Team:Carnegie Mellon/Week3

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'''Monday, June 24'''
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{{:Team:Carnegie_Mellon/Templates/header}}
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9:20 am started new culture with 1088, 1089, and 1090 in LB with maltose and Mg (in incubator on 2nd floor)<br>
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0.5 L LB/ amp agar <br>
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1 ml amp/ 1000 ml agar<br>
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Start new overnights from plates for VSC537, 1088, 1089, and 1090<br>
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'''Monday, June 17th'''
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Streaked XL10 Gold Ultracompetent cells<br>
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-Oligos arrived<br>
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streak VSC537, 1088, 1089, and 1090<br>
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-mRFP on plate 3 12N (BBa_E1010) <br>
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Need to start overnight cultures of KR and RFP<br>
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<html><img src="https://static.igem.org/mediawiki/2013/c/c6/Cmuplate1.png"></html><br>
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-Top Row (left to right): ladder, KillerRed with BB promoters (SpeBBKRR and XbaBBKRF)  x3, control without DNA<br>
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---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<br>
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-Purified PCR products (KR biobrick, RFP sequence)
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'''Tuesday, June 25th'''<br>
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'''Tuesday, June 18'''<br>
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Didn’t start RFP or KR cultures. <br>
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-Verified sequence of plasmid with ptac (H17) and WT lac (D1)<br>
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Streaked plates look excellent (single colonies)<br>
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-Digest WT lac (D1), RFP, EGFP and KR with PstI and SpeI (2 hour digestion at 37ºC)<br>
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Need to start overnights of the plates for tomorrow<br>
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ends at 12:30pm<br>
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Packaging extract started: 9:46 end: ~11:30<br>
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-Gel purify fragments in 1% agarose<br>
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Need chloroform, IPTG/X-gal (from Cheryl)<br>
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---D1 - RFP - EGFP - KR - Ladder - Cheryl’s<br>
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Started new culture of VCS257 in LB w/ Mg and Maltose for afternoon infection ~12:30<br>
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-Media-preparing supplies arrived<br>
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Nanodrop results:<br>
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-Purified by GeneJet<br>
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RFP: 5.8ng/µL 260/280=1.88 260/230=.04<br>
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-Ligation by Thermo-Fisher  T4 ligase kit<br>
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KR: 26.4ng/µL 260/280=1.85 260/230=.1<br>
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---6:2 µL RFP and EGFP ligations<br>
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Possible carbohydrate contamination indicated by low 260/230 ratios (possible agarose contamination?)
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---2:2 µL KR since KR band was very strong<br>
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Transformation<br>
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---2 min on ice<br>
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---5 min at 42ºC<br>
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---2 min  on ice<br>
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---add 500 µL LB and incubate at 37ºC for 1 hr<br>
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---Plate on LB/Amp plates and incubate overnight
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'''Wednesday, June 19'''<br>
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RFP and KillerRed plates have many colonies EGFP has about 10 colonies.
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Start infection strain cultures at 12:15pm<br>
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Colony PCR for 4 EGFP, 4 KillerRed and 1 control of the plasmid DNA <br>
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Stored 100 10-3 10-4 10-5 dilutions at 4ºC<br>
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---Use VF2/VR primers<br>
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At 4pm, dilute, infect and plate<br>
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---PCR ends at 4:50pm<br>
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to top agar, add 15 µL IPTG and 25µL X-gal to 2.5mL top agar
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---Top Row (left to right): eGFP in pSC103 x4, KillerRed in psc103 plasmid x4, pSC103, ladder<br>
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<html><img src="https://static.igem.org/mediawiki/2013/1/1b/Cmugel3.png"></html><br>
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Use gel to confirm the sequence<br>
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pSC103 is smaller than pSC103 with KR insert-transformation verified
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'''Wednesday, June 26'''<br>
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'''Thursday, June 20'''<br>
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KR cells cultured in tubes covered in aluminum foil<br>
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Observations:<br>
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---Streaked plates grew successfully. Several colonies to choose from<br>
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---KR cultures are very turbid (no color)<br>
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---RFP cultures are bright red<br>
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---EGFP culture is turbid (no color)<br>
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---Negative control (VCS257) has no growth<br>
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---Phage strains are all turbid<br>
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Digestion of EcoKR<br>
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Find tube of KR from 6/13<br>
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Digestion started: 10:40<br>
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Digestion started RFP 11:33<br>
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End digestion around 1:30<br>
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'''Friday, June 21'''<br>
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KR cells kept covered in aluminum foil until photobleaching <br>
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Diluted cells at 12:45pm in LB+amp<br>
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---Note: amp should be added as 1/1000 dilution<br>
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Induction with IPTG: 1:45pm<br>
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---Note: IPTG should be added as 1/500 dilution (6µL added to 3mL)<br>
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---Check RFP for color change at 2:30 (no significant difference between induced and uninduced at 2:30 and 3pm)
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Photobleaching: 4-5pm?
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Only one potential plaque was observed on 10^-3 plate made on Tuesday. Hopefully it is actually a plaque and not just an irregularity in the top agar. <br>
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RFP + (light, 2 duplicates)<br>
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VSC257 cells infected with phage (undiluted and 10^-1) ~1:00 pm. <br>
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---1. induced<br>
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inoculated 1 ml SM buffer with picked phage <br>
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---2. not induced<br>
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200 μL of VSC257 cells from fridge with 10μL picked phage in SM<br>
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200 μL of VSC257 cells with 10μL of 10^-1 dilution of picked phage<br>
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200 μL of VSC257 cells with  10 μL of packaging control (iptg and x-gal added to top agar) <br>
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PCR RFP/KR<br>
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7x20µL reactions of RFP and KR with one tube of control for each (must repeat)<br>
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Induced RFP/KR cells at 1:45pm. No red color in either tube<br>
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2:50, no color in either tube<br>
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5:50, no color in either tube<br>
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PCR started again ending around 4:15<br>
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RFP - (no light)<br>
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5-minute digests were set up for 7 minutes<br>
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---not induced
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Ligation is sitting at 12ºC overnight using Thermo T4 ligase<br>
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KR + (light, 2 duplicates)<br>
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---1. induced<br>
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---2. not induced
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RFP - (no light)<br>
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---not induced
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photobleaching measured with UV/Vis (where?) <br>
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'''Thursday, June 27th'''<br>
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---6 LB/ amp plates at 10-5 dilution
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30 µL 1088 (lytic) cell line in 3 ml LB supplemented with maltose and Mg inoculated at 11am<br>
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@12pm checked OD600: 0.673  <br>
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Pelleted, diluted in 4 ml 10mM Mg<br>
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Packaging will be complete at 12:50 (add SM and chloroform to stop reaction)<br>
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Added 500µL 1M Magnesium sulfate to packaging extracts by accident, added 500µL of SM to help correct. Extra magnesium shouldn’t cause problems with infection since the magnesium will be diluted in the cells and top agar.
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Inactivation of KillerRed is about 20 minutes according to Evrogen<br>
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---http://www.evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml
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Inactivation of mRFP is about 10x faster than mCherry (which takes ~1.5-2 minutes)<br>
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---http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf
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Inactivation of RFP takes >30 minutes at 100% light<br>
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'''Friday, June 28th'''
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---Inactivation of KillerRed tubes were irradiated for 5 minutes at 100% light<br>
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---All tube were plated (100µL) without dilution. KillerRed should have lower viable cell counts.
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'''Saturday'''
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Phage plates from June 27th:<br>
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Rfp light, induced<br>
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RFP plates look good-both clear and blue plaques on IPTG/X-gal plates<br>
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Some uneven spreading but basically a lawn, fairly red<br>
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need to screen for phage which have KR in correct direction<br>
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<html><img src="https://static.igem.org/mediawiki/2013/a/ae/Cmuplate2.png"></html><br>
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approx. 20 clear plaques on 10^-2 dilution plate of RFP phage<br>
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1:35 pm started 1089 and 1090 for infection.<br>
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@2:19pm no observable increase in turbidity<br>
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@3:10pm.... still nothing :(<br>
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4:15pm...yay!<br>
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1089: OD600 of 0.366<br>
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1090: OD600 of 0.650<br>
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5:20: resuspend in 4 ml 10 mM Mg<br>
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Infected at 6pm with first 3 picked phages from 10^-2 dilution RFP plate from 6/27 in 1089 and 1090<br>
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Made more 10mM Mg<br>
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Make LB agar/pour plates/ autoclave 1ml tips<br>
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8x PCR of KillerRed ending 3:20pm. <br>
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NanoDrop results from yesterday’s PCR and digests:<br>
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KR digest: 54.6 ng/µL<br>
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260/280=2.01<br>
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260/230=.11<br>
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RFP digest: 72.1 ng/µL<br>
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260/280=1.83<br>
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260/230=.15<br>
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Possible lack of DNA for KR ligation although not likely. Will try to increase DNA concentration for subsequent digestions and ligations. In order to do this, the tube from yesterday’s undigested DNA (14µL left) and the 160µL from today’s PCR can be put onto a PCR purification column, cleaned and eluted in 30µL. Then digested and cleaned and then Nanodropped before the ligation step so achieve the .2µg requirement for the ligation.
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118 ng/µL for digested clean EcoKR 1.7µL was used for the overnight ligation to achieve the ideal 200ng.
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Rfp light, uninduced<br>
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More evenly spread, very small individual colonies observable near edges of plate. Both pink and non pink colonies
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Rfp no light uninduced<br>
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Some uneven spreading, lawn that is quite pink.
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'''Saturday, June 28th'''
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Kr no light uninduced<br>
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Eric came in and began packaging ~9am<br>
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Complete lawn, no color
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Kathy stopped the packaging and plated new KR phages
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�
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Kr light, induced<br>
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More or less a lawn (a few individual colonies observable), no observable color.<br>
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Kr light, uninduced<br>
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More uneven spreading than other two kr plates, some individual colonies observable, but it's basically a lawn. No color.

Latest revision as of 23:34, 27 September 2013

Killer Red



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.

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