User:Kchang17/notes

February 28, 2006

• characterization of penI QPI in I13537.pSB1A3 with three replicates (replicate 1) (concatenated) (MATLAB analysis)
• rough measurement of original (unmutated) tetR inverter, Q04400 (one promoter SP1.0) (two promoter SP (I13537))
• empty screening plasmids at 0%, 1e-4%, and 3e-4% arabinose inductions
  • I13534.pSB1A2 (picture)
  • I13537.pSB1A2 (picture)
  • I13538.pSB1A2 (picture)
  • I13534.pSB4A3 (picture)

March 09, 2006

• Q04400 inverter library in I13534.pSB1A2 (SP1.0) under a range of inductions (...details?) (uncalibrated) (calibrated)

March 15, 2006

• first measurement of mutated tetR QPI Q04400.007 (obtained from inverter library) in SP1.0 (calibrated) (concatenated) (MATLAB analysis)

June 22, 2006

• tested everything in SP1.0 at 6 arabinose levels (0%-10^-4%)
• Q04400.007 innoculated at OD 0.0005 and grew for ~10 hrs (picture) (MATLAB analysis)
  • note: 10 hrs probably wasn't enough time for it to reach steady state esp. at high induction, assuming inverter was working better on July 11
• Q04740 exptl cultures grew too dense - performed an extra dilution and redid the innoculation - innoculated at OD 0.0005 and grew for ~14 hrs
• I14103 - grew very slowly - innoculated at OD 0.001 and grew for ~14 hrs (grew a little too dense)

June 29, 2006

• devices in SP1.0, six arabinose levels (0%-10^-4%)
• Q04720 - innoculated at 0.0003 - grew ~15 hrs (too dense) (picture) (uncalibrated)
• Q04730 - innoculated at 0.0003 - grew ~16 hrs (too dense) (picture)
  • during construction, a point mutation occurred in the ORF of the repressor protein (C0073) resulting in a change from Phe to Leu
• Q04740 - innoculated 0.0003 - grew ~16 hrs (too dense) - weirdness, where did GFP and RFP go?
• I14103 - innoculated at 0.0005 - grew ~15 hrs (too dense) (picture)
• I14104 - having trouble cloning this, have to wait on testing

July 6, 2006

• picture
• induced ~7 hrs at no and high induction
• 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.
• devices in SP1.0, six arabinose levels (0%-10^-4%)
• Reshma's constructs:
  • B0030.C2002.B0015.p8 (A) - innoculated at 0.0001 - grew ~14.5 hrs (picture) (uncalibrated)
  • B0030.C2003.B0015.p8 (B) - innoculated at 0.0001 - grew ~15 hrs (picture) (uncalibrated)
• 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
    • i.e.: this isn't what we want
• empty SP1.0 (E) - innoculated at 0.0001 - grew ~14.5 hrs (picture)
• Q04400.007 (F) - innoculated at 0.0001 - grew ~14.5 hrs (picture) (concatenated) (MATLAB analysis)

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) (uncalibrated)
  • 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 library

July 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
• Stratagene's GeneMorph® II Random Mutagenesis kit
• using VF2-VR (24 pmol each per 50 ul reaction)
• 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
• grew for ~7 hrs, took OD's:
  • Q04400 = 0.39 (made 1 glycerol, labeled L.Q04400.1)
  • 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
  • initial amt target DNA = 20 ng
• PCR cleanup and elution with 30 ul EB; nanodrop:
  • Q04720 PCR reaction #1 = 120.7 ng/ul
  • Q04720 PCR reaction #2 = 110.6 ng/ul
  • Q04400 PCR reaction = 62.5 ng/ul, looked bad, contamination?
  • C2002 QPI PCR reaction = 120.5 ng/ul
  • C2003 QPI PCR reaction = 124.3 ng/ul
• 50-ul digest with all 30 ul of PCR product
  • 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
• Q04720#1: grew ~12 hrs, harvested 1 ml
  • 0% final OD = 0.44
  • 1e-4% final OD = 0.46
• Q04720#2: grew ~12 hrs, harvested 1 ml
  • 0% final OD = 0.58
  • 1e-4% final OD = 0.57
• C2002 QPI: grew ~13 hrs
  • 0% final OD = 0.50
  • 1e-4% final OD = 0.54
• C2003 QPI: grew ~13 hrs
  • 0% final OD = 0.60
  • 1e-4% final OD = 0.60

MoFlo

• collected data for all 4 libraries (picture)
• sorted into 1 ml M9
  • C2002 low in/high out (66 cells), high in/low out (230 cells)
  • C2003 low in/high out (985 cells)
  • Q04720 #1 low in/high out (983 cells), high in/low out (1100 cells)
  • Q04720 #2 low in/high out (1118 cells)

Second round

• added the collected cells to 5 ml M9+AG, plus the same arabinose condition under which they were sorted
• grew overnight and throughout the day until cultures were dense (OD roughly 0.3)
• put 1 ml samples on ice (just looking at these samples to see how well they sorted)
• for C2002 QPI sorted low in/high out (~66 cells), added 10 ul to a new 5 ml culture at 1e-4% arabinose

MOFLO

• results
• the ~66 cell sublibrary of C2002 QPI looked all negative fluoresence at no and high induction --> gate too stringent?
• sorted Q04720#1 low in/high out (>1000 cells) and high in/low out (~750 cells) under the same conditions, trying to tighten up the sublibraries

August 09, 2006

Restriction Digests

• overnight digests of:
  • P1010 in SP1.0, XP, 10 ug
  • B0032.C2002.B0015 in SP1.0, SP, 10 ug
  • B0030.C2003.B0015 in SP1.0, SP, 10 ug
  • Q04740 library, XP, 3.5 ug (all of PCR product)
  • Q04400 library, XP, 2.4 ug (all of PCR product)
• gel extraction of SP1.0 backbone
• PCR cleanup of the rest, into 30 ul EB

August 22, 2006

Day 1 (Aug 22)

Restriction Digests

• 1 hr, 50 ul digests with 1 ul each enzyme
  • P1010 in SP1.0, XP, 10 ug (PCR clean only, to be compared to O/N digest & gel extracted backbone)
    • x2 --> using a lot of the vector (8.5 ul per ligation)
    • DNA concentration after PCR cleanup = 328 ng/ul
• forgot to put in warm rm, incubated at rm temperature for 1 hr instead but it seemed to work ok

  

  on the left is 0.6 ul (out of 30 ul elution) of O/N, gel extracted backbone from August 10; on the right is 1 ul (out of 50 ul digest reaction) of 1 hr digest from today, not PCR cleaned yet

on the left is <0.6 ul (out of 30 ul elution) of O/N, gel extracted backbone from August 10; on the right is 1 ul (out of 30 ul elution) of 1 hr, PCR cleaned digest from today (meant to do 0.6 ul)

Ligations

• compare undiluted (400 units of ligase) and 1:10 dilution of T4 DNA ligase for Q04720
  • for all others use the 1:10 dilution --> 40 units of ligase
• digests from August 8 (PCR cleaned only)
  • C2002 QPI library (97.8 ng/ul)
  • C2003 QPI library (96.7 ng/ul)
  • Q04720 library #1 (93.2 ng/ul)
• digests from August 10 (PCR cleaned only)
  • Q04400 library (71.1 ng/ul)
  • Q04740 library (109.2 ng/ul)
• use 8.5 ul of digested insert + 8.5 ul vector
• 6 total reactions
• >30 min incubation at rm temp
• heat inactivation 65 C for 10 min
• dialysis for 30 min

Transformations

• electroporated 3 ul of dialyzed ligation reaction and incubated 1 hr
• plating:
  • LB+AG = 50 ul (1:20 dilution)
  • LB only = 1:1e6 dilution

Day 2 (Aug 23)

• spun down the library O/N cultures and resuspended in 10 ml of M9, took OD's
• undiluted ligase had ~3.5x more transformants --> proceed with this, throw out the other Q04720 library

plating, etc.

library	LB only	LB+AG	OD	lib size	x lib size	innoc. vol
Q04720 (undil.)	167	438x4	0.34	3.5e4	60	13 ul*
Q04740	128	777	0.33	1.6e4	60	6 ul
Q04400	174	162	0.37	3.2e3	275	5 ul
Q04720 (dil.)	142	505	N/A	1.0e4	N/A	N/A
C2003 QPI	100	231	0.23	4.6e3	200	9 ul
C2002 QPI	179	378	0.34	7.6e3	125	6 ul

• innoculated into 50 ml expt'l cultures, low & high induction, except Q04720 library into 100 ml cultures
• grew overnight at 37 C

Day 3 (Aug. 24): MoFlo

• harvested 1 ml from libraries after 16.5 hrs

OD's

	Q04400	Q04720	C2003 QPI	C2002 QPI	Q04740
0%	0.02	0.36	0.33	0.09	0.31
1e-4%	0.03	0.37	0.37	0.11	0.30

• picture (calibrated)
  • sorted Q04400 library under 0% arabinose (~6,000 cells)
  • sorted Q04740 library under 0% arabinose (~10,000 cells)
  • sorted Q04740 library under 1e-4% arabinose (~8,000 cells)
• sorted into 1 ml of M9+AG, put into warm room at noon
• also looked at 2 colonies of Q04740 in SP1.0 (obtained from stock plate) that had been grown directly from plate for ~24 hrs (growing very slowly, took this long to achieve a a good density) (picture)
  • put what's left of the 5 ml cultures back into warm room to prep/sequence

Day 4 (Aug. 25)

• grew sorted cells ~22 hrs, added 5 ml M9+AG, put into warm rm at 10:30 a.m.
• took out at 5 p.m.
• OD's:
  • Q04740 sorted low in/high out = 0.31
  • Q04740 sorted high in/low out = 0.29
  • Q04400 sorted low in/high out = 0.32
• made 2 glycerols of each, put rest in fridge
• (I think...)
  • Q04740 low in/high out labeled L.Q04740.1.0
  • Q04740 high in/low out labeled L.Q04740.1.1
  • Q04400 low in/high out labeled L.Q04400.1.0

Day 5 (Aug. 28)

• innoculated experimental cultures with ~9e5 cells into 50 ml M9+AG (0% and 1e-4% arabinose) using libraries stored in the fridge over the weekend (6 ul of the library into each culture, OD's were about the same as on Aug. 25)
  • 5 p.m.
• sequencing of Q04740 in SP1.0 colony#1 all came up OK (VF2, VR, E0040F, E0040R, E1010F, E1010R)
  • a point mutation present in pSB1A2 backbone (pMB1 replication origin)--also showed up in sequencing for I14103 and all other times I sequenced Q04740 in SP1.0 with E1010F--probably came from a stock of SP1.0--don't know if this affects anything

Day 6 (Aug. 29): MoFlo

• grew sublibraries ~17 hrs
• OD's came up around 0.52
• moflo run
• sorted L.Q04400.1.0 for high in/low out --> L.Q04400.1.0.1
  • 7268 cells
• grew in 1 ml M9+AG until next day

Day 7 (Aug. 30)

• added 5 ml to the sublibrary, grew ~7.5 hrs
  • OD = 0.83
• made 1 glycerol: L.Q04400.1.0.1
• added 1.4e6 cells to 25ml experimental cultures (0%, 1e-4%)

August 28, 2006: Reshma's promoter libraries

• double-stranded library oligos (25 pmol each)
  • L.RS.P8
  • L.RS.P9
  • L.RS.R2000
• saved 1 ul (out of 100 ul PCR reaction) for gel analysis

  

  1ul of PCR reaction compared next to 1ul of 1:40 dilution of original single-stranded library (lanes alternate)

• PCR cleaned the rest into 30 ul EB

Restriction Digests (August 29)

• 20min, 37 C restriction digests on libraries (XP), heat inactivation
• PCR cleanup & elution into 30ul
  • L.RS.P8 = 42.1ng/ul * 30ul
  • L.RS.P9 = 41.5ng/ul * 30ul
  • L.RS.R2000 = 39.6ng/ul * 30ul

• backbones from August 09: cut SP
  • B0032.C2002.B0015 in SP1.0 = 319.9ng/ul * 30ul
  • B0030.C2003.B0015 in SP1.0 = 300.5ng/ul * 30ul

• used 1ul of these samples for gel analysis

Ligation

• 6 ligation reactions with 8.5ul insert + 8.5ul vector
• undiluted ligase (1ul) in 20ul reaction volume
  • messed up with L.RS.P8 into C2002 backbone reaction --> had to bump up the volume to 47ul, but there's still just 1ul ligase
• 30min rm temp incubation, heat inactivation
  • save 1ul for gel analysis
• dialysis 30min

comparisons of restriction digests, ligations, and dialyzed ligations (1ul volumes of each); left to right: C2002 backbone (SP), C2003 backbone (SP), P8 libary (XP), P9 library (XP), R2000 library (XP), P8/C2002 ligation, P8/C2002 dialysis, P9/C2002 ligation, P9/C2002 dialysis, R2000/C2002 ligation, R2000/C2002 dialysis, P8/C2003 ligation, P8/C2003 dialysis, P9/C2003 ligation, P9/C2003 dialysis, R2000/C2003 ligation, R2000/C2003 dialysis

same gel with longer UV exposure to help see the insert better

Transformation

• electroporated 3ul, except for the one I messed up, did 6.3ul for that one
• on ice, then incubate at 37 C for 1hr
• plated:
  • LB+AG 1:40 dilution (25ul of transformation directly)
  • LB only 1:1e6 dilution
• added 10ml M9+AG, grew overnight ~12.5 hrs

Library sizes (August 30)

Plating Results

library	LB+AG	LB only	library size
P8/C2002	1391x2	305	1.1e5
P9/C2002	353x8	185	1.1e5
R2000/C2002	411x8	177	1.3e5
P8/C2003	385x8	178	1.2e5
P9/C2003	515x8	194	1.6e5
R2000/C2003	512x8	156	1.6e5

Induction

• spun down, resuspended in M9+AG

Plating Results

library	OD
P8/C2002	0.53
P9/C2002	0.45
R2000/C2002	0.49
P8/C2003	0.43
P9/C2003	0.43
R2000/C2003	0.34

• made glycerols (labeled with L.RS.P8/P9/R2000 and B0032.C2002.B0015/B0030.C2003.B0015)
• added 1.6e6 cells to 25ml experimental cultures (0%, 1e-4%)
  • this is about 10x the library sizes

MOFLO

• fire alarm at flow cytometry center, no data :(
• libraries didn't seem to have working clones