in the phosphate buffer was used as control (●). Samples were taken at 10-min intervals for a total of 60 min, and the reaction was stopped by boiling for 3 min. The samples were assayed for AI activity as described

mRFP1
All the basic information about the part is here.
https://parts.igem.org/Part:BBa_E1010

Characterization

Unlike as reported by team UNIPV-Pavia iGEM 2011, even when aiiA was put under low copy plasmid there was considerable degradation of AHL. We found that degradation rates were sufficient enough to drive the oscillations.

Plasmid constructed by Danino et al[5] have included aiiA on low copy number plasmid for stable oscillations. Through modelling and then simulation of its results, we understood that if aiiA production rate was same as that of AHL, oscillations were not possible. It was important part of the design to put luxI gene on psb1C3 and aiiA on psb3T5.

Individual Characterization of k2173000

1. We did the fluorescence imaging of the positive control (self-amplifying mRFP1-K2173002/sfGFP under the control of the lux promoter) and the negative control (K2173000) for both the Danino oscillator and the iDanino oscillator 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. The negative control showed negligible fluorescence (figure 3). All images were taken on the Nikon Eclipse Ti-S fluorescence microscope under 10X and 20X magnification. ***
   
   
   
   Fluorimetry
   1. Fluorimetry in bulk culture shows oscillatory behavior and characteristic burst

   We characterized the circuit using the spectramax M2 e 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.

   
   
   
   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), plotting 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 37^oC

   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 4).

   The images were analyzed using ImageJ. Within each image, to test synchronization, we picked up 6-10 sample squares of similar dimensions, and measured the mean fluorescence within each square, and then took the average and standard deviation and variance from all of these squares combined. The values of each individual square was very close to the moving average of all the squares combined.

   
   Figure – Oscillations observed in the microfluidic chamber. Cells were pumped into the device first by loading a culture onto the cell loading port by applying a positive pressure at the port and blocking the waste port directly below it. Cells were diluted such that the density (O.D. 600) values did not exceed a certain threshold, and thus produced oscillations. Images were taken at 6 different times and visually, we see that there are oscillations of mRFP1 levels. Images were also analyzed using ImageJ to confirm synchronization following this.

   i)
   
   ii)
   

   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, ie all the cells were in the same/similar phases at every time point. The results are shown in table 2.

   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 37^oC until it reached a cell concentration (O.D.₆₀₀) value of ~1, following which the temperature was switched to 30^oC. Samples from this culture were taken at different times, and fluorescence microscopy for these samples was done at 10X and 20X magnification (figure 5). We saw the following trends in the images:

   a. If the reporter (mRFP1) was placed downstream of a P_luxpromoter, when the temperature was brought down to 30^oC, 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.

   b. If the reporter (mRFP1) was placed downstream of a P_lux-ʎ hybrid promoter, when the temperature was brought down to 30^oC, the oscillations stopped similar to case (a) and an decrease in the level of mRFP1 was seen, until a near zero level of protein n expression was reached. This corresponds to a constant “OFF” 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 formulated

   
   

   Verification of Part

   1. We did agarose gel electrophoresis of the undigested plasmid, singly-digested plasmid and the doubly-digested plasmids.
   
   
   Gel run of singly-digested plasmid
   
   2. For further confirmation, we got the part sequenced from Thermo Fisher Scientific.
   
   
   

   References:

   • Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia
   • carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000).
   • Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24.