Team:elan vital korea/Result

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PROJECT
-Result-

Result

Our project had two main stages: in the first stage, we made the relevant plasmids, and in the second stage, we tested the cells transformed with the resulting plasmids. On the second stage, we used control experiments to make sure that the results we got was our actual results. For the control tests, we used untransformed E. coli and pure AHL as control groups.

Through the experiments, we confirmed the successful recombination of the test plasmid and the reporter plasmid, and the successful transformation of the test cell and the reporter cell. To make sure everything goes as planned, we tested the plasmids not only at the end, but also after the addition of each part.

For our project, we engineered two main plasmids: the reporter plasmid and the test plasmid. The reporter cell, which are competent E. coli transformed by reporter plasmids, produces LuxR, and has an inducible promoter that is activated by a LuxR-AHL complex. This promoter activates the transcription of GFP, which is easily detectable. The test cell, which are competent E. coli transformed by test plasmids, produces lactonase, which breaks down AHL. For the test plasmid, we tried two different types of enzymes: AiiA, and LacZ or beta-galactosidase.

When the test cell is treated with AHL, it breaks down AHL. When the reporter cell is added to the mix, no GFP is produced, as there is no AHL, whereas GFP is produced when a control group is used instead of a test cell.

We used the plasmids J61100, C0060, C0062, K823017, J37032, R0062, I732006 We transformed and raised the cells in solid plates to observe growth.

Picture A: C0062

Picture B: C0060

Picture C: I732006

Picture D: R0062

Picture E: J37032

Picture F: K823017

We went through miniprep, then electrophoresis to confirm that the plasmids were the ones we wanted.

For our initial electrophoresis results for the individual plasmids, look at: Picture H, Picture I, Picture J, Picture K Lane 8, Picture L Lane 5, Picture M Lane 1.

After we confirmed that all the plasmids were as they should be, we started the recombination process.

We also checked on every step of the recombination process by electrophoresis.

Picture G: Ladder reference

Picture H: Lane 1 : DNA ladder marker,
Lane 3 : C0060+EcoRI+PstI,
Lane 6 : I732073+EcoRI+PstI,
Lane 7 : J37032+EcoRI+PstI,
Lane 8 : K823017+EcoRI+PstI

Picture I: Lane 1-3: C0062+EcoRI+PstI,
Lane 9: DNA Ladder Marker

Picture J: Lane 1: DNA Ladder Marker,
Lane 2-3: R0062+EcoRI+SpeI

Picture K: Land 1: DNA Ladder Marker,
Lane 2-7: (J61100+R0062)+EcoRI+PstI,
Lane 8: J61100+EcoRI+XbaI,
Lane 9-14: (J61100+R0062)+XbaI

Picture L: Lane 1: DNA Ladder Marker,
Lane 2-4: (R0062+J61100+I732006)+XbaI+PstI,
Lane 5: I732006+XbaI+PstI

Picture M: Lane 1 : DNA Ladder marker,
Lane 2 : J61100+EcoRI,
Lane 3 : (R0062+J61100)+EcoRI,
Lane 4 : (R0062+J61100+I732006)+EcoRI,
Lane 5 : (K823017+ R0062+J61100+I732006)+EcoRI,
Lane 6 : (R0062+J61100)+EcoRI+PstI,
Lane 7 : (R0062+J61100+I732006)+EcoRI+PstI,
Lane 8 : (K823017+R0062+J61100+I732006)+EcoRI+PstI

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After the construction of the plasmids, we ran some experiments with our model to make sure it worked.

Picture N: Final Results

We confirmed that GFP was exhibited in reporter cells to which AHL was added.

We confirmed that if the test cell was added to the GFP beforehand, GFP was not exhibited.