Difference between revisions of "Part:BBa K594015:Design"

Revision as of 02:54, 6 October 2011

A device that can accepts the 3--O-C6-HSL and then produces lasI and EYFP

Design Notes

The device do not contain the genes that will arouse biosafety problems.

Device inducing

This device includes a plac, which is inhibited by lac I gene in E.coli. IPTG can relieve it from inhibition.

For the inducing, 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 this device 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.

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, geting a more clear image. 4.Switches 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

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

This proves that this circuit has worked.

Source

It's a composite part. All the basic parts are from the iGEM.

References

1.PC Seed, L Passador and BH Iglewski. Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy.J. Bacteriol., Feb 1995, 654-659, Vol 177, No. 3