the single red colony on a plate (probably got mixed with some white colony) from Q04740 in SP1.0 glycerol - got high RFP signal and GFP tracked induction - streaked this culture for July 11 testing
one white colony from the same plate - GFP showed this time - background RFP
empty SP1.0 - showed GFP and RFP
July 11, 2006
Colony PCR of Q04740 colonies, E0040F-E1010R. Two red and two white colonies. Region should be ~1.6 kb. Red colonies ~3 kb (transposable element jumped in). White colonies ~1.6 kb.
grew to stationary, then diluted back 100x, grew for ~8 hrs (cultures were all very light except for the empty SP and Q04400.007), innoculated at ~11:30 p.m.
clones from July 6's penI red no induction culture, streaked on plate:
Q04740 red colony #1 (C) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
Q04740 white (pink) colony #4 (D) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
sequencing results: red colony had IS5 element jump in after the hairpin / before the prefix; white colony, when grown up/prepped/sequenced had a population with the insertion element in the same spot and a population without IS5
July 14 mutagenic PCR, lanes left to right: 188 ng, 75 ng, 12.5 ng target DNA, 1.1 kb standard (100ng)July 18 gel of restriction digest (XP) of Q04720 libraryJuly 20 MoFlo run of library under no induction
Day 1 (July 14): mutagenic PCR
trying out mutagenic PCR on Q04720.pSB1AK3 (mnt) - the prep for this was taken from MC4100, so pJat8 isn't in there and I know how much DNA I'm adding
three 50 ul PCR reactions: (note, I goofed, meant to do 4x these amts so I could cover low-med-high mutation frequencies but forgot to take the vector into account)
~188 ng target DNA (medium range mutation freq)
~75 ng target DNA (medium range mutation freq)
~12.5 ng target DNA (high range mutation freq)
PCR cleanup
Day 1.5 (July 17): restriction digest
did overnight double digest (XP) on PCR reactions
Day 2 (July 18): ligation and transformation
ran digest on a gel, did gel extraction - eluted into 30 ul EB total
performed a 40-ul ligation reaction using all of the high range mut freq digest + 1 ul cut SP1.0
dialyzed - after dialysis, total volume was 13 ul
used 6 ul for a transformation that arced
used 3 ul for a transformation that worked (~4 ul left over)
dialyzed 40 ul of positive control (2 ng/ul?) - took 1 ul of the diaylzed plasmid and transformed it
did a negative control electroporation with CW2553/pJat8
grew in 1 mL SOB for 1.25 hrs
plated 50 ul (1:20) of the experimental (AG), positive control (Amp, could have used AG in CW2553/pJat8), and negative control (AG) on antiobiotic plates
plated 1:1e7 of expt'l and controls on LB only
put the rest of the transformation 1 mL - 65 ul into 9 mL of supplemented M9 in buffeted (?) flasks around 7:30 or 8 p.m.
put plates in the warm room around 9 p.m.
Day 3 (July 19): arabinose induction
the 10 ml culture grew ~15-16 hours overnight: end OD = 0.27
set up 25 ml experimental cultures at 0% and 1e-4% arabinose
added 1 ml or 0.5 ml of overnight (4 flasks total)
plate results:
+ control:
1:1e7 dilution on LB only = 11 colonies
1:20 dilution on LB+AG = lawn of cells --> replate with a 1:1e5 , 1:1e4
- control:
1:1e7 on LB only = no colonies --> replate 1:1e5, 1:1e4
1:20 on LB+AG = no colonies
Q04720 library:
1:1e7 on LB only = no colonies on LB only --> replate at 1:1e5, 1:1e4
1:20 on LB+AG = 1 colony
Day 4 (July 20): MOFLO
ran the no induction library culture on the MoFlo
forgot about the high induction culture (ooops)
replating results:
+ control: 1 colony on LB+AG at 1:1e4 dilution, none at 1:1e5 dilution
- control: no colonies on LB only at 1e4 or 1e5
Q04720 library: no colonies on LB only at 1e4 or 1e5
July 19, 2006: inverter libraries
July 24 gel of restriction digests (XP) left-right, top-bottom (2 lanes per reaction): P1010.SP1.0, Q04400 library, Q04720 library, Reshma's QPI (C2002) library, Reshma's QPI (C2003) library
Day 1 (July 19): mutagenic PCR
started with 20 ng target DNA
used VF2-VR
inverters:
Q04400
Q04720 (also try BioBricks primers I ordered)
Reshma QPI C2002
Reshma QPI C2003
BioBricks primers didn't work -- melting temp too high? ran at 45 C, ~20 C below Tm
Day 1.5 (July 20): restriction digest
overnight restriction digest (XP) on all PCR reactions and on SP1.0 with P1010 insert
Day 2 (July 24): restriction digest cleanup
separated inserts on a gel and extracted
SP1.0 digest looked low, didn't use enough of the prep - didn't extract this
Day 2.5 (July 25): ligation and transformation
Ligations
20 ul ligations with 1 ul T4 DNA ligase and ~6:1 molar ratio insert:vector, assuming PCR insert ~17 ng/ul and vector ~25 ng/ul
Q04400 library (6.7 ng DNA total)
Q04720 library (6.7 ng DNA total)
Reshma QPI C2002 (6.7 ng DNA total)
Reshma QPI C2003 (6.7 ng DNA total)
20 ul ligations with old ~188 ng mnt library (July 14), probably ~4:1 molar ratio insert:vector since I used 1/4 of the PCR product for a gel
20 ng DNA total (max for efficiency range suggested by NEB)
6.7 ng DNA total
2 ng DNA total (min for efficiency range suggested by NEB)
Transformations
Q04400: used 3 ul and it arced, used 1 ul that worked
Q04720: used 1 ul worked
Reshma QPI C2002: used 1 ul arced, 0.5 ul arced repeatedly, finally 0.25 ul worked
Reshma QPI C2003: used 1 ul worked
old mnt 20 ng (A): 1 ul arced, 0.5 ul worked
old mnt 6.7 ng (B): 1 ul worked
old mnt 2 ng (C): 1 ul worked
Plating
plating on LB only:
all 1:1e5
plating on LB+AG:
(+) 1:200
(-) 1:20
expt'l 1:20
Overnight cultures
put the 1-mL SOC transformations into 25 mL supplemented M9 + AG at 9:40 p.m.
initial OD for all cultures = 0.04, except Reshma's QPI C2002, which was 0.03
Day 3 (July 26): harvest cells
at 9:25 a.m., OD ~0.19 (cultures all looked similar)
Plating Results
plate
(+)
(-)
Q04400
Q04720
Resh(C2002)
Resh(C2003)
mnt A
mnt B
mnt C
LB+AG
875
0
3
1
0
11
1
1
0
LB only
1102
~1000
~1000
~1000
~1000
~1000
~1000
~1000
~1000
since none of the libraries were very big, just took 1 mL of the overnight (grew ~15 hrs) and put on ice for the MOFLO
Day 4 (July 27): MOFLO
ran all 7 libraries (no induction) and collected data (picture)
sorted Q04400 (200 cells) and mnt A (150 cells) for high RFP into 1 mL M9
put in 5 ml M9+AG in warm room around noon
July 20, 2006
everything except GFP only control (and negative control) in SP1.0
grew overnight, diluted back 50x around 2 p.m., grew ~6 hrs
at this point, the first 3 penI cultures (1,2,3) gave an OD of 0.00 (used 50 ul), the fourth (4) 0.05 (10 ul)
Q04400 (40) OD = 0.01 (50 ul)
Q01400 (14) OD = 0.16 (3.125 ul)
put experimental cultures in at 9 p.m.
Q04740 (from a plate from original glycerol) (no induction and 1e-4%) (picture)
colony 1 - innoculated at ??? no OD reading - grew 16.5 hrs
colony 2 - innoculated at ??? no OD reading - grew 16.5 hrs
colony 3 - innoculated at ??? no OD reading - grew 16.5 hrs
colony 4 - innoculated at 0.0001 - grew 16.5 hrs
tetR inverters (started overnights from glycerols) (all 6 induction levels)
Q04400 - innoculated at 0.0001 - grew 13.5 hrs (picture)
Q01400 - innoculated at 0.0001 - grew 13.5 hrs (picture)
GFP only control (I13522) - started overnight ~5 p.m. - harvest at 10:30 a.m. (picture)
CW2553/pJat8 negative control - started overnight ~5 p.m. - harvest at 10:30 a.m.
July 27, 2006: ligation controls
(+) control:
1 ng straight electroporation (diluted in MilliQ H20)
20 ul "ligation" with 12 ng of pUC18 DNA and 1 unit (1:400 dilution) of T4 DNA ligase (2 hrs)
(+) control single cut (PstI):
20 ul "ligation" with 12 ng of DNA and no ligase (2 hrs)
20 ul ligation with 12 ng of DNA and 10 units (1:40 dilution) of T4 DNA ligase (2 hrs)
20 ul ligation with 12 ng of DNA and 1 unit of T4 DNA ligase (30 mins, 1 hr, 2 hrs)
mnt library:
PCR insert from July 19 cut XP (~17 ng/ul? did QiaQuick gel extraction and nanodrop gave ~23 ng/ul)
SP1.0 vector from an XP digest that was extracted using Qiaex II kit (nanodrop gave ~9.3 ng/ul)
used 2.3 ul insert and 6.7 ul vector for every ligation, which is maybe ~6:1 ratio insert:vector, ~100 ng DNA total
20 ul ligation with ~100 ng DNA and 1 unit of T4 DNA ligase (30 mins, 1 hr, 2 hrs)
used MilliQ H20 for ligation reactions
dialyzed 30 mins
electroporated with 0.75 ul of dialyzed ligation
(+) uncut with 1 unit ligase I accidentally drowned - electroporated 2 ul of this
mnt 30 min ligation arced, worked 2nd time
(+) PstI with 10 units ligase arced, worked 2nd time
