Team:Carnegie Mellon/Week9


Killer Red

Monday, July 29

Get filter installed in lamp. Do practice run with Fluorescein/HCy5 dye?

1.9 mM Fluorescein
25mM HCy5

in 400µL with SM:
1µL HCy5 dye=62.5µM
25µL Fluorescein= 119µM

25µL Fluorescein
1µL HCy5
374µL SM
Started at 12:35pm

Take ratio of peaks in Tecan

Also conduct excitation/emission scans
UV is damaging Cy5 dye produced from the fluorescein photobleaching. The filter will help cut out these damaging wavelengths.

Tuesday, July 30

Repeat fluorescein/HCy5 experiment but take 5 minute time points for 20 minutes.

50µL Fluorescein = 95 nM
2µL HCy5 = 50 nM

Filter setup:
375 LP filter in front of the lamp. Will test with Fluorescein/HCy5 dye.
Cy5 Ex: 250,000 M-1 cm-1 QY: .3
Brightness= 75,000
Fluorescein Ex: 92,300 M-1 cm-1 QY=.79
Brightness= 72,917
Brightnesses are comparable. Therefore, we should see a distinct change in solution color when HCy5 is converted to Cy5.
3 hours is too long because it destroys the Cy5 dye (at least with the UV range). Perhaps the filter will eliminate the bleaching problem of the Cy5 dye.
375 LP filter is in place of the .3ND filter (which is currently in the box where the 375LP was.

Wednesday, July 31
Week on week he toiled at complicated phage experiments. His work--his hands, his technique--became more adept, and his days more steady, less fretful. -Arrowsmith

Updated MATLAB model for KillerRed
Fluorescein/HCy5 experiment:
1000µL total volume (SM as a solvent)
50µL = 1.9 mM Fluorescein = 95µM
2µL = 25 mM HCy5 = 50µM
Time points:
0 5 10 60 180
Results- significant signal obtained from dye after just 10 minutes of fluorescein reaction.... longer exposure results in profound bleaching of dye, with virtually no signal remaining after 180 min.�

Low amount of fluorescein photobleaching due to high concentrations.

Thursday, August 1

Overnight cultures (lysogens) were diluted 1:100 in SM buffer for imaging in Andor.

Imaging protocol:
25µL of Overnight cultures were diluted to 100µL and 100µL were plated on a MayTEK glass-bottom dish.
Microscope setup used: Andor Revolution XD System with Spinning Disk Confocal/TIRF/FRAPPA Microscope.

No fluorescence visible from the microscope that was directly attributed to RFP or KillerRed inside a bacterial cell. To test whether copy number is the issue here, Kathy is setting up the lysogens and inducing them with heat (42ºC), to produce a lot of phage. They will then be tested on the Tecan.

Used TECAN on 7th floor to scan for fluorescence. Insignificant difference between induced and uninduced KR. Levels of fluorescence of cultures grown at 30C and 42C were equivalent.

Friday, August 2

Kathy is running colony PCR to determine if the lysogen phages have the inserts. This might explain why we’re not seeing expression in our lysogens. Should be done around 1:45.

Modification to the positive control experiment:
1.5 mL
6 µL 1.9 mM Fluorescein 7.6 µM
.5 µL 25 mM HCy5 = 8.3 µM