Team:Carnegie Mellon/Week3

Monday, June 17th -Oligos arrived
-mRFP on plate 3 12N (BBa_E1010)

-Top Row (left to right): ladder, KillerRed with BB promoters (SpeBBKRR and XbaBBKRF) x3, control without DNA
---Bottom Row (left to right): RFP with EcoRFPfor and EcoRFPstR x3, control without DNA, ladder, RFP with SpeRBSRFPfor and PstSTRFPrev primers x3, control without DNA
-Purified PCR products (KR biobrick, RFP sequence)

Tuesday, June 18
-Verified sequence of plasmid with ptac (H17) and WT lac (D1)
-Digest WT lac (D1), RFP, EGFP and KR with PstI and SpeI (2 hour digestion at 37ºC)
ends at 12:30pm
-Gel purify fragments in 1% agarose
---D1 - RFP - EGFP - KR - Ladder - Cheryl’s
-Media-preparing supplies arrived
-Purified by GeneJet
-Ligation by Thermo-Fisher T4 ligase kit
---6:2 µL RFP and EGFP ligations
---2:2 µL KR since KR band was very strong
Transformation
---2 min on ice
---5 min at 42ºC
---2 min on ice
---add 500 µL LB and incubate at 37ºC for 1 hr
---Plate on LB/Amp plates and incubate overnight

Wednesday, June 19
RFP and KillerRed plates have many colonies EGFP has about 10 colonies.

Colony PCR for 4 EGFP, 4 KillerRed and 1 control of the plasmid DNA
---Use VF2/VR primers
---PCR ends at 4:50pm
---Top Row (left to right): eGFP in pSC103 x4, KillerRed in psc103 plasmid x4, pSC103, ladder

Use gel to confirm the sequence
pSC103 is smaller than pSC103 with KR insert-transformation verified

Thursday, June 20
KR cells cultured in tubes covered in aluminum foil
Observations:
---Streaked plates grew successfully. Several colonies to choose from
---KR cultures are very turbid (no color)
---RFP cultures are bright red
---EGFP culture is turbid (no color)
---Negative control (VCS257) has no growth
---Phage strains are all turbid
Digestion of EcoKR
Find tube of KR from 6/13
Digestion started: 10:40
Digestion started RFP 11:33
End digestion around 1:30

Friday, June 21
KR cells kept covered in aluminum foil until photobleaching
Diluted cells at 12:45pm in LB+amp
---Note: amp should be added as 1/1000 dilution
Induction with IPTG: 1:45pm
---Note: IPTG should be added as 1/500 dilution (6µL added to 3mL)
---Check RFP for color change at 2:30 (no significant difference between induced and uninduced at 2:30 and 3pm) Photobleaching: 4-5pm?

RFP + (light, 2 duplicates)
---1. induced
---2. not induced

RFP - (no light)
---not induced

KR + (light, 2 duplicates)
---1. induced
---2. not induced

RFP - (no light)
---not induced

photobleaching measured with UV/Vis (where?)
---6 LB/ amp plates at 10-5 dilution

Inactivation of KillerRed is about 20 minutes according to Evrogen
---http://www.evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml

Inactivation of mRFP is about 10x faster than mCherry (which takes ~1.5-2 minutes)
---http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf

Inactivation of RFP takes >30 minutes at 100% light
---Inactivation of KillerRed tubes were irradiated for 5 minutes at 100% light
---All tube were plated (100µL) without dilution. KillerRed should have lower viable cell counts.

Saturday

Rfp light, induced
Some uneven spreading but basically a lawn, fairly red

Rfp light, uninduced
More evenly spread, very small individual colonies observable near edges of plate. Both pink and non pink colonies

Rfp no light uninduced
Some uneven spreading, lawn that is quite pink.

Kr no light uninduced
Complete lawn, no color

Kr light, induced
More or less a lawn (a few individual colonies observable), no observable color.
Kr light, uninduced
More uneven spreading than other two kr plates, some individual colonies observable, but it's basically a lawn. No color.