Team:Nanjing-China/wetlab

Our wet lab experiment is consisted of three major parts, construction and testing of our circuit, testing of mlrD and mlrA, testing of riboswitches. Photos of electrophoresis[For specific procedures, please refer to the protocols.] and results of sequencing will be listed as basic results of construction and results of HPLC and fluorescence measurement will work as efficiency verification of our project.

In actual experiment, our project is composed of three sections, the detector, the positive feedback circuit, the suicide circuit which are all used to test our design in experiment. In the process of construction, we first construct small devices from parts provided in the kits and then put all these devices together step by step.

The detector circuit is used to measure concentration of AHL and present fluorescence at certain concentration. It is made up of two plasmids.

          1 Plasmid 1: Pcons-rbs-luxR-T-Prlux-rbs-lacI-T-Plac-rbs-rfp-T

          Linking of Pcons2-rbs-luxR-T-Prlux-rbs-lacI-T and Plac-rbs-rfp-T

Figure*: From left to right, Marker(1Kb), Pcons2-rbs-luxR-Ter-Prlux-rbs-lacI-Ter, Pcons2-rbs-luxR-Ter-Prlux-rbs-lacI-Ter-Plac-rbs-rfp-Ter

          2 Plasmid 2: Prlux-rbs-cI-T-PcI-rbs-lacI-T

Figure*: From left to right, Marker(DL2000), Prlux-rbs-cI-T, PcI-rbs-lacI-T, Prlux-rbs-cI-T-PcI-rbs-lacI-T. The substances of electrophoresis are made from PCR of plasmids with VF2 and VR as their primers.

3.Testing of function

We have two plasmids to sense specific concentration of AHL. The first is Pcons2-rbs-luxR-Ter-Prlux-rbs-lacI-Ter-Plac-rbs-rfp-Ter and the second is Prlux-rbs-cI-Ter-PcI-rbs-lacI-Ter. Bacterial in the first tube contains plasmid1, bacterial in the second contains plasmid2, and bacterial in the third contains both plasmid1 and plasmid2.

1 Construction of plasmid
    Positive feedback system is consisted of four similar plasmids to test the efficiency of positive feedback and compare with results of modeling.

Figure*: From left to right, Marker(DL10000), Prlux, Prlux-rbs-rfp,
Prlux-rbs-rfp-Ter, Prlux-rbs-rfp-rbs-luxI, Prlux-rbs-rfp-rbs-luxI-Ter,
Prlux-rbs-rfp-rbs-luxR-Ter, Prlux-rbs-rfp-luxR,
Prlux-rbs-rfp-luxI-rbs-luR, Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter.

Figure*: From left to right, Marker(1Kb),
Pcons2-rbs-lacI-T-Plac-rbs-luxI-T-Pcons2-rbs-luxR-Ter,
Pcons2-rbs-lacI-T-Plac-rbs-luxI-T-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-Ter,
Pcons2-rbs-lacI-T-Plac-rbs-luxI-T-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxI-Ter,
Pcons2-rbs-lacI-T-Plac-rbs-luxI-T-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter.

2 Testing of function

As is shown in the picture, plasmid with “LuxR-LuxI-T”(Yellow) shows high Fluorescence intensity, which means bacteria with such plasmid will express more enzyme than others. Besides plasmid with “LuxI-T”(purple) also shows higher Fluorescence intensity than none(blue), so this kind of plasmid can also be more efficient.

1 Construction of plasmid

Figure*: From left to right, Marker(DL2000), Ptet, Ptet-rbs-rfp,
Ptet-rbs-rfp-T, Marker(DL2000), Plac, Plac-rbs-tetR,
Plac-rbs-tetR-T, Marker(1Kb),
PgolTS-golB-PgolB-tetR-Ter-Ptet-rbs-rfp-Ter,
PgolTS-golB-PgolB-tetR-Ter-Ptet-rbs-rfp-Ter-Plac-rbs-tetR-Ter-
Pcons2-rbs-lacI-Ter.

2 Testing of function

It is a system to make sure that our bacterial will not cause any damage to the environment after functioning.

We measured the intensity of fluorescence qualitatively and quantitatively. Bacterial with plasmid displayed different color with different situation of Au and IPTG. When there was enough Au or IPTG respectively, the bacterial remained white. If there was no Au or IPTG, the bacterial turned red. The intensity of fluorescence also showed this. The experiment result is consistent with our respect, which means our suicide system actually works.