Team:Carnegie Mellon/Week13

Monday, August 26th
Overnight cultures made of the KR12 and RFP10 lysogens (heat induced for 1 hour at 42ºC and grown overnight in 10mM IPTG)
Overnight plasmids (XL10 strains) grown overnight,

Tuesday, August 27th
Plasmid cultures diluted at 9:30 and IPTG added at 10:45
Overnight lysogens checked on Tecan and scanned for the presence of RFP/KR

Conditions needed:
Lysogens:
KR12 IX = KR12 42, IPTG 37 IPTG
RFP10 IX = RFP10 42, IPTG 37 IPTG
KR12 = KR12 -IPTG
< RFP10 =RFP1-IPTG
Plasmids:
KR/RFP IX, KR/RFP X, KR/RFP

Tecan results: it seems the RFP lysogens that have IPTG after the 42ºC heat induction have the same levels of RFP (10x lower than the XL10 plasmid induced for 2 hours at mid-log).
Photobleaching started: 1:10pm
RFP expression from the induced plasmid is enough to see fluorescence in front of the lamp

Wednesday, August 28th

New protocol for photobleaching and KillerRed/RFP expression from lysogens:
Start overnight cultures of the lysogens at 30ºC
Dilute cells 1/10 and add 10mM IPTG and incubate at 30ºC for 1 hour
Heat induce at 42ºC for 1 hour
Incubate at 37ºC for 2-3 hours
Place tubes in 4ºC refrigerator overnight to allow proteins to fold and mature

Transformation of KillerRed BioBrick plasmid into the XL10 K strain for submission to iGEM.

Thursday, August 29th

Protocol for photobleaching and KillerRed/RFP expression from lysogens:
Start overnight cultures of the lysogens at 30ºC
Dilute cells 1/10 and add 10mM IPTG and incubate at 30ºC for 1 hour
Heat induce at 42ºC for 1 hour
Incubate at 37ºC for 2-3 hours
Place tubes in 4ºC refrigerator overnight to allow proteins to fold and mature

Duplicates of the KillerRed-containing plasmids and lysogens are made during the induction step. The plasmids follow the same protocol but grow entirely at 37ºC and do not have a heat induction step.

Friday, August 30

Photobleached 6 cultures for 5 hrs: RFP plasmid and lysogen, duplicate KR plasmid, duplicate KR lysogen. Excitation scans show subtle (but visible) presence of RFP in the lysogen and KR in the XL10 (and, of course, not-so-subtle RFP in the XL10).Each culture (both exposed and unexposed) were plated.