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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
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 14, 2006: trial library (mnt)
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 baffled 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
Ligations into SP1.0
library
insert
vector
Q04400
2 ul
1.3 ul
Q04720
1.5 ul
1.7 ul
Resh (C2002)
1.5 ul
1.7 ul
Resh (C2003)
1.5 ul
1.7 ul
mnt A
4.6 ul
5 ul
mnt B
1.5 ul
1.7 ul
mnt C
0.46 ul
0.5 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, grew for >48 hrs (got very dense), and made 1 glycerol of each
Day 5 (August 6): second-round induction
thawed glycerols, spun down, aspirated media, and added to 10 ml M9+AG
mnt A = 0.33 (made 1 glycerol, labeled L.Q04720C.1)
innoculated 25 ml of M9+AG at 0% and 1e-4% arabinose with 20 ul of each culture (4 cultures total)
Day 6 (August 7): harvest
Q04400: grew ~17 hrs, harvested 1 ml & put on ice
0% final OD = 0.06
1e-4% final OD = 0.07
mnt A: grew ~12 hrs, harvested 1 ml & put on ice
0% final OD = 0.41
1e-4% final OD = 0.42
Day 7 (August 8): MoFlo
looked at libraries under no induction again (see how well it sorted last time) and under 1e-4% arabinose, didn't sort (picture)
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
plating
LB only: 1:1e6
LB+AG: 1:20, except 1:200 for uncut (+) control
Plating Results
plate
(+)
(+)
(+) PstI
(+) PstI
(+) PstI
(+) PstI
(+) PstI
mnt
mnt
mnt
amt ligase
N/A
1 unit
0 units
10 units
1 unit
1 unit
1 unit
1 unit
1 unit
1 unit
time
120 min
120 min
120 min
120 min
30 min
60 min
120 min
30 min
60 min
120 min
LB+AG
972
123 (drowned)
64
thousands
thousands
thousands
thousands
0
2
0
LB only
131
<--about same
<--about same
<--about same
<--about same
<--about same
<--about same
<--about same
<--about same
<--about same
July 31, 2006: more ligation (mnt library)
used the Q04720 library made on July 19
20-ul ligations with 10 units (1:40 dilution) of ligase per reaction
forgot to use MilliQ H20
also did a 10-ul ligation using 8.5 ng of cut (+) control (not gel extracted)
note: the numbers for the insert:vector ratio and the max library size below are calculated based on assuming I'm adding cut insert at ~27 ng/ul and cut vector at ~9 ng/ul -- I have no idea how accurate these numbers are
Ligations
insert
vector
ratio
max library size
0.2 ul
1 ul
~6
~1.6e9
2 ul
1 ul
~60
~1.6e9
16 ul
1 ul
~470
~1.6e9
1.25 ul
4 ul
~9
~6e9
let ligation go for 30 min before heat inactivating
dialyzed ligations for 30 min
electroporated 1 ul of dialyzed ligation into CW2553/pJat8
ran out of electrocompotent cells, only transformed using 0.2 ul, 2 ul, and 16 ul (insert amt) ligations
plating
LB only: 1:1e6
LB+AG: 1:20
put the rest of the 1 mL SOC transformation into 25 mL M9+AG and grew overnight
Plating results
plate
0.2 ul insert
2 ul insert
16 ul insert
LB only
230
<--about same
<--about same
LB+AG
1
2
2
August 01, 2006: electrocompetent cells
made new electrocompetent CW2553/pJat8 (~45 tubes, 40-ul aliquots)
final OD at 1:100 dilution = 0.56
stock at ~3e10 cells/ml --> ~1.2e9 cells per 40 ul tube
(+) control transformation:
LB+AG (1:2000 dilution) = 155 colonies
LB only (1:1e6 dilution) = 258 colonies
survival rate = 21.5%
transformation efficiency: 3.1x108 transformants / ug DNA
transformation frequency: 9.13x10-4 transformants / molecule DNA
August 03, 2006: ligations again
vector: SP1.0 (20-ul XP digest with 1.5 ug DNA, gel extracted and eluted with 30 ul EB)
insert:
(A) Q04720 (20-ul XP digest with 1.5 ug DNA, gel extracted and eluted with 30 ul EB)
(B) Q04400 (~20 ng target DNA mutagenic PCR VF2-VR, cleanup with 30 ul EB, 50-ul XP digest, gel extracted and eluted with 30 ul EB)
ligation with 400 units (no dilution) of ligase in 20-ul reaction for ~30 min
ligations with the following vector+insert amts (18 total reactions, A1-A9 and B1-B9):
0.02 ul vector
0.2 ul vector
2.0 ul vector
0.02 ul insert
1
2
3
0.2 ul insert
4
5
6
2.0 ul insert
7
8
9
30 min dialysis
electroporation with 1 ul dialyzed ligation
plating:
LB only = 1:1e6
LB+AG = 1:20 (50 ul)
Plating Results: Q04720 (miniprep)
LB+AG
0.02 ul vector
0.2 ul vector
2.0 ul vector
LB only
0.02 ul vector
0.2 ul vector
2.0 ul vector
0.02 ul insert
0
1
0
0.02 ul insert
0.2 ul insert
0
0
12 (drowned)
0.2 ul insert
363
2.0 ul insert
2
0
158
2.0 ul insert
229
Plating Results: Q04400 (mutagenic PCR)
LB+AG
0.02 ul vector
0.2 ul vector
2.0 ul vector
LB only
0.02 ul vector
0.2 ul vector
2.0 ul vector
0.02 ul insert
0
0
1
0.02 ul insert
0.2 ul insert
2
1
5
0.2 ul insert
smeared
2.0 ul insert
1
0
23
2.0 ul insert
323
plan: use more DNA!
August 04, 2006: inverter libraries
Preparation of vector
three 50-ul XP digests of SP1.0+P1010 with 5.5 ug of DNA in each
two gel lanes per digest
one spin column (max binding capacity is 10 ug) for every two lanes
try to keep agarose under 400 mg
elute all three columns with the same 30 ul of EB
use 8.5 ul of eluted digest in ligation
Preparation of insert
mutagenic PCR on Q04720.pSB1A2 / Q04400.pSB2K3 / B0030.C2002.B0015.p8.pSB1AC3 / B0030.C2003.B0015.p8.pSB1AC3
Q04720#1: PCR cleanup, elute with 30 ul, use 8.5 ul in ligation
Q04720#2: gel extraction, elute with 30 ul, use 8.5 ul in ligation
Q04400: PCR cleanup, elute with 30 ul (haven't used)
C2002 QPI: PCR cleanup, elute with 30 ul, use 8.5 ul in ligation
C2003 QPI: PCR cleanup, elute with 30 ul, use 8.5 ul in ligation
Ligation
8/5/06: Q04720#1 and Q04720#2 ligations & transformations
2 ul 10X T4 DNA ligase buffer
8.5 ul vector digest
8.5 ul insert digest
1 ul (400 units) T4 DNA ligase
8/6/06: C2002 QPI and C2003 QPI ligations & transformations
2 ul 10X T4 DNA ligase buffer
4.4 ul vector digest (all I had left)
8.5 ul insert digest
1 ul (400 units) T4 DNA ligase
H20 to 20 ul
note: if the estimated efficiencies of previous steps are correct, this amt of DNA should be below 200 ng, the maximum recommended by NEB
used 3 ul of dialyzed ligation product (recovered ~10-15 ul from dialysis) for transformations
Transformation
put on ice after electroporation for a few minutes before moving to 37 C, as per Knight lab protocol
after 1 hr incubation, added 25 ml M9+AG and grew ~17 hrs overnight (~12 hrs for Reshma's QPI's)
plating:
LB only = 1:1e6
LB+AG = 1:20
Plating Results
Q04720#1 (PCR cleanup)
Q04720#2 (gel extraction)
C2002 QPI
C2003 QPI
LB only
smeared, maybe ~600?
164
304
269
LB+AG
lots
171
smeared
smeared
replate Q04720#1
LB only = 1:1e7 --> none, maybe serial dilution went bad in 4 C overnight
LB+AG = 3:1000 (plate 3 ul of original transformation) --> 47 colonies
replate C2002 QPI and C2003 QPI, LB+AG = 1:200
C2002 QPI --> ~83 colonies, cells were clumpy
C2003 QPI --> ~247 colonies, cells were clumpy
Library size
Q04720#1 = 1.5e4
Q04720#2 = 3.4e3
C2002 QPI = 1.6e4
C2003 QPI = 4.9e4
Induction
made 3 glycerols of both of Reshma's QPI libraries from the overnight cultures (labeled L.C2002.0 and L.C2003.0, note: not part names, names of the repressor protein)
spun down overnight cultures, aspirate off SOC/M9 media, resuspend in 10 ml M9, took OD's:
Q04720#1 = 0.41
Q04720#2 = 0.43
C2002 QPI = 0.42
C2003 QPI = 0.38
added 1 ml of resuspension to 10 ml M9+AG (should be more than enough cells to cover our library size at this OD)
grew ~7-8 hrs, took OD's:
Q04720#1 = 0.29 (made 3 glycerols of this labeled L.Q04720A.0)
Q04720#2 = 0.40 (made 1 glycerol of this labeled L.Q04720B.0)
C2002 QPI = 0.14
C2003 QPI = 0.17
innoculated 25 ml M9+AG at 0% and 1e-4% arabinose with 50 ul of each culture
