Team:Carnegie Mellon/Week2


Killer Red

Monday June 10th:
- Met with Natasa about modeling
- Made 0.5 L LB Agar and 0.5 L LB broth
- Autoclaved
- Added chloramphenicol to the agar
- Poured plates (remember to put plates in fridge!)
- Looked through registry for Lac promoters
- Transformed 3 promoters (BBa_R0011, BBa_K864400, BBa_R0010) and killer red into E. coli
- BBa_R0010 and killer red on ampicillin, other two on chloramphenicol

Promoters plate 3, 5C (inconsistent) (chloramphenicol) -lambda pL hybrid plate 1, 17H (confirmed) (chloramphenicol) -ptac/trp/lac plate 5, 1D (confirmed) (Ampicillin) -WT lac promotor

Tuesday June 11th:
-Colonies on chloramphenicol plates had non-uniform size
---possible that chloramphenicol was rather old
-Left LB/cam plates out all night :(
---plated with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 to see if plates are still ok
-Ordered promoters and lambda arms/ packaging extract
-We inoculated 4 cell cultures and stored them in 37 degree room
-Cheryl is developing primers for cloning

Wednesday, June 12
-Plates with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 confirmed that plates are ok. Plate with strain that was only amp resistant had no colonies, two plates with chloramphenicol resistant plasmids had individual colonies, not a bacterial lawn (yay!)
-Mini-preps for all four plasmids
---Ran 2 uL DNA on gel- lanes right to left- standard; 5C lambda pL; 1D WT lac; 17H ptac/trp/lac; killer red

---Poor yield of 1D lac believed to be caused by pipetting error
---Multiple bands in KillerRed lane probably because plasmid is large and was nicked during prep

Thursday, June 13 -PCR of KillerRed and Primers
--Gel of PCR products:
Top row (left to right): ladder, Killer Red x3 with EcoKRF and EcoKRstR primers, eGFP x3 with EcoEGFPfor and EcoEGFPstR primers, Killer Red x3 with SpeRBSKRF and PstStKRrev primers
Bottom row (left to right): first two are Cheryls, no DNA controls x4 (For: KillerRed1, eGFP, KillerRed2, promoters from left to right), pTac/trp/lac, WT lac, lambda pl, ladder (SpeRBSKRF, PstStKRrev primers for promoters)

Friday, June 14
-repeated PCR with KillerRed and primers SpeRBSKRF and PstStKRrev
---primers were long and were at room temp for several minutes before cycler started, so we think they formed primer dimers based on gel from June 13
-made gel: ---0.4g agarose, 40ml TE buffer, swirl to break up agarose. microwave 1 min 30s in erlenmeyer
---Swirl again to prevent agarose settling and add 2 \micro\ liters ethidium bromide and swirl to mix
---Pour gel in taped mold
-Decided to clone KillerRed into plasmid (high copy?) first because phage parts won’t be here until next Wednesday
-Sent ptac promoter DNA (H17) and WT EColi lac promoter to be sequenced (s/b here by tues)
---5 μL H_2O, 5 μL DNA, 1 μL 10 μM primer (VF2)
---iGEM1: ptac promoter
---iGEM2: WT E. Coli lac promoter
-From left to right: ladder, KillerRed template with primers SpeRBSKRF and PstStKRrev x2, control and KillerRed with primers as first 2 rows (whoops) and control