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

Testing Biobrick BBa_M30011 (ompR controlled mRFP)

Status: Success

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) 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.

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.