Detection of AHL

Gate 1 of the alpha system should produce AHL molecules when aTc is present in the bacteria environment. In order to check if our gate works as expected we collaborated with BGU_Israel iGEM team.
The BGU team conducted an AHL detection experiment (Marks R.S.and Kushmaro A., 2011) to help us test the gate.

Positive control:
For a positive control the indicator strain, CV026, was incubated with a synthetic AHL (3-OXO-C6). When this strain senses AHL it changes color to purple. A plate was covered with soft agar containing the indicator strain, and then synthetic AHL was added. After incubation the plates were purple as a response for the high levels of the AHL.



Picture 1. Positive control- soft agar containing the detector strain CV026 after incubation with synthetic AHL


TOP10 bacteria containing gate 1
The TOP10 bacteria containing Gate 1 were spread on LB agar plate and after half an hour the plate was covered by soft agar containing the indicator strain CV026. After incubation, a purple color appeared on the plates.
Although aTc wasn't added to the bacteria, the leakiness of the Ptet promoter enabled sufficient expression of AHL. The results means gate 1 was assembled correctly and functions as expected.



Picture 2. TOP10 bacteria with gate 1 on LB agar plate which was covered by a soft agar containing the indicator strain



In a different assay a soft agar with the indicator strain was spread on the plates. 100µl of the supernatant from the TOP10 growth medium were spread on top of the soft agar. After incubation, a color change didn't appear. This time the low expression of AHL caused by the leakiness of the Ptet promoter wasn't sufficient in the supernatant in order to change the color of the indicator strain. The reason for the results is that the induction of gate 1 by aTc was missing.



Picture 3. 100µl of the supernatant from the TOP10 containing gate 1 growth medium on soft agar with the indicator strain

Characterization of Gate 2 of the alpha system

Objective

The goal is to characterize the second gate (Plac-LuxR) of the alpha system, using a reporter gene (GFP) under the Plux promoter and varying concentrations of AHL.

Description

In this experiment, varying concentrations of 3o6c-AHL (3-oxohexanoyl-homoserine lactone) are to be added to a culture of E. coli K-12 Top 10 containing two different plasmids: pSB1C3-Plac-LuxR and pSB1A2-Plux-GFP.
When adding the inducer IPTG to the bacteria we expect to get the protein LuxR in excess and by adding AHL to the bacteria, the complex LuxR-AHL supposed to form. If the complex forms properly it will bind to the Plux promoter and GFP will be expressed. We expect higher GFP fluorescence when higher AHL concentration is administered, until the system will be saturated.
The GFP fluorescence of the bacteria (emission peak: 511nm) can be measured using a plate-reader.
To test the leakiness of the Plac promoter, we used the same bacteria without IPTG induction.

for full protocol follow the link

Results

As can be seen from the graph, there was a rise in the GFP fluorescence according to the rise in AHL concentrations until 0.1 µM AHL. At higher concentrations of AHL, saturation in the GFP expression can be seen.
The experiment results showed that a relative high GFP fluorescence was received without IPTG induction, an indication for the leakiness of the Plac promoter. A higher GFP expression was seen with the IPTG induction, as expected.

Future work

Although the results of the experiment were successful, there are points need to be checked in order to improve the gate and the whole system function:
1. Checking gate 2 in combination with gate 1 of the alpha system. In this case, gate 1 will hopefully be the AHL source and external AHL source won't be needed.
2. Trying to change the promoter of gate 2 to a less leaky one.

Testing Biobrick BBa_M30011 (ompR controlled mRFP)

Status: Success

For full protocol click the follwing link

We used the biobrock BBa_M30011 (ompR controlled mRFP) to test the Taz construct (BBa_K1343016). Since the page in the parts registry had no record of any experience with the part, we decided to test it.
Two isogenic strains of E. coli K12, BW25113 (parent strain from the Keio collection) and JW3367-3 (with ΔEnvZ mutation) were transformed with pSB1C3 carrying the BBa_M30011 reporter. The bacteria were cultured in growth media containing varying concentrations of NaCl. After two hours of growth the relative RFP fluorescence of the cultures was determined (fluorescence/OD).
As a positive control we used E. coli Top10 transformed with biobrick BBa_J04450 (RFP under Plac).
OD was measured at 600nm.
Fluorescence excitation wavelength: 560nm
Fluorescence emission wavelength: 612nm

Figure 1: Relative fluorescence dependent on NaCl concentration (mM)

We expected that the mutant strain (ΔEnvZ) would show a constant level of relative fluorescence which is lower than that of the parent strain. This is because histidine kinase protein which detects osmolarity changes in the cells environment (EnvZ) is not present in the mutant. The EnvZ does not phosphorylate the ompR. The low level of fluorescence could be due to another mechanism (such as an acetyl phosphate dependent mechanism) which phosphorylates the ompR, leading to activation of the PompC promoter. In Figure 1 we see that the mutant showed the expected constant low level of relative fluorescence.
Since the parent strain (+EnvZ) is sensitive to osmolarity changes in the cell’s environment, we expected that an increase in NaCl concentration would cause an increase in relative fluorescence. This is because at high osmolarity, more ompR is phosphorylated, leading to increased activation of the PompC promoter. However, the parent strain also showed a constant (but high) level of expression (see Figure 1). We repeated the experiment three times with different ranges and dilutions of NaCl concentration but all showed a similar result.



Figure 2: (A) E. coli BW25113 with no plasmid. (B) E. coli BW25113 containing ompR controlled mRFP (BBa_M30011) on pSB1C3. (C) E. coli JW3367-3 (ΔEnvZ) containing ompR controlled mRFP (BBa_M30011) on pSB1C3. (D) E. coli JW3367-3 (ΔEnvZ) with no plasmid.

Taz Results

Status: Success

For full protocol click on the following link

To test the activity of the Taz construct we created, varying concentrations of L-aspartatic acid were added to a culture of E. coli expressing Taz construct on plasmid pSB1AK3 and RFP under the promoter PompC (Bba_M30011) on plasmid pSB1C3.

Two isogenic strains of E. coli K-12, BW25113 (parent strain for the Keio collection ) and JW3367-3 (with ΔEnvZ mutation) were transformed with pSB1AK3 carrying our Taz construct (BBa_K1343016) and pSB1C3 carrying the BBa_M30011 reporter. The bacteria were cultured in growth media containing varying concentrations of L-aspartic acid. After two hours of growth the relative RFP fluorescence of the cultures was determined (fluorescence/OD).

The strains used were given to us by Lior Zelcbuch, Elad Hertz from Ron Milo’s lab at the Weizmann Institute of Science.
OD was measured at 600nm.
Fluorescence excitation wavelength: 560nm
Fluorescence emission wavelength: 612nm

The goal was to compare the expression in the wild type and in the ΔEnvZ mutant. We expected that in the wild type the expression will be greater than in the mutant since the natural EnvZ/ompR system will cause expression of the RFP.


Figure 2: Relative fluorescence dependent on L-aspartic acid concentration (mM)

Figure 2 shows that at low concentrations of L-aspartatic acid (below 1mM), there is a steady, constant expression of the reporter in both the wild type and the mutant strain. The relative fluorescence observed by the mutant strain (ΔEnvZ) is lower than that of the parent strain.
The mutant shows an increase in relative fluorescence is observed at 1.33mM L-aspartic acid, with a peak at 2mM followed by a drop in relative fluorescence at 4mM. The drop in relative fluorescence in both the mutant and wild type indicates a toxic concentration level.
In the mutant strain, the relative fluorescence increases 202% in comparison to the basal level. This occurs over a narrow concentration range, which reflects the sensitivity of the two-component signaling system. This sensitivity is critical for our system to be able to function as a low-concentration detector.

Pcat_luxR_Plux_mCherry_luxI

Determine the quantitative expression of mcherry under the promoter Plux with and without Vibrio fischeri AHL synthase(LuxI)

Objective

We aspire to determine the kinetics of the promoter Plux (using the reporter gene mcherry), and whether or not the presence of the gene luxI enhances its signal.

Description

In this experiment, varying concentrations of 3o6c-AHL (3-oxohexanoyl-homoserine lactone) are to be added to a culture of E. coli K-12 Top 10, expressing mcherry, a red fluorescent protein, under the promoter Plux. The bacteria were engineered to contain a plasmid with the gate Pcat-luxR-Plux-mcherry-LuxI.
In this experiment we will compere Pcat-luxR-Plux-mcherry-LuxI and Pcat-luxR-Plux-mcherry in order to determine whether or not the presence of AHL synthase (LuxI) causes signal enhancement.
Pcat is a constitutive promoter- therefore, luxR is expressed in excess in the bacteria, creating a dimer with the added AHL. This dimer binds to the Plux promoter, resulting in expression of mcherry, and fluorescence (emission peak: 608nm) which is to be measured using a plate-reader.

Materials and Method:

E. coli strains :
•K-12 Top 10 containing the gate Pcat-luxR-Plux-mcherry-LuxI (meant to check whether we have enhancement or not)
•K-12 Top 10 containing the gate Pcat-luxR-Plux-mcherry (no AHL enhancement bacteria)
•K-12 Top 10 containing the gate Plux-mcherry (luxR negative control)
•Top 10 containing Plac-RFP (positive control)
•Top10 containing pSB-1C3 ( mCherry negative control, blank)

A starter was prepared by growing the cells in LB medium + appropriate antibiotics at 37°C overnight. Then, 0.5ml of the starter was transferred into a 250ml Erlenmeyer flask containing 50ml LB with the appropriate antibiotics.
The bacteria were grown at 37°C until 0.6 OD600nm.
The bacteria were centrifuged for 2 minutes, and the pellet re-suspended in Bioassay medium (BA). They were then transferred to a 48 well plate containing various concentrations of AHL, as seen in figure 1. It was then incubated for 3 hours in a shaker ( 37°C, 250 RPM). After 3 hours, absorbance (OD600 – for cell concentration) and fluorescence (excitation peak: 584nm, emission peak: 608nm) were measured using a plate reader. A measurement was taken every 30 minutes for 2 hours (to a total of 4 times).

Results and conclusions

It can be seen that the relative fluorescence of the strain K-12 Top 10 : Plux-mcherry (in green in fig.1) is low and constant.
Since this strain does not have the gene luxR, there is no formation of the dimer luxR-AHL, and therefore, no induction of Plux. This strain represents the basal level of transcription under the promoter Plux.
In a similar way, the strain K-12 Top 10 : Pcat-luxR-Plux-mcherry (in blue in fig. 1), which does contain luxR, shows low levels of relative fluorescence. Therefore, it is safe to assume that the promoter in this strain is not induced in the presence of AHL.
Since we did not sequence the plasmid inserted into this strain of bacteria, but used restriction instead to verify the presence of the insert (time was short), it may possible that a mutation occurred, that the insert was not properly inserted into the plasmid, or that the scar ligation that was conducted in order to build this construct, failed.
On the other hand, the strain K-12 Top 10: Pcat-luxR-Plux-mcherry-LuxI (red in fig.1), which contains an insert that was constructed in a separate batch of scar ligation, did show a steady and moderate induction by AHL.
In addition, it can be seen that the basal level of transcription of mcherry under Plux is double in the strain that contains LuxI.
We suspect that the presence of AHL synthase (LuxI) in our bacteria causes induction enhancement, but since the strain Pcat-luxR-Plux-mcherry did not show any induction by the presence of AHL, we cannot fully compare between the results.
It is impossible to determine whether the presence of AHL synthase (LuxI) in our bacteria causes induction enhancement without repeating the experiment using a strain of Pcat-luxR-Plux-mcherry that contains an intact construct.

Fig.1: Relative florescence as a function of AHL concentration

Determine the qualitative expression of AmilCP under the promoter Plux

Objective

We aspire to verify the expression of the reporter pigment protein AmilCP under the promoter Plux.

Description

In this experiment, a culture of E. coli K-12 Top 10 expressing AmilCP (a dark blue pigment protein) under the promoter Plux was grown overnight.
The bacteria were engineered to contain a plasmid with the gate Plux-AmilCP.
We originally aspired to determine the activity of the promoter Plux using the reporter gene AmilCP, by cloning and testing bacteria containing the gate Pcat-luxR-Plux-AmilCP.
Pcat is a constitutive promoter- therefore, luxR is expressed in excess in the bacteria, creating a dimer with AHL. This dimer binds to the Plux promoter, resulting in expression of AmilCP.
However, we ran out of time, so we decided to at least show that the construct Plux-AmilCP functions properly while relying on basal levels of transcription. Our next step would have been a scar ligation, similar to the one conducted to create Pcat-luxR-Plux-mcherry-luxI from the previous experiment.

Protocol

A starter was prepared by growing the cells in LB medium + appropriate antibiotics at 37°C for 19 hours. The bacteria were then centrifuged for 10 minutes, and a picture of the pellet was taken.

Results and conclusions

The pellet was dark blue, it is highly probable that the bacteria contain the gate Plux-AmilCP.

Azo Benzene Experiments

After learning the theory behind Azo-Benzene, and modeling the behavior of cells combined into a bio-film by it, we decided to step into the wet-lab. This was not very easy, as we have not found any previous attempts to test the effects of AB on bacteria. However, guided by our simulations, we knew that a positive result would be clearly visible to an OD-meter, and maybe even to the naked eye. Since there is no existing protocol, and since there are many different variables to be changed (the frequency of excitation, the amount of light in the excitation, the concentration of Azo-Benzene, the initial OD of the bacteria, and the concentration of EDC), we decided to use a high-throughput experimental system. At the center of our system was a TECAN made Freedom EVO® wet-lab robot:



We programmed the robot to fill several 96-well plates, so that the conditions of each of the wells will be different. We then activated the AB in the different wells to different degrees, using different wavelengths. When presenting the results of a high-throughput experiment, there is clearly a tradeoff between the number of data-points presented, and the amount of data that can be conceivably presented per data-point. That is why we have chosen three main techniques of obtaining data, going from the high resolution to the many data-points:

Microscopic Imaging

Initially, we just grew the bacteria with/without AB, and shone light on it. We then took deposits of cells from the bottom of the wells, and imaged them using a microscope. We also used a function of the micro-scope called a z-stack, which takes several images while changing the focal point in between. Using this function, we can see the three-dimensionality of the constructs imaged by the microscope.


These results are clearly in agreement with the model which predicts very large clusters containing thousands of cells, which brought us to the next step:

High-Resolution Plate Reader Imaging

At the end of an experiment where we grew cells on one plate with AB and on another without, we noticed that both the plates with AB and the ones without had large deposits of cells at the bottom of plate, but that their shapes were different: in the plate without AB, we saw round symmetric deposits for an a plate left in an incubator, and crescent shaped sediments for a plate that was left on the workbench. Meanwhile in the plate with AB we saw that all the deposits had the same shape - one or two lines, which suggests some kind of symmetry breaking, due to some external factor.
To showcase these deposits, and their differences, we took some images of these deposits with a normal CCD camera, while providing lighting with a common tablet:

However, it is difficult to focus the lens well, and any analysis of the results would require advanced image processing. To obtain more accurate and easily accessible data, we used an unmodified Infinite 200 PRO Microplate Reader's function allowing for the measurement of OD within the wells in 225 different spots inside the each well arranged in a 15x15 grid. We used this as an ad-hoc imaging technique, through which we could determine the structures of the deposits get what is essentially an image of the sediments in every well.


As you can see, some of the wells on the 2nd, 3rd and 4th plate images (which are of the 2 plates without AB) appear to have patterns similar to those found in the wells with AB, despite the fact that conventional imaging showed otherwise. A closer look at which wells show the circular patterns seen by the naked eye, and which ones don't, show that the earlier a well was measured the more circular their pattern appeared.
Moreover, for the "Plate left out" (3rd and 4th images) we took 2 pictures, a few minutes apart, the second time, more of the wells had restructured into a linear shape as with the AB plates. In addition, in the second image, we saw that the wells which had already restructured themselves during the first image maintained their shape during the second measurement, which leads us to believe that this structure has a certain stability preference over the symmetric ones.
We believe this is caused by the AB being volatile (and therefore moving from one plate to the other by air, and thus contaminating our plate reader for the last 3 images), but more experiments are necessary to be sure of this conclusion. Either way, we are definitely witnessing some kind of external force working on the cells to restructure into a shape which is asymmetric.

High-Throughput Plate Reader Imaging

We proceeded with our high-throughput experimental design whereby we checked different concentrations of AB, mixed with cells with a different initial OD. We began by running letting the cells grow for a while, then measuring OD, activating the AB using specific wavelengths of photons, measuring OD again and again over constant time-lapses, and then shining on the cells with the activation wavelength again. Here are some of the results we got from select wells:

When we saw these results, we decided to look closer at the data, and bring to our aid the fact that the plate readers measured the OD at 16 different points (a 4x4 grid) within each well. We were then able to see what happens to the standard variation during the spike, and we saw that it actually plummets:

This means that the OD in all of the well suddenly becomes uniform, as opposed to having some variance between the concentration of bacteria for different locations throughout the well. We believe this was caused by the fact that the clusters were partially broken by the temporary change in the form of the AB caused by the shining of light – causing the OD to increase overall, while becoming more uniform.

Conclusion

From these experiments we have concluded that there could definitely be some kind of interaction between the AB and the bacteria, causing them to form unusually large clusters, our results were far from neat and orderly (some wells without any AB to begin with showed the formation of clusters) but that might just be due to the volatile nature of Azo-Benzene.
To find out more about this phenomenon we first need to be able to show a clean control run of the protocol we formulated for the robot. We plan to do just that in the time leading up to the competition and come closer to characterizing the nature of the affect AB has on bacteria like E.Coli.