Team:Carnegie Mellon/Week6


Killer Red

Monday, July 8
--Repeat photobleaching stuff (longer outgrowth after photobleaching)
--Flooded RFP 10^0 titer plate with 10 ml SM to be left at 4 deg C until tomorrow
--Started diluted cultures (Y1088, Y1090) for infection at 10:45
--checked 11:22am - no visible increase in turbidity
--12:30 - OD600 of 1088 was 0.301 - check in approx. half hour
--Started diluted cultures at 10:50 (for photobleaching)
--Split overnights tubes for photobleaching (exposed/unexposed)
--All 8 conditions plated
--Last time point diluted to 3 mL and 200µL plated at 12:20. Cultures are in the incubator ready to grow (grow for 4 hours)
--Overnight cultures prepared and induced for tomorrow
--PCR of phage samples
--Phage KR6 has KR in the forward direction!
--Gel from 7/5:

1st gel from 7/08: KillerRed forward sample visible in lane 6
^ lane 6 has a positive result from PCR. This is stored in the 4ºC refrigerator (ready for infection)

2nd gel from 7/08: (first 5 lanes are not ours)

Overnights started at 5pm (.5mM IPTG for induction) total of 6 mL media (colonies picked from original XL10 plate.

Tuesday, July 9
--Video conference with Purdue and other iGEM teams (10am)
--Digestion of C5 and BB KR started at 11:30. 2 hour digestion at 37ºC by SpeI and XbaI
--Gel started post-digestion at 1:44pm. Check at 2:30 (Not done at 2:30. Possibly by 2:45-3)
--Infected 1088 with KR positive phage
--Infected 1088 with RFP high-titer stock to determine titer: 10^0, 10^-4, 10^-7, 10^-8, 10^-9, 10^-10, 10^-11
--10µL ligation reaction started at 4:50pm
--Ligation transformed at 5:20pm by Cheryl
--Overnight cultures prepared by Andrew at 5:05pm
--Data from Photobleaching:

Wednesday , July 10
--RPM on shaker was too low; therefore low expression of RFP. 2 mL diluted to 4mL (2µL of IPTG and Amp added). Started at 10:45. Incubate for 2 hours at 37ºC (check at 12:45)
--KR positive phage in 1088: Small, clear plaques in center of 10^-3 dilution plate. Probably means that single plaques could have been counted on a 10^-4 dilution or 10^-5 dilution plate.
--Flooded 10^-3 plate with 10 ml SM: 10:45am
--Cell density info:
--Overnight: 10^10 cells/ml
--OD600 =1: 8x10^8 cells/ml
--dilutions linear between 0.1 and 1.0
--protein concentration in the cell 135 µg/mL
--RFP phage in 1088 titer
--Plaques are obvious and almost confluent on 10^-10 dilution plate, but no obvious plaques on 10^-11 plate
--Number of cells is potentially too high- make sure OD is 0.5 with another spec?
--Overnights are expressing protein at 11:45am but protein might not be totally folded.
--12:45pm: the uninduced RFP cells show some pink color and the induced look like they grew overnight. Higher RPMs significantly increase the maturation rate
--RFP consistently is noticeably bleached after about 30 minutes of 100% power
--GAP (growth after photobleaching) started at 2:40pm
--Growth was stopped at 4:50pm
--BioBrick KillerRed transformation
--Ligation run for ~10 minutes
--1hr step ends at 4:54 (plate tube labeled BBa_KR ligation in incubator on Cheryl’s bench) onto Chloramphenicol plate
--Kathy collected the high titer stock and plated to determine the actual titer.
Overnights prepared

Thursday, July 11

Data from 7/10:

--Note: KR-I outgrowth may be low because too much ampicillin added
--KR+ high titer stock
--As expected, 10^0 stock had too many phage- no visible plaques
--10^-6 through 10^-11 all had about the same number of plaques. May mean we just have a ton of phage.
--Streak XL10
--Let’s make a lysogen!
--Y1089 is hfl (high frequency lysogeny)
--infect with KR+ phage for 15 min at room temp
--add LB, allow to grow for 1 hr
--plate on LB amp plates
--Make dilutions and plate: 10^-2, 10^-4, 10^-6
--Photobleach for 3 hours and grow for 5 hours, put plate and cultures in 4ºC for measurements tomorrow.
--3mL starting volume (200µL per time point) should allow for longer exposure times
--10µL of cells from photobleaching experiment added to 1000µL of LB+amp (no IPTG for any cells)
--After 4 hours, (started at 3:05pm), put cells in the 4ºC refrigerator

Friday, July 12
--RFP lysogenic infection @ 11:10am
--Fetch tube at 12:10am, make dilutions (10^-2, 10^-4, 10^-6), and plate
--Test whether KR lysogen from yesterday is actually a lysogen
--Streak KR high titer phage stock in one direction
--Streak potential lysogens perpendicular to phage
--true lysogens should be immune to phage infection
--non-lysogens will be infected by phage and won’t grow past streaked phage
--Mini-prepped BioBrick KillerRed
--Sent lambda RFP, lambda KR, 2 BioBrick KR samples out for sequencing

RFP Photobleaching t0.5=99 minutes (1.65 hr) (R2=.99)
KR Photobleaching t0.5=866 minutes (14.4 hr) (R2=.19)