Team:Tsinghua

Experiment and Data

In this part we will show the experiment we have designed and carried out along with the results. Only valid and positive results will be displayed.
Our project is based on three modules, so is the part of experiment. So we will introduce our work to you according to different modules.

Module I. Signal transmission

In this module, we hope we can apply LuxI protein to produce AHL which can keep the signal transmitting.
Construct 1: luxI on pet15b

To make sure the protein LuxI was produced we carried out a SDS-page after purification using a Ni-column (LuxI was with a His tag).

Figure0. A SDS-page showing the purification of LuxI.
The production of AHL needs LuxI protein as an enzyme and a substrate molecule which is supposed to exist originally in E.coli

Module II. Signal sensing and releasing

According to the project design, we are using quorum sensing system in gram negative bacteria as our signaling system.
Our first construct consists of Lux Promoter which can be activated by AHL and RFP.
Construct 2: Plac-luxr-Plux-RFP on pSB1A2

the part 1-9H in the igem 2012 kit was used

Figure 1. A PCR result to confirm the accomplishment of construct 1.

Then we did a few tests to see if this construct could successfully sense the signal of AHL and produce RFP as an indicator. To test the efficiency of this AHL trigger, we directly added AHL into the E.coli and the photo below is taken 12h after adding AHL.

Figure2. Tests of AHL trigger. Different AHL molecules and different concentration were set as the photo shows.

Next, we use fluorescence microscope to visualize the appearance of RFP as the time goes.

Figure3. Without AHL, the bacteria didn’t tend to produce much RFP.

Figure4. 1h after AHL was added (bright and dark).

Figure5. 2h after AHL was added.

Figure6. 3h after AHL was added.

After all these tests, we are convinced that this construct does function as both a signal sensor and a signal releaser.

After confirming construct2, we naturally want to combine module I and II to see what will happen.
At the first, we just mix up these two kinds of bacteria(one can produce AHL, the other sense it), the result was as below(negative control was not red which isn’t shown here).

Then, we decide to separately locate the two kinds of bac and see if it could appear red in the contacting area.

After all these tests, we decided to move on, so we designed the next construct.
Construct 3: Plac-luxr-Plux-RFP-Plac-LuxI on pet15b

Figure8. A enzyme digestion test showing that the sample on lane 4 is positive.
However, we didn’t see any sign of AHL signaling through this construct and we believe the arrangement of promoters is somehow inappropriate. So we redesigned the plasmid and built construct 5.

Construct 5:Plac-luxr-Plux-RFP-LuxI on pet15b

Figure9. A PCR screen showing that lane 5 is positive.

Then we made dishes with AHL in the middle of the plate only and spread E.coli with construct 5 on the whole plate hoping we can see the red firstly appear in the middle and then spread out to the whole plate.

Figure10. AHL in the middle. We can see that only the central part of the plate turned red.

And we have known that concentration of AHL needs to be higher than some certain value to activate the expression. So we also tried to add a low level of AHL all over the plate and still with a high level of AHL in the middle of the plate.

Figure11. a, E.coli with construct 2, a red patch appears where there is AHL(yet the photo is not clear); b-f, 200μl water with 0, 0.05, 0.2, 0.5, 2μl AHL(10-4mol/L). RFP appear all over the plate in c-f, while some on the edge in b.

The results were not perfect and not able to prove the successful transmission of AHL. However, the result show us some hope that the system will work as long as the circumstances are appropriately modified.

Module III. Signal Center

The work done before was not just for seeing some RFP, we build this system to see if we can get some amplified signal transmitting through some distance that can be processed in a signal center. Then we design construct 6&7.

Construct6: Plux-LacI-LuxI-Plac-LuxR-LasR-Plac-LacO-cI-Plas-LasI-Plas/cI-RFP

The advanced version of our design consists of too many parts which can hardly assembled one by one. So we decided to use overlap PCR to get the construct. However, we have only finished part of the plan and are concentrating on the rest.

Figure12. Some results of a complex overlap PCR to get construct 6.

Construct7:  And-not Gate

And-not gate works when one signal exists while the other one not. We designed this part based on two kinds of AHL：C6HSL(signal A) and C12HSL(signal B).

 

As the picture above shows, C6HSL could activate the promoter Plux and the promoter Plas is activated by C12HSL. In the design of this part, we also added cI operator downstream of Plas. As a result, the promoter Plas could be activated by signal B but repressed by cI protein. The expression of cI protein is under the control of Plux, which could be activated by signal A. RFP is controled by Hybrid promoter(Plas and cI operator), which would work as a reporter.

In a word, the expression of RFP is only on the condition that A is absent and B is present.

Figure13. Results of overlap PCR to get construct 7. The picture on the left shows the acquirement of two of the single parts (Hybrid promotor (HP) and RFP). The Picture on the right shows that we obtained the fusion product of HP and RFP. (F means Failure, while S means Success. the fusion product is marked by red arrow)