Team:WHU-China/Measurement
This year, in the quenching module of our project, we have chosen quorum quenching enzymes to
degrade
AHLs, the quorum sensing signals of Pseudomonas aeruginosa. We hope in this way, the toxicity of
these
pathogens can be blocked and they will be less harmful to the human bodies. There are two classes of
such quenching enzymes, AHL lactonases and AHL acylases, and a great number of certain enzymes
belonging
to the two family. So the key subject of our quenching module is to select one or more quenching
enzymes
that are best for inhibiting the pathogens. To achieve this goal, although some data about several
quenching enzymes degrading different AHLs exist (which we submit to the registry), we still plan to
conduct our experiments to measure the enzyme activity suitable to our situation.
We decide to use HPLC (High Performance Liquid Chromatography) to analyze the experimental results.
The
procedure is as follows:
In spite of the samples, we also need to create a standard curve of AHL concentrations as the control group to the results of our samples. To achieve this, we simply dissolve certain amount of AHL dry powder in chromatographic grade methanol to create a series of AHL solutions with different concentrations, analyze them by HPLC and draw the standard curve of AHL concentration versus HPLC data. And by measuring the AHL concentration of our samples versus time with the help of the standard curve, we can then use excel to draw the Lineweaver-Burk plot and obtain the Km (Michaelis constant) value of the enzymes towards this kind of AHL. And the same operation can be done to attain different Km values towards different kinds of AHLs, especially towards 3-oxo-C12-HSL and C4-HSL in Pseudomonas aeruginosa. Combined with our quorum dynamics model, we can finally decide which enzyme(s) to use.
As a proof-of-concept, we used 3-oxo-C12-HSL and C4-HSL bought from Sigma-Aldrich as the materials to try this method because they are the quorum signaling molecules of Pseudomonas aeruginosa. We dissolved 2mg dry powder in 2ml chromatographic grade methanol to reach a final concentration of 1mg/ml for each of them and tested the solutions by HPLC (Figure 1). The results show that there are two peaks for C4-HSL and one peak for 3-oxo-C12-HSL. This makes sense as the AHLs we bought have both “D” and “L” configurations mixed together and this chemical property may influence the time the peaks occur. But for 3-oxo-C12-HSL, which has a longer carbon chain, the different configuration may not influence its property intensely so the expected two peaks didn’t separate and appeared as one peak. The preliminary experiment results shown below successfully prove that we can use this method to quantify the AHL concentration and provide us the conditions needed to conduct HPLC for them, which will provide very useful information to the next season.
(A)
(B)
Figure 1: HPLC analysis of the standard solutions of C4-HSL(A) and 3-oxo-C12-HSL(B) (1mg/ml). A: the two peaks for C4-HSL occur at about 6 min and 8.4 min. B: the peak for 3-oxo-C12-HSL occurs at about 4.3 min.
Besides, we use the procedure described above on the crude cell extract which may contain pSB1C3+aiiC. And the cells were induced by IPTG and cultured for AiiC production. In fact we weren’t sure whether the enzymes were produced as we didn’t have time to verify it. But we still tried to conduct preliminary experiments on our samples to get information for the next season. The results are not shown here, but you can see the Engineering part of our wiki for more details.
Since we wanted to select 1 or 2 chemokines for our engineering bacteria to recruit immune cells, such as monocytes, we need to test the chemotaxis of several chemokines to these cells. We choose THP1 cell line as our model of monocytes and transwell migration assay is conducted[Figure 2].
Figure2: Corning® Transwell®
For each kind of chemokines, 3 parallel experiments were performed, in which 0.2 ml THP1 cell
culture medium of certain density was added to the upper chamber and 0.6 ml chemokine solution of 10
nmol/l (dissolved in pure culture medium) was added to the lower chamber. We set 3 parallel control
groups as well, in which the pure culture medium was added to the lower chamber. After about 15
hours of incubation, the number of cells in the lower chambers were counted. The chemotactic
index(CI) of each chemokine was furtherly calculated to evaluate the chemotaxis ability of
chemokines. The details of results are shown in Results.
The chemotactic index(CI) is defined as:
Flow-cytometry experiment was conducted in order to count the number of cells in the lower chambers of transwell boards[Figure 3], since THP1 cells are suspended in the medium and difficult to be counted by counting the cells on the bottom side of upper chamber direct;y.
Figure 3: Beckman Coulter, Inc. CytoFlex (Model No. : A00-1-1102)
The standard THP1 cell culture medium was first transferred to the special flow tube. The cells were
washed for three times, with 1 ml PBS for each time and finally resuspended in PBS. The tube was
then put into the cytometer to record the forward scatter(FSC) and side scatter(SSC) of each cell.
FSC reflects the size and activity of a cell and SSC indicates the granulation degree of a cell. The
combination of this two parameter in this experiment set standards for THP1 identifidation. The cell
culture medium from each lower chamber of transwell boards was measured by the flow cytometer later
in the same way. The THP1 cells are recognized based on the data measured from the THP1 cell culture
medium before. Cells from all these samples were recorded and counted by a software named
CytExpert.
Result
The records of combination of FSC and SSC of each cell were displayed in the form of a scatter
diagram[Figure 4]. Red marked dots located in the red frame were identified as records of target
cells while others were not. The number of these indicated the real number of THP1 cells in each of
the lower chambers. The number of cells of each transwell chamber were furtherly collected and
analyzed, which are shown in details in Results.
Figure 4: The flow-cytometry of one of a lower chamber added with 10 nmol/L CCL2(labeled as CCL2 2). The number of cells identified is 9396.P1 refers to the percentage of identified target of all events was 97.84% .
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