Team:Carnegie Mellon/Week5


Killer Red

Monday, July 1

Results from Weekend:
---Killer Red packaging experiment was seemingly successful with hundreds of plaques. Will need to image on Typhoon to screen plaques.
---RFP picked plaques that were chosen to infect both the 1089 and 1090 strains revealed between 10-50 plaques. Plaque morphology is different between the two strains. 1089 plaques tend to be circular and clear, the 1090 plaques look like “dents” and are more turbid. 1089 is used for large-scale protein expression and 1090 is the lysogen strain.
Things to do today:
--Dilute RFP/KR plasmid cultures and induce after 2 hours for photobleaching. Streak these on LB/amp plates
--started 11:45 am
--Infect RFP/KR plaques into XL10 that was prepared overnight with maltose/MgSO4
---1/100 dilution of 1M MgSO4 and 20% maltose and start log phase
---Started 11:45 am
---1:00 pm OD600 of 0.413
---didn’t infect into XL10 because no plaques were clearly positive for KR
Infect KR phage into 1088 strain
Kathy made more top agar tubes for infection
for Typhoon (exc/em)
--mRFP: 584/607 (~75% absorption at 570nm)
--KillerRed: 585/610 (85% absorption at 570nm)
Safire experiment
--scrape off ¼ of the 1090 RFP plates and suspend in 250µL of SM buffer
--let sit for 15 minutes and centrifuge for 1 minute
--Refrigerate at 4ºC until ready to plate
--Centrifuge again if necessary to pull off supernatant without getting solid agar
--Take 100-200µL and put into 96 well plate
--Induce RFP/KR cells at 1:45pm
--Check cells again at ~2:00pm (at 1:45 they were almost in mid-log)
---Induced (3µL) at 2pm
---Not much color after 3pm, noticed mistake with adding too little IPTG. Added 80µL at 3pm and added 1mL of LB. (IPTG
stock is .5M)
---Tecan results:
---Overnight cultures had lots of RFP and KR. KR is fairly stable in the dark (after sitting for a few days on a bench at RT)
---Induced phages on plates did not produce much RFP
--Second Tecan result (photobleaching)
---Start fresh overnight cultures (dilute 1/10 in the morning)
---photobleaching (10 minute time points)
---RFP fluorescence was clearly photobleaching after 30-40 minutes
--infect in 1089
--grow for several hours
--induce w/ iptg
--spin down and wash
--measure fluorescence

Tuesday, July 2

Diluted cultures: 10:45am (1/10)
Induce at 11:45 and photobleach around 12:45-2
--Take out 1mL of each photobleached culture, dilute to 3mL (take OD600)
--Grow for 2 hours and compare OD600
--KR should have a lower OD600 because the photobleaching should have killed off a generation of growth
--Cannot start photobleaching until protein has begun to mature. No color visible after approximately 1 hour since induction. (See note)
--Since photobleaching must be done at RT and in water, possibly try icing the cells before photobleaching to ensure maturation of translated proteins
Streaked plates from yesterday (of RFP/KR in the XL10 strains) have several single colonies to use for later Re-streaked host strains
Kathy is setting up multiple infections
--1088 with lambda RFP phage #4
--dilutions: 10^0, 10^-1, 10^-2, 10^-3, 10^-4 (perhaps overkill. will see)
--KR phage in 1088 w/ IPTG and X-gal to try to determine whether it’s producing KR
--RFP phage #6 in XL10 cells
LB plate preparation

mRFP1 Ex. Coefficient: 44,000 QY: .25 Brightness: 11,000
Killer Red Ex. Coefficient: 45,000 QY: .25 Brightness: 11,250
Killer Red is actually brighter than RFP and has similar excitation/emission (Ex. Co • QY). We should find out why RFP is being synthesized more than KR
KR might be underexpressed due to lack of codon optimization
--Photobleaching experiments seem to work as long as the bandwidths are 10nm.
--Photobleaching RFP for 1 hr at maximum power bleached it 72%
--Photobleaching KR for 1 hr at maximum power bleached it 33%

At 5:30pm, OD’s were taken.
--Unexposed cells grew by .05 units and the exposed cells maintained their OD within .01 units. The KillerRed cells to be effective at killing when even a small percentage of the protein is photobleached. :D

Wednesday, July 3
Restreaked 1088, 1089, 1090 and VSC257 all have single colonies to pick from.
Titer plates: 10^-4 dilution: 2 PFU10^-3 dilution: 3 PFU10^-2 dilution: 46 PFU10^-1 dilution: ~300 PFU10^0 dilution: ----
XL10 and RFP infection
---no plaques on any plate
---possibly because lambda needs recA?
Imaged KillerRed plates infected on 7/2 using Typhoon
--again, no clearly fluorescing plaques- will use PCR to determine whether any phage are producing KR
Cheryl ordered PCR primers last night
picked 7 more plaques from new plates to screen for KR gene
< Photobleaching of induced overnights of KillerRed, RFP
--Split into two cultures of 2 mL
--Exposed/unexposed for each at 0, 15, 30, 45, 60 min
--Forgot to dilute until 1hr20 after photobleaching... samples of each at this time and at 3hr5 after photobleaching (1hr45 after diluting)