"

From 2011.igem.org

For effectiveness of Devices 1 2 3

Device inducing mechanism

Device I includes a plac, which is inhibited by lac I gene in E.coli. IPTG can relieve it from inhibition. Device II and III is promoted by ptetR. As the chassis E.coli we used do not contains tetR, it could have a constitutive expression.

Protocols
For device I, we will,
1.Get the AHLs 3-O-C6-HSL. The BBa_I751250 is a device that can produce 3-O-C6-HSL. Transform this part into competent cell, then inoculate the bacteria into sterilized LB medium, cultivating at 30℃ and 180 r/min. 2. Add the AHLs 3-O-C6-HSL to sterilized LB medium according to the proportion of 1:4,
3. Inoculate the bacteria with device I and then incubate them with shake cultivation at 37℃ for 3~4 hours, until the OD600=0.3.
4.Add the IPTG, whose initial concentration is 1M, its final working concentration is 1mM.
5. Incubate bacteria with shake cultivation at 30℃ for 12 hours
6. Filter the culture with 0.22 um film, and get the supernatant, inside which exists the AHLs 3-O-C12-HSL produced by lasI, it can be used to induce the device 2
7. Making slides and detect the fluorescent protein under fluorescence microscope.


For device II, we will,
1. Inoculate the bacteria with device II and then incubate them with shake cultivation at 37℃ for 3~4 hours, until the OD600=0.3.


2.Add the AHLs 3-O-C12-HSL to culture according to the proportion of 1:4, then incubate bacteria with shake cultivation at 30℃ for 12 hours.Since AHL filtrate composition is not a absolute one, ex. for device 2, the filtrate from device 1 contains mainly AHLs 3-O-C12-HSL, but also the remaining AHLs 3-O-C6-HSL, antibiotics, medium and so on, so we also added the AHLs 3-O-C6-HSL to another device 2 tube, working as a controlled group, and made sure all other containment would not interfere with the result----A antibiotics, same LB liquid medium.
3. Filter the culture with 0.22 um film, and get the supernatant, inside which exists the AHLs 3-OH-C14:1-HSL produced by cinI, it can be used to induce the device 3 .
4. Making slides and detect the fluorescent protein under fluorescence microscope.

Then we could use the AHLs 3-OH-C14:1-HSL to induce device III, the process is same to device II.
The experiment method above uses Peking University’s protocol as a reference.

Fluorescent protein detection

1. Make slides before detection, the shelf time should not be too long.
2. Turn the microscope’s ordinary light and fluorescent light’s power on. Preheat the fluorescent light for 5~10 minutes.
3. Find the moving bacteria layer under the ordinary microscope.
(1) First, find the bacteria under low power len.
(2)Second, convert the objective lens to a higher magnification, then find the more clear moving bacteria.
(3) Use oil len, getting a more clear image.
4. Switch the ordinary light mode to fluorescent light mode, choose proper exciting light wave and optical filter.
5. Adjust the exposure time, then find a suitable fluorescence figure.



6. Save the pictures.
7. Turn off the software and switch the fluorescent light mode to ordinary light mode.
8. turn off the fluorescent light power and ordinary light power.
9. Wipe the objective len with len’s wiping paper. Cover the fluorescence microscope with cloth.
Attention: During the process of using mercury lamp supply, we need to be careful not to switch its power off at will. We must wait for 15 to 20 minutes before turnning mercury lamp on everytime after we have turn off the power, or it may result as a mercury lamp explosion)

The exciting and emiting light wave

The Olympus fluorescence microscope in QIBEBT has four mirror units.

Result

Device I result
The EYFP in device I have a successful expression, it can be detected from 1:U-MWU2 to 4.U-MW1BA2. Especially good in 2:U-MWB2




E.coli image with No exciting light



Excited by 2:U-MWB2

EYFP did not show its yellow, that is because of overlapping effect of fluorescence microscope’s optical filters.This proves that the promoting circuit in device I has worked.
Controlled group didn’t show any fluorenscence
As for the promoting circuit in device II and III, we could not detect any fluorescent protein.

Retrieved from "http://2011.igem.org/Team:OUC-China/Result/fv/fpe"