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From 2013.igem.org

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:

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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
Photobleaching:
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)