Difference between revisions of "Template:SJTU-BioX-Shanghai/Notebook/Testing/w10"

Revision as of 00:45, 19 September 2015

Testing

first desalination test of pcpcG2-HR

Sep. 7

Inoculated 15 wildtype & 15 pcpcG2-HR (use pcpcG2-HR from colony 2) at 10pm.

Sep. 9

Induced with green light at 10pm, added 22g/L NaCl & retinene, then took the first 6 samples (3 parallel samples for each).

Sep. 10

Cultivated with red light at 10am, then took the second 6 samples,

Took the third 6 samples at 2pm.

Took the fourth 6 samples at 10pm.

Sep. 11

Took the fifth 6 samples at 10am.

Detection of Membrane Localization of Halorhodopsin by Immunocytochemistry

To examine whether the HR expressed successfully onto the cell membrane, we designed an immunocytochemical experiment. For that the C terminal of the HR sequence we designed contains a His-tag, we used anti-His mAb as the primary antibody and goat anti-mouse IgG Alexa Fluor 488(green fluorescence) as the second antibody. And if a loop of green fluorescence can be seen under the confocal microscope, we will ensure that the HR expressed successfully onto the cell membrane. A few things should be paid attention to:

1. The incubating condition can be overnight at 4℃ or 2 hours at 37℃. But if a better result is wanted, use the former condition.
2. Be careful that the procedures involving fluorescent antibodies need to be operated under lucifugal condition.

Sep. 15

1ml cyanobacteria liquid for each sample was prepared, including four experiment groups--Pdark-HR(green), Pdark-HR(red), PcpcG2-HR(green) and PcpcG2-HR(red), which had been cultured to OD730=0.6 and induced for 12h, and controls—wildtype(green) and wildtype(red), which were not been induced.

Anti-His mAb was applied with PBS in proportion of 1:100 as the primary antibody. After 2 hours’ incubating(at 37℃) and washing steps, goat anti-mouse IgG Alexa Fluor 488(green fluorescence) and goat anti-mouse IgG Alexa Fluor 594(red fluorescence) were applied respectively with PBS in proportion of 1:1000 as the second antibodies.

After 2 hours’ incubating(at 37℃) and washing steps, the cyanobacteria pellets were resuspended with 50μl PBS. Six slides were prepared. While observing under a confocal microscope, we found out that the cyanobacteria has spontaneous red fluorescence. Several positive results can be seen but the signal is not strong enough. So we decided to optimize the protocol and redo it.

Sep.15 evening to Sep. 16

1ml cyanobacteria liquid for each sample was prepared, including four experiment groups--Pdark-HR(1:100), Pdark-HR(1:50), PcpcG2-HR(1:100) and PcpcG2-HR(1:50), which had been cultured to OD730=0.6 and induced for 16h, and controls—wildtype(1:100) and wildtype(1:50), which had been induced for 16h as well.

The primary antibody anti-His mAb was applied with PBS in proportion of 1:100 or 1:50 as mentioned. The samples were incubated at 4℃ overnight. The second antibody goat anti-mouse IgG Alexa Fluor 488(green fluorescence) was applied with PBS in proportion of 1:300 and the samples were incubated at 37℃ for 2 hours.

After washing the samples, the cyanobacteria pellets were resuspended with 20μl PBS. Six slides were prepared. The results were better than the former one. Concerning of time, we only got the micrographs of the samples containing the primary antibody with the concentration of 1:50.

In the experiment groups (Pdark-HR and PcpcG2-HR), many loops of green fluorescence can be seen under the confocal microscope while the control groups (wildtype) cannot, which indicates that the halorhodopsin expressed successfully on the cell membrane of the genetic cyanobacteria instead of any other position in the cells.