Team:Duke/Notebook/Test

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Revision as of 04:43, 17 October 2014

9/10/14

Objective: assemble decoy binding site arrays

Setting up two PCRs with same cycling program as last time:

one with 4x 50uL reactions using the same concs of everything as the 0.5 uL rx from 9/9/14 (blue labels)
one with equimolar oligo1 and oligo2 and varying amounts of reaction 0.5 from 9/9/14 (green labels)

So something’s happening. PCR cleanup’d reactions 1-4 (green) and 5-8 (blue) in to one tube each labeled Decoy array (1-4 or 5-8) PCR1. Tomorrow will PCR with the end oligos and try gibson, dig/lig in to pSB1C3 and/or pSB1A2

9/11/14

Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays

Faw setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space as oligos on 0.5-2.0 ng of PCR cleanup’d green and blue rx from 9/10/14 and running with the same cycling conditions.
CBC did not take a picture and doesn’t remember why. PCR looked bad for some reason.

9/12/14

Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays

Garima setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space as oligos on higher concs than yesterday ofPCR cleanup’d green and blue rx from 9/10/14 and running with the same cycling conditions.
PCR cleanup to pool tubes 2-4 for both series and labeled Decoy Array PCR2

9/13/14

Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone

CBC attempts a SLIC with 0.5 uL of the “Super Clean” XbaI/PstI pSB1C3 backbone. Note that puts 15 bp of nonhomology in the way of successful SLIC. I did this because sometimes it works despite mismatches and there was no EcoRI/PstI dig’d pSB1C3 for me to work with. Green series looks like there is a bigger difference between no-template and +template tubes, so I will try that

set up tubes for backbone-only, backbone + 0.5, 1, 2, 3uL of insert from Green tube at 88.8 ng/uL, and 3uL insert-only.

Didn’t work.

Objective: observe fluorescence changes with antitracr time course over time

Last night antitracr strains started in LB+AmpKanCm and let shake overnight. Diluted this morning in to +/- aTc and began collection at time indicated. Replaced LB with PBS before flowing
Isolated “singlets” which are probably not singlets (smalls) or left alone (bugs) and got mean GFPA or gfpa/fsca or /ssca to normalize roughly to size.

time point	n/a	n/a	1	2	3	4	5
time (min)	n/a	n/a	120	180	240	300	360
GFP1-9	+ aTc	gfpa (smalls)	1.52	2.74	3.17	3.16	2.81
GFP1-10	+ aTc	gfpa (smalls)	1.39	2.8	3.18	3.42	2.88
GFP1-11	+ aTc	gfpa (smalls)	1.45	2.92	3.31	3.53	2.88
GFP1-12	+ aTc	gfpa (smalls)	1.53	3.13	3.72	3.6	2.89
blankpdCas9-9	+ aTc	gfpa (smalls)	45.6	56	68.4	76.8	74.9
blankpdCas9-10	+ aTc	gfpa (smalls)	46.9	54	67.8	73.8	74.7
blankpdCas9-11	+ aTc	gfpa (smalls)	48.4	53.1	65.8	73	76.2
blankpdCas9-12	+ aTc	gfpa (smalls)	45.6	54.2	68.2	74.3	78.4
pdCas9	+ aTc	gfpa (smalls)	0.109	0.16	0.185	0.139	0.143
GFP1-9	- aTc	gfpa (smalls)	1.43	3.08	3.65	3.67	3.07
GFP1-10	- aTc	gfpa (smalls)	1.32	3.17	3.67	3.88	3.07
GFP1-11	- aTc	gfpa (smalls)	1.73	3.3	4.15	3.99	3.18
GFP1-12	- aTc	gfpa (smalls)	1.57	3.47	4.16	3.83	3.29
blankpdCas9-9	- aTc	gfpa (smalls)	41.7	53.4	67	75.8	75.6
blankpdCas9-10	- aTc	gfpa (smalls)	41.4	51.3	67.4	74.3	71.7
blankpdCas9-11	- aTc	gfpa (smalls)	39.4	52.4	66	73.9	60.8
blankpdCas9-12	- aTc	gfpa (smalls)	40	52.7	67	76	73.2
pdCas9	- aTc	gfpa (smalls)	0.0949	0.156	0.162	0.129	0.122
GFP1-9	+ aTc	gfpa/fsca*1000 (smalls)	83.9	157	188	189	172
GFP1-10	+ aTc	gfpa/fsca*1000 (smalls)	70.6	159	186	205	177
GFP1-11	+ aTc	gfpa/fsca*1000 (smalls)	72.8	169	193	208	175
GFP1-12	+ aTc	gfpa/fsca*1000 (smalls)	80.1	172	206	199	166
blankpdCas9-9	+ aTc	gfpa/fsca*1000 (smalls)	2526	3227	3986	4592	4520
blankpdCas9-10	+ aTc	gfpa/fsca*1000 (smalls)	2700	3078	4044	4486	4543
blankpdCas9-11	+ aTc	gfpa/fsca*1000 (smalls)	2892	3028	3795	4291	4553
blankpdCas9-12	+ aTc	gfpa/fsca*1000 (smalls)	2641	3131	4038	4531	4614
pdCas9	+ aTc	gfpa/fsca*1000 (smalls)	6.26	9.36	11.1	8.92	9.11
GFP1-9	- aTc	gfpa/fsca*1000 (smalls)	72.8	177	221	228	194
GFP1-10	- aTc	gfpa/fsca*1000 (smalls)	60.5	187	220	241	193
GFP1-11	- aTc	gfpa/fsca*1000 (smalls)	94.8	192	245	245	199
GFP1-12	- aTc	gfpa/fsca*1000 (smalls)	79.6	193	235	222	193
blankpdCas9-9	- aTc	gfpa/fsca*1000 (smalls)	2184	3100	4012	4663	4666
blankpdCas9-10	- aTc	gfpa/fsca*1000 (smalls)	2188	2957	4160	4554	4386
blankpdCas9-11	- aTc	gfpa/fsca*1000 (smalls)	2004	3047	4035	4565	3842
blankpdCas9-12	- aTc	gfpa/fsca*1000 (smalls)	2039	3051	4107	4630	4512
pdCas9	- aTc	gfpa/fsca*1000 (smalls)	5.64	9.13	9.66	8.24	7.79
GFP1-9	+ aTc	gfpa/ssca*1000 (smalls)	47.7	75.1	78.8	75.1	64.8
GFP1-10	+ aTc	gfpa/ssca*1000 (smalls)	46.3	76.2	79.1	81	66
GFP1-11	+ aTc	gfpa/ssca*1000 (smalls)	48	78.7	82.6	83.3	66.4
GFP1-12	+ aTc	gfpa/ssca*1000 (smalls)	51.5	85.9	96.7	93.4	71.1
blankpdCas9-9	+ aTc	gfpa/ssca*1000 (smalls)	1273	1469	1666	1779	1718
blankpdCas9-10	+ aTc	gfpa/ssca*1000 (smalls)	1226	1413	1636	1719	1688
blankpdCas9-11	+ aTc	gfpa/ssca*1000 (smalls)	1182	1382	1605	1704	1739
blankpdCas9-12	+ aTc	gfpa/ssca*1000 (smalls)	1182	1391	1645	1714	1790
pdCas9	+ aTc	gfpa/ssca*1000 (smalls)	3.43	4.38	4.82	3.89	3.69
GFP1-9	- aTc	gfpa/ssca*1000 (smalls)	46.3	80.9	89.5	87.5	71
GFP1-10	- aTc	gfpa/ssca*1000 (smalls)	45.6	80.6	89.6	91.3	71.4
GFP1-11	- aTc	gfpa/ssca*1000 (smalls)	50.4	84.4	101	95	74
GFP1-12	- aTc	gfpa/ssca*1000 (smalls)	55.3	93.8	108	100	83.8
blankpdCas9-9	- aTc	gfpa/ssca*1000 (smalls)	1250	1375	1606	1758	1715
blankpdCas9-10	- aTc	gfpa/ssca*1000 (smalls)	1215	1320	1606	1734	1635
blankpdCas9-11	- aTc	gfpa/ssca*1000 (smalls)	1203	1319	1563	1718	1425
blankpdCas9-12	- aTc	gfpa/ssca*1000 (smalls)	1230	1338	1595	1754	1661
pdCas9	- aTc	gfpa/ssca*1000 (smalls)	3.05	4.55	4.63	3.67	3.35
GFP1-9	+ aTc	gfpa (bugs)	2.17	3.7	5.12	5.27	5.68
GFP1-10	+ aTc	gfpa (bugs)	2.1	3.82	5.06	5.77	5.85
GFP1-11	+ aTc	gfpa (bugs)	2.17	3.92	5.48	6.04	5.89
GFP1-12	+ aTc	gfpa (bugs)	2.07	4.2	5.49	5.95	5.85
blankpdCas9-9	+ aTc	gfpa (bugs)	70.3	82.2	105	120	133
blankpdCas9-10	+ aTc	gfpa (bugs)	74.4	78.2	102	122	137
blankpdCas9-11	+ aTc	gfpa (bugs)	75.6	76.9	102	114	130
blankpdCas9-12	+ aTc	gfpa (bugs)	75.3	79.9	103	120	136
pdCas9	+ aTc	gfpa (bugs)	0.169	0.26	0.396	0.319	0.34
GFP1-9	- aTc	gfpa (bugs)	2.26	4.28	6.12	6.62	6.7
GFP1-10	- aTc	gfpa (bugs)	2.01	4.53	6.07	7.13	6.9
GFP1-11	- aTc	gfpa (bugs)	2.72	4.59	6.58	7.36	7.43
GFP1-12	- aTc	gfpa (bugs)	2.16	4.63	6.39	7.15	7.22
blankpdCas9-9	- aTc	gfpa (bugs)	63.4	76.5	107	127	141
blankpdCas9-10	- aTc	gfpa (bugs)	64.1	74.2	112	127	134
blankpdCas9-11	- aTc	gfpa (bugs)	61.2	77.2	110	127	126
blankpdCas9-12	- aTc	gfpa (bugs)	63.3	78.4	110	128	138
pdCas9	- aTc	gfpa (bugs)	0.157	0.26	0.357	0.331	0.287
GFP1-9	+ aTc	gfpa/fsca*1000 (bugs)	88.5	159	205	206	216
GFP1-10	+ aTc	gfpa/fsca*1000 (bugs)	75.6	163	197	228	220
GFP1-11	+ aTc	gfpa/fsca*1000 (bugs)	77.3	172	210	232	217
GFP1-12	+ aTc	gfpa/fsca*1000 (bugs)	83.4	174	215	227	223
blankpdCas9-9	+ aTc	gfpa/fsca*1000 (bugs)	2559	3229	4012	4818	5003
blankpdCas9-10	+ aTc	gfpa/fsca*1000 (bugs)	2751	3078	4107	4768	5130
blankpdCas9-11	+ aTc	gfpa/fsca*1000 (bugs)	3006	3014	3787	4456	4927
blankpdCas9-12	+ aTc	gfpa/fsca*1000 (bugs)	2695	3129	4076	4738	5032
pdCas9	+ aTc	gfpa/fsca*1000 (bugs)	6.33	10.1	15.8	12.9	14.1
GFP1-9	- aTc	gfpa/fsca*1000 (bugs)	78.4	182	242	263	257
GFP1-10	- aTc	gfpa/fsca*1000 (bugs)	64.6	194	236	278	259
GFP1-11	- aTc	gfpa/fsca*1000 (bugs)	101	195	263	287	277
GFP1-12	- aTc	gfpa/fsca*1000 (bugs)	82.5	195	250	270	274
blankpdCas9-9	- aTc	gfpa/fsca*1000 (bugs)	2213	3106	4099	5045	5411
blankpdCas9-10	- aTc	gfpa/fsca*1000 (bugs)	2212	2976	4289	5022	5084
blankpdCas9-11	- aTc	gfpa/fsca*1000 (bugs)	2022	3060	4164	4964	4574
blankpdCas9-12	- aTc	gfpa/fsca*1000 (bugs)	2061	3053	4158	4990	5205
pdCas9	- aTc	gfpa/fsca*1000 (bugs)	5.59	10	14.4	13.3	12.3
GFP1-9	+ aTc	gfpa/ssca*1000 (bugs)	49	73.4	74.8	71.9	69
GFP1-10	+ aTc	gfpa/ssca*1000 (bugs)	48.4	74.5	74.4	77.2	68.9
GFP1-11	+ aTc	gfpa/ssca*1000 (bugs)	49.8	77.1	79.2	80.5	69.6
GFP1-12	+ aTc	gfpa/ssca*1000 (bugs)	53	84.8	90.9	88.6	74.1
blankpdCas9-9	+ aTc	gfpa/ssca*1000 (bugs)	1267	1433	1538	1591	1583
blankpdCas9-10	+ aTc	gfpa/ssca*1000 (bugs)	1210	1380	1489	1535	1541
blankpdCas9-11	+ aTc	gfpa/ssca*1000 (bugs)	1170	1359	1497	1514	1583
blankpdCas9-12	+ aTc	gfpa/ssca*1000 (bugs)	1174	1356	1512	1531	1634
pdCas9	+ aTc	gfpa/ssca*1000 (bugs)	3.25	4.27	5.57	4.17	4.13
GFP1-9	- aTc	gfpa/ssca*1000 (bugs)	48.5	77.5	84	84.1	77.8
GFP1-10	- aTc	gfpa/ssca*1000 (bugs)	47.8	77.4	83.9	88.1	76.9
GFP1-11	- aTc	gfpa/ssca*1000 (bugs)	51.9	80.9	94.2	92.9	82.8
GFP1-12	- aTc	gfpa/ssca*1000 (bugs)	56.8	90.5	101	94.2	90.5
blankpdCas9-9	- aTc	gfpa/ssca*1000 (bugs)	1246	1323	1446	1575	1613
blankpdCas9-10	- aTc	gfpa/ssca*1000 (bugs)	1208	1269	1413	1553	1529
blankpdCas9-11	- aTc	gfpa/ssca*1000 (bugs)	1196	1255	1395	1529	1366
blankpdCas9-12	- aTc	gfpa/ssca*1000 (bugs)	1231	1282	1420	1561	1553
pdCas9	- aTc	gfpa/ssca*1000 (bugs)	2.82	4.23	4.92	4.26	3.62

It looks like the decrease in fluor for pdCas9-GFP1 coli manifests fairly early and persists for 6 hours. Lutz & Bujard’s paper use slightly less aTc for full induction of pTet, but if the decrease we see here were due to aTc inhibiting translation then we would expect to see a decrease in +aTc cultures with pdCas9. If pTet expression of aTc was removing trasncription/translation resources away from GFP production then you would also expect that to manifest in pdCas9. However it might be possible that the pdCas9 strains are experiencing the same decrease from aTc or growht burden but the strong GFP expression is masking the effect.
The other possibility is that something scientifically interesting, but not useful for iGEM purposes, is going on.

9/15/14

Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays (retry)

CBC sets up PCR with 0.5 ng/uL green or blue PCR1 template with 0, 0.4, 0.7, 1.0 uL in 50uL reactions with same cycling conditions and oligo concs etc as before. Maybe with less template the dominant species in the tubes will be that with prefix & suffix added. Maybe
No difference between + template and notemplate tubes. No picture taken.

9/16/14

Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone

CBC prays, puts PCR2 products used on 9/13/14 in a digestion reaction with EcoRI/PstI and incubates 37C for 4h. PCR cleanup and CIP (buffer 2) for 20m then cleanup again. Both tubes have ~35 ng/uL and Dusseldorf ligation calculator calls for 12 ng of 100 (ish) bp insert for 86 ng 2000 bp bb. Assuming that the large majority of the inserts lack the prefix and suffix, I will try 1 and 2 uL of insert for 1 of the bb. Also bb only and insert only, of course. Ligating 90m at RT
Shocking in to Z1; 30m on ice with 10uL of the ligation mix, 80m recovery before plating on LB+Cm
next morning: no colonies on any plates. Cm works

9/17/14

Objective: PCR to assemble decoy binding site array

MZ does PCR on 4 tubes: 0 um, 0.5 um, 1 um, and 2 um of ~200 ng/uL prefix oligo respectively + 50 uL standard mix (Q5 buffer and polymerase, repeat-spacer-repeat and spacer-repeat-spacer oligos, dNTPS, H2O)
PCR for 75 min
Gel to confirm (picture)
From left to right, 0, 0.5, 1, 2 uL of prefix oligo. Shortening of schmear is consistent with intended effect but not conclusive evidence thereof.
CBC takes 0.3 uL out of wells 0.5, 1, 2 to serve as template for new PCR2 with prefix & suffix oligos. MCF runs the gel

9/18/14

Objective: PCR to assemble decoy binding site array

CBC sets up 3 new PCR series, each with 0.3, 0.6, 0.9, 1.2 uL of tubes 2, 3, 4 from previous reaction as template.
Left to right is series from reactions templated with tube 2, 3, and 4. Gels look more like small pieces are dominating (primer dimer??) but we will try gibson anyway

Objective: PCR to assemble decoy binding site array

MZ with CBC get Mert’s homemade 1.33x gibson assembly mix and make reactions with backbone only, then backbone + insert
1uL of (conc) backbone (=EcoRI/PstI/CIP pSB1C3-R0040 made by CBC on [date] conc = [conc]) and 0.3uL of PCR products from series 2, 3, 4 at concs [conc conc conc] respectively.
Next morning: 100s of colonies.

Objective: PCR to assemble decoy binding site array

Master mix X13: (of a standard 50 uL reaction)

494 uL dH2O
130 uL buffer (Q5)
13 uL dNTPs
3.25 uL each oligo (prefix & suffix oligos)
6.25 uL enzyme (Q5)
0.3 uL of each template (0.5, 1, 2)

Ran PCR with iGEM protocol (previously saved)
Some uneven loading in gel or at addition of template to tube step; either way it’s fine. Pool with PCR cleanup protocol and then can use this for GA. 0.5 and 1.0 series look the most promising for this.

9/19/14

Objective: confirm insert presence & size of decoy array inserts from gibson assembly yesterday

100s of colonies on bb+insert plates 2, 3, 4. None on insert-only plates or backbone-only.
Colony PCR with pSB1C3-up and -dn with 62C annealing temp (see 7/25/2014 entry, Farnitano figured this temp out for these oligos)
1ng pSB1C3-R0040, no template control, then 4 colonies from plate 2, 4 from 3, 6 from 4.
No amplification on anything. No picture

Objective: assemble decoy arrays. Insert putative arrays in to pSB1C3

Delta pools reactions from Garima’s PCR/gel produced yesterday in to three tubes. CBC sets up gibson assembly. 1 uL backbone and 0.3 insert as appropriate, H2O to 5 uL, vortexed briefly then added 2.5uL of this to 7.5 uL of master mix. Only enough master mix was in the freezer for 6 of these half-sized reactions so there is no insert-only control for reactions labeled 2 in Garima’s gel.
Transforming all 10 uL in to 80uL of competent Z1s.
Next morning, lots of colonies on all the places there should be colonies. Huzzah

9/21/14

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

Picked 18 colonies from gibson/transformation performed on 9/19/14 yesterday and miniprepped. EcoRI/PstI digest for 3h then run 15m on 0.8% gel

Unadorned pSB1C3 is 41bp between EcoRI and PstI, 107 for minimal intended length of the four assembly oligos. It looks like there might be some incomplete digestion happening on these maybe, or just overloaded.
taaggatgatttctgg aattcgcggccgcttctagagCCATCTAATTCAACAAGAATTGGtgatgttaatCCATCTAATTCAACAAGAATTGGtgatgttaattactagtagcggccgctgca gtccggcaaaaaagggc

Left to right is 1-18. Two probably aberrant clones but they’re getting sequenced anyway. Sent to bio sequencing core seqd from pSB1C3-up-1 and -dn-1. Clones 1-4, 15-18.

9/22/14

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

Mike picks 12 more colonies for miniprepping tomorrow

9/23/14

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

Mike starts miniprep for 12 new colonies, charlie takes over after N3 addition & spin. Mike sets up EcoRI-HF/PstI-HF digest (cutsmart) for each clone.
Garima loaded this 1.3% TAE gel. Lane 7 (ie clone 25) has one big band at ~600 but also looks to have a small band down at the 1x size. Contamination? Unstable repeats? Another gel 0.8% has the same thing, but smearier.
Sequencing results come back!! As expected and consistent with the gel above, almost all colonies have a single insert, but clone 17 has 5! It works, kind of!!! The chromatograms taper off in quality around the repeats which makes it difficult for seqman to put it assemble properly, but manually getting called bases and finding repeats makes it clear. Clone 2 from yesterday was trash.

ctrl-F in text editor highlights each decoy site. To the left and right are prefix & suffixes

Objective: transfer decoy arrays to pSB1A2

CBC sets up prep scale digest of EcoRI/PstI of clones 17 & 18 & pSB1A2-R0040-antitracr. PCR cleanup and CIP treatment of clones 17 & 18 followed by a second PCR cleanup. Gel extraction of pSB1A2 backbone leaves only 22 ng/uL of dirty DNA by nanodrop. Tomorrow will try ligating, and also will set up another EcoRI/PstI digest of more pSB1A2-R0040-antitracr

Objective: optimize colony PCR conditions for testing pSB1C3-decoy arrays

Garima sets up master mix with 9x50uL rx. pSB1C3-up-1 and -dn-1 included at 0.25*9uL from 100uM stock.

45 uL Taq buffer
2.25 uL Taq polymerase
9 uL dNTPs
9 uL of each pSB1C3-up-1 and -dn-1
375.75 uL dH2O
9 uL template in positive tube/ 0 uL template in negative tubes

Ran colony pcr using saved protocol:

edited annealing step of protocol from 62 degrees to a gradient : 58-65 degrees
extension time of 15s (was 30)
Fairly crappy snapshot (crapshot?) but you can see that the primer dimers on the - gel are all at ~100bp and the product on +template are at 2-300 which is consistent with R0040-antitracr size. the best amplification took place at higher temps. New protocol: 65C with 15s extension for short pieces, next try with some actual colonies.

9/24/14

Objective: transfer decoy arrays to pSB1A2

Setting up ligation reaction with EcoRI/PstI/CIP treated clone 17 and 18 from yesterday (5x and 1x decoys respectively) and EcoRI/PstI/gel-purified pSB1A2 bb. Also including controls below
used ligation calculator from dusseldorf

	17 (5x)	18 (1x)	1A2 bb
length	~500	~100	2252
conc	84.1	63.8	22.8
ng needed	66	13	100
using uL	1	0.5	5

thing	bb+ins 17	insert only 17	bb+ins 17	insert only 17	bb only
backbone	5uL	0	5	0	5
insert	1uL	1	0.5	0.5	0
T4 buffer	2uL	2	2	2	2
enzyme	1uL	1	1	1	1
h2o	11uL	16	11.5	16.5	12

Mixed andl et sit for 3h at room temp on bench. Took 10uL and transformed with 80uL of frozen competent Z1s
next day: lots of colonies on bb+insert plates, none on bb-only or insert-only. huzzah

Objective: find more long decoy arrays

miniprepping 12 more colonies from plate 1.0, middle one from 9/18/14
EcoRI/PstI digest for 3h gives all small bands at 1x size. No picture taken, all tubes discarded.

9/25/14

Objective: find more long decoy arrays

colony PCR on 4 colonies from plate 1.0 next to no-template, 1ng of pSB1C3-R0040-antitracr, pSB1C3-5xGFP1 decoy, and pSB1C3-1xGFP1 decoy. 10s extension at 64C
No amplification on any of the colonies?

no template, pSB1C3-R0040-antitracr, 5x, 1x, then 4 colonies. At first I thought this was no amplification but actually I think it worked but all of the colonies are 1x or maybe a 2x for colony 4. Threw them all away in any case and before I had the realization that they amplified I already set up the next PCR with 8 colonies on a 58-65C gradient.

left to right is 65 to 58. My strong suspicion is that the long band in lane 1 is from a long template rather than 65. Picking colonies 1 and 3 for further investigation. Labeled 43 and 44 on the tubes, because that’s where we are in the screening.

Objective: confirm insert presence & size of decoy array inserts from gibson assembly

sequencing clone 25 with pSB1C3-up and -dn, dugsim job 1827

It might be better to take the 5x repeat and then expand it with some other scheme.

It is possible to do this by stepwise dig/lig/confirm cycles with XbaI/PstI dig’d insert and SpeI/PstI dig’d backbone (gel purified).
Another possible trick to speed up the expansion would be to cycle from 16C to 37C a couple of times with XbaI/SpeI dig’d pSB1C3-5xGFP1 and also add XbaI/SpeI & T4 ligase and a third enzyme that cuts somewhere else in the backbone.

When XbaI and SpeI ligate it will destroy the recognition site. Any sites re-ligating to self will be cleaved in the next 37C cycle. The last step in the cycling reaction will be a long digest.
Follow that with a ligation to XbaI/SpeI/CIP treated backbone.
Circularization is possible with this scheme

Third possibility: cycle XbaI/PstI/CIPd insert with backbone, SpeI, PstI, ligase and cycle

XbaI ligs to SpeI and destroys site. XbaI ligs to XbaI and gets re-cut, same with PstI. On re-cutting this would restore the phosphate removed by CIP so that doesn’t help much. Hmm

…..or just the stepwise dig/lig/shock will work. I think the problems of circularization will be there no matter what I do with CIP & including restriction enzymes the ligase.

9/26/14

Objective: Confirm pSB1A2-GFP1 decoy arrays

Yesterday picked 4 colonies from transformation of 5x and 1x repeat arrays and set up with EcoRI/PstI digest, alongside colonies 43 and 44 from yesterday’s colony PCR and pSB1C3 5x and 1x parents.
Slightly hard to read on the picture, but they’re loaded in the order mentioned above. 43 might be longer than 5x, 44 looks like 1x so discarding.
Zoom in on the 5x on 1A2 and Colony 1 might be slightly shorter than colony 4? Making bacteria with both of these just in case. Should probably sequence as well. pSB1C3-up-1 binds to 1A2, but not pSB1C3-dn
Shocking Z1 to make a couple of strains:

pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1
pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4
pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1
pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1
pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4
pdCas9, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1

9/27/14

Objective: extend decoy arrays

XbaI/PstI digest of 1C3-5xGFP1decoy followed by CIP, then SpeI/PstI of 1C3-5xGFP1 (same plasmid) with NO GEL PURIFICATION makes the backbone. I am hoping that since SpeI to PstI is only 18bp that the PCR cleanup kit gets rid of the fragment. Ligating with 3-fold molar excess of CIP-treated insert for 20m then shocking 10uL in to 70uL Z1. This is rushing the protocol; the other 10uL of the ligation mix will sit at RT for 90m in case the rushed version fails.

Update: it looks like it did not fail; there are some colonies on bb-only and ins-only but ~10x as many on b+i.

9/28/14

Objective: test 1x and 5x decoy arrays

4 colonies each picked from plates from

pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 G51
pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 G54
pdCas9-GFP1, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 G11
pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 C51
pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 C54
pdCas9, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 C11

….and grown overnight (started 9/27/14). 9:15am today diluted 1/200 in 3 mL fresh LB+AmpKanCm. 3h45m in 37C shaker. 1mL collected, spun down, and resuspended in PBS. Flowed 75000 events and got something that looks super promising. Means here are written down from stats view on MQ during acquisition and might change after full processing. There is a lot of variability in G51

2014-09-28 flow data, not fully processed

(constitutive GFP reporter)	pdCas9 with or without GFP-targeting crRNA	decoy binding site count & colony	expected GFP if working	tube label	unprocessed mean gfp	avg	std
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-1	derepressed	G51-1	42	69.25	28.14
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-1	derepressed	G51-2	50
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-1	derepressed	G51-3	83
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-1	derepressed	G51-4	102
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-4	derepressed	G54-1	64	68.33	5.13
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-4	derepressed	G54-2	67
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-4	derepressed	G54-3	nan
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-5xGFP1decoy-4	derepressed	G54-4	74
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-1xGFP1decoy-1	near repressed levels	G11-1	16	16.00	0.82
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-1xGFP1decoy-1	near repressed levels	G11-2	15
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-1xGFP1decoy-1	near repressed levels	G11-3	17
pSB4K5-K608012	pdCas9-GFP1	pSB1A2-1xGFP1decoy-1	near repressed levels	G11-4	16
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-1	unrepressed	C51-1	131	141.75	7.46
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-1	unrepressed	C51-2	143
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-1	unrepressed	C51-3	145
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-1	unrepressed	C51-4	148
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-4	unrepressed	C54-1	153	149.00	3.16
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-4	unrepressed	C54-2	150
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-4	unrepressed	C54-3	146
pSB4K5-K608012	pdCas9	pSB1A2-5xGFP1decoy-4	unrepressed	C54-4	147
pSB4K5-K608012	pdCas9	pSB1A2-1xGFP1decoy-1	unrepressed	C11-1	150	153.25	3.95
pSB4K5-K608012	pdCas9	pSB1A2-1xGFP1decoy-1	unrepressed	C11-2	152
pSB4K5-K608012	pdCas9	pSB1A2-1xGFP1decoy-1	unrepressed	C11-3	159
pSB4K5-K608012	pdCas9	pSB1A2-1xGFP1decoy-1	unrepressed	C11-4	152

Saved G51-1 through -4, then colony 1 for one of each different strain as glycerol stocks. Labeled very minimally with the bolded labels in first sub-bullet list.
Next steps:

repeat
try with more decoy site numbers: 10x is on the way, need to also make 0x

@@ Line 1: / Line 1: @@
 <html>
-<span>9/10/14</span></h2><p class="c6"><span class="c16">Objective: assemble decoy binding site arrays</span></p><ul class="c3 lst-kix_j8vf5upxzyho-0 start"><li class="c6 c10"><span>Setting up two PCRs with same cycling program as last time: </span></li></ul><ul class="c3 lst-kix_j8vf5upxzyho-1 start"><li class="c1"><span>one with 4x 50uL reactions using the same concs of everything as the 0.5 uL rx from 9/9/14 (blue labels)</span></li><li class="c1"><span>one with equimolar oligo1 and oligo2 and varying amounts of reaction 0.5 from 9/9/14 (green labels)</span></li></ul><ul class="c3 lst-kix_j8vf5upxzyho-0"><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 10, 2014 6:28:14 PM.jpg" src="images/image56.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>So something&rsquo;s happening. PCR cleanup&rsquo;d reactions 1-4 (green) and 5-8 (blue) in to one tube each labeled Decoy array (1-4 or 5-8) PCR1. Tomorrow will PCR with the end oligos and try gibson, dig/lig in to pSB1C3 and/or pSB1A2</span></li></ul><p class="c4"><span></span></p><h2 class="c6 c29"><a name="h.dmhnzg83ygzw"></a><span>9/11/14</span></h2><p class="c6"><span class="c16">Objective: assemble decoy binding site arrays. Step 2: add prefix &amp; suffix to arrays</span></p><ul class="c3 lst-kix_lyxpt4rd7frr-0 start"><li class="c6 c10"><span>Faw setting up two series of PCRs with </span><span class="c68 c7">1c337prfGfp1Space and 1c337sufGfp1Space</span><span>&nbsp;as oligos on 0.5-2.0 ng of PCR cleanup&rsquo;d green and blue rx from 9/10/14 and running with the same cycling conditions.</span></li><li class="c6 c10"><span>CBC did not take a picture and doesn&rsquo;t remember why. PCR looked bad for some reason.</span></li></ul><p class="c4"><span></span></p><h2 class="c6 c29"><a name="h.knhldp48br8h"></a><span>9/12/14</span></h2><p class="c6"><span class="c16">Objective: assemble decoy binding site arrays. Step 2: add prefix &amp; suffix to arrays</span></p><ul class="c3 lst-kix_1zm68rntz6ky-0 start"><li class="c6 c10"><span>Garima setting up two series of PCRs with </span><span class="c7 c68">1c337prfGfp1Space and 1c337sufGfp1Space</span><span>&nbsp;as oligos on higher concs than yesterday ofPCR cleanup&rsquo;d green and blue rx from 9/10/14 and running with the same cycling conditions.</span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 16, 2014 11:21:26 AM.jpg" src="images/image06.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>PCR cleanup to pool tubes 2-4 for both series and labeled Decoy Array PCR2</span></li></ul><p class="c4"><span></span></p><h2 class="c6 c29"><a name="h.qg2pv53zrivc"></a><span>9/13/14</span></h2><p class="c6"><span class="c16">Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone</span></p><ul class="c3 lst-kix_1zm68rntz6ky-0"><li class="c6 c10"><span>CBC attempts a SLIC with 0.5 uL of the &ldquo;Super Clean&rdquo; XbaI/PstI pSB1C3 backbone. Note that puts 15 bp of nonhomology in the way of successful SLIC. I did this because sometimes it works despite mismatches and there was no EcoRI/PstI dig&rsquo;d pSB1C3 for me to work with. Green series looks like there is a bigger difference between no-template and +template tubes, so I will try that</span></li></ul><ul class="c3 lst-kix_1zm68rntz6ky-1 start"><li class="c1"><span>set up tubes for backbone-only, backbone + 0.5, 1, 2, 3uL of insert from Green tube at 88.8 ng/uL, and 3uL insert-only.</span></li></ul><ul class="c3 lst-kix_1zm68rntz6ky-0"><li class="c6 c10"><span>Didn&rsquo;t work.</span></li></ul><p class="c6"><span class="c16">Objective: observe fluorescence changes with antitracr time course over time</span></p><ul class="c3 lst-kix_1zm68rntz6ky-0"><li class="c6 c10"><span>Last night antitracr strains started in LB+AmpKanCm and let shake overnight. Diluted this morning in to +/- aTc and began collection at time indicated. Replaced LB with PBS before flowing</span></li><li class="c6 c10"><span>Isolated &ldquo;singlets&rdquo; which are probably not singlets (smalls) or left alone (bugs) and got mean GFPA or gfpa/fsca or /ssca to normalize roughly to size.</span></li><li class="c4 c10"><span></span></li></ul><a href="#" name="504882174bdbc3a066b549e955cdc3b34f759d86"></a><a href="#" name="7"></a><table cellpadding="0" cellspacing="0" class="c61 c66"><tbody><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">time point</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">n/a</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">n/a</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">time (min)</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">n/a</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">n/a</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">120</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">180</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">240</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">300</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">360</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.52</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.74</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.17</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.16</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.81</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.39</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.18</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.42</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.88</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.45</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.92</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.31</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.53</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.88</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.53</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.13</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.72</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.89</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">45.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">56</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">68.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">76.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.9</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">46.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">54</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">67.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">73.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.7</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">48.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">53.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">65.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">73</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">76.2</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">45.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">54.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">68.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">78.4</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.109</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.16</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.185</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.139</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.143</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.43</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.08</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.65</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.67</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.07</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.32</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.17</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.67</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.88</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.07</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.73</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.15</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.99</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.18</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1.57</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.47</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.16</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.83</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.29</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">41.7</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">53.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">67</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">75.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">75.6</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">41.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">51.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">67.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">71.7</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">39.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">52.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">66</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">73.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">60.8</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">40</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">52.7</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">67</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">76</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">73.2</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.0949</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.156</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.162</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.129</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.122</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">83.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">157</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">188</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">189</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">172</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">70.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">159</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">186</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">205</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">177</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">72.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">169</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">193</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">208</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">175</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">80.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">172</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">206</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">199</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">166</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2526</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3227</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3986</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4592</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4520</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2700</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3078</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4044</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4486</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4543</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2892</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3028</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3795</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4291</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4553</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2641</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3131</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4038</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4531</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4614</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.26</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">9.36</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">11.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">8.92</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">9.11</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">72.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">177</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">221</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">228</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">194</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">60.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">187</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">220</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">241</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">193</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">94.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">192</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">245</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">245</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">199</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">79.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">193</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">235</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">222</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">193</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2184</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3100</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4012</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4663</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4666</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2188</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2957</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4160</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4554</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4386</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2004</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3047</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4035</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4565</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3842</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2039</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3051</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4107</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4630</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4512</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.64</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">9.13</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">9.66</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">8.24</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">7.79</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">47.7</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">75.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">78.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">75.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">64.8</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">46.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">76.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">79.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">81</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">66</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">48</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">78.7</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">82.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">83.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">66.4</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">51.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">85.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">96.7</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">93.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">71.1</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1273</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1469</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1666</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1779</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1718</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1226</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1413</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1636</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1719</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1688</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1182</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1382</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1605</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1704</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1739</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1182</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1391</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1645</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1714</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1790</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.43</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.38</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.82</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.89</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.69</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">46.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">80.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">89.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">87.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">71</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">45.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">80.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">89.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">91.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">71.4</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">50.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">84.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">101</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">95</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">55.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">93.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">108</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">100</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">83.8</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1250</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1375</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1606</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1758</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1715</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1215</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1320</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1606</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1734</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1635</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1203</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1319</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1563</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1718</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1425</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1230</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1338</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1595</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1754</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1661</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (smalls)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.05</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.55</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.63</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.67</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.35</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.17</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.7</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.12</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.27</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.68</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.82</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.06</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.77</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.85</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.17</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.92</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.48</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.04</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.89</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.07</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.49</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.95</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.85</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">70.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">82.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">105</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">120</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">133</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">78.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">102</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">122</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">137</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">75.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">76.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">102</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">114</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">130</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">75.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">79.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">103</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">120</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">136</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.169</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.26</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.396</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.319</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.34</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.26</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.28</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.12</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.62</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.7</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.01</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.53</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.07</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">7.13</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.9</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.72</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.59</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.58</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">7.36</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">7.43</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.16</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.63</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.39</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">7.15</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">7.22</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">63.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">76.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">107</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">127</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">141</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">64.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">112</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">127</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">134</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">61.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">77.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">110</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">127</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">126</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">63.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">78.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">110</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">128</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">138</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.157</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.26</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.357</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.331</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">0.287</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">88.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">159</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">205</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">206</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">216</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">75.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">163</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">197</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">228</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">220</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">77.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">172</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">210</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">232</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">217</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">83.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">174</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">215</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">227</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">223</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2559</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3229</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4012</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4818</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5003</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2751</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3078</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4107</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4768</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5130</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3006</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3014</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3787</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4456</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4927</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2695</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3129</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4076</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4738</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5032</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">6.33</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">10.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">15.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">12.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">14.1</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">78.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">182</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">242</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">263</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">257</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">64.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">194</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">236</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">278</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">259</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">101</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">195</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">263</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">287</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">277</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">82.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">195</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">250</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">270</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">274</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2213</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3106</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4099</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5045</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5411</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2212</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2976</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4289</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5022</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5084</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2022</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3060</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4164</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4964</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4574</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2061</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3053</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4158</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4990</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5205</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/fsca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.59</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">10</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">14.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">13.3</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">12.3</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">49</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">73.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">71.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">69</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">48.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">77.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">68.9</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">49.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">77.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">79.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">80.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">69.6</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">53</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">84.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">90.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">88.6</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">74.1</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1267</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1433</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1538</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1591</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1583</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1210</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1380</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1489</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1535</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1541</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1170</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1359</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1497</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1514</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1583</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1174</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1356</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1512</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1531</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1634</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">+ aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.25</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.27</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">5.57</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.17</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.13</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">48.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">77.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">84</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">84.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">77.8</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">47.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">77.4</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">83.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">88.1</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">76.9</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">51.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">80.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">94.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">92.9</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">82.8</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">GFP1-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">56.8</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">90.5</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">101</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">94.2</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">90.5</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1246</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1323</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1446</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1575</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1613</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-10</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1208</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1269</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1413</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1553</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1529</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-11</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1196</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1255</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1395</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1529</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1366</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">blankpdCas9-12</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1231</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1282</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1420</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1561</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">1553</span></p></td></tr><tr class="c33"><td class="c8" colspan="1" rowspan="1"><p class="c9"><span class="c0">pdCas9</span></p></td><td class="c18" colspan="1" rowspan="1"><p class="c9"><span class="c0">- aTc</span></p></td><td class="c39" colspan="1" rowspan="1"><p class="c9"><span class="c0">gfpa/ssca*1000 (bugs)</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">2.82</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.23</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.92</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">4.26</span></p></td><td class="c34" colspan="1" rowspan="1"><p class="c9"><span class="c0">3.62</span></p></td></tr></tbody></table><ul class="c3 lst-kix_1zm68rntz6ky-0"><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 585.00px; height: 925.00px;"><img alt="" src="images/image49.jpg" style="width: 585.00px; height: 925.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>It looks like the decrease in fluor for pdCas9-GFP1 coli manifests fairly early and persists for 6 hours. Lutz &amp; Bujard&rsquo;s paper use slightly less aTc for full induction of pTet, but if the decrease we see here were due to aTc inhibiting translation then we would expect to see a decrease in +aTc cultures with pdCas9. If pTet expression of aTc was removing trasncription/translation resources away from GFP production then you would also expect that to manifest in pdCas9. However it might be possible that the pdCas9 strains are experiencing the same decrease from aTc or growht burden but the strong GFP expression is masking the effect.</span></li><li class="c6 c10"><span>The other possibility is that something scientifically interesting, but not useful for iGEM purposes, is going on.</span></li></ul><h2 class="c6 c21"><a name="h.oz7brdgn00cg"></a><span>9/15/14</span></h2><p class="c6"><span class="c16">Objective: assemble decoy binding site arrays. Step 2: add prefix &amp; suffix to arrays (retry)</span></p><ul class="c3 lst-kix_1zm68rntz6ky-0"><li class="c6 c10"><span>CBC sets up PCR with 0.5 ng/uL green or blue PCR1 template with 0, 0.4, 0.7, 1.0 uL in 50uL reactions with same cycling conditions and oligo concs etc as before. Maybe with less template the dominant species in the tubes will be that with prefix &amp; suffix added. Maybe</span></li><li class="c6 c10"><span>No difference between + template and notemplate tubes. No picture taken.</span></li><li class="c4 c10"><span></span></li></ul><p class="c4"><span></span></p><h2 class="c6 c21"><a name="h.bvksvj6lqq8i"></a><span>9/16/14</span></h2><p class="c6"><span class="c16">Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone</span></p><ul class="c3 lst-kix_66g213lrqxp4-0 start"><li class="c6 c10"><span>CBC prays, puts PCR2 products used on 9/13/14 in a digestion reaction with EcoRI/PstI and incubates 37C for 4h. PCR cleanup and CIP (buffer 2) for 20m then cleanup again. Both tubes have ~35 ng/uL and Dusseldorf ligation calculator calls for 12 ng of 100 (ish) bp insert &nbsp;for 86 ng 2000 bp bb. Assuming that the large majority of the inserts lack the prefix and suffix, I will try 1 and 2 uL of insert for 1 of the bb. Also bb only and insert only, of course. Ligating 90m at RT</span></li><li class="c6 c10"><span>Shocking in to Z1; 30m on ice with 10uL of the ligation mix, 80m recovery before plating on LB+Cm</span></li><li class="c6 c10"><span>next morning: no colonies on any plates. Cm works</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/17/14</span></p><p class="c6"><span class="c16">Objective: PCR to assemble decoy binding site array</span></p><ul class="c3 lst-kix_l8zt1ty00aa9-0 start"><li class="c6 c10"><span>MZ does PCR on 4 tubes: 0 um, 0.5 um, 1 um, and 2 um of ~200 ng/uL prefix oligo respectively + 50 uL standard mix (Q5 buffer and polymerase, repeat-spacer-repeat and spacer-repeat-spacer oligos, dNTPS, H2O) </span></li><li class="c6 c10"><span>PCR for 75 min</span></li><li class="c6 c10"><span>Gel to confirm (picture)</span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:40:25 PM.jpg" src="images/image03.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>From left to right, 0, 0.5, 1, 2 uL of prefix oligo. Shortening of schmear is consistent with intended effect but not conclusive evidence thereof.</span></li><li class="c6 c10"><span>CBC takes 0.3 uL out of wells 0.5, 1, 2 to serve as template for new PCR2 with prefix &amp; suffix oligos. MCF runs the gel</span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:40:52 PM.jpg" src="images/image15.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/18/14</span></p><p class="c6"><span class="c16">Objective: PCR to assemble decoy binding site array</span></p><ul class="c3 lst-kix_l8zt1ty00aa9-0"><li class="c6 c10"><span>CBC sets up 3 new PCR series, each with 0.3, 0.6, 0.9, 1.2 uL of tubes 2, 3, 4 from previous reaction as template. </span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:44:02 PM.jpg" src="images/image14.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>Left to right is series from reactions templated with tube 2, 3, and 4. Gels look more like small pieces are dominating (primer dimer??) but we will try gibson anyway</span></li></ul><p class="c6"><span class="c16">Objective: PCR to assemble decoy binding site array</span></p><ul class="c3 lst-kix_e2abf5cxqh-0 start"><li class="c4 c10"><span></span></li><li class="c6 c10"><span>MZ with CBC get Mert&rsquo;s homemade 1.33x gibson assembly mix and make reactions with backbone only, then backbone + insert</span></li><li class="c6 c10"><span>1uL of (conc) backbone (=EcoRI/PstI/CIP pSB1C3-R0040 made by CBC on [date] conc = [conc]) and 0.3uL of PCR products from series 2, 3, 4 at concs [conc conc conc] respectively.</span></li><li class="c6 c10"><span>Next morning: 100s of colonies.</span></li></ul><p class="c6"><span class="c16">Objective: PCR to assemble decoy binding site array</span></p><ul class="c3 lst-kix_q28ky2oxurzk-0 start"><li class="c6 c10"><span>Master mix X13: &nbsp;(of a standard 50 uL reaction)</span></li></ul><ul class="c3 lst-kix_q28ky2oxurzk-1 start"><li class="c1"><span>494 uL dH2O</span></li><li class="c1"><span>130 uL buffer (Q5)</span></li><li class="c1"><span>13 uL dNTPs</span></li><li class="c1"><span>3.25 uL each oligo (prefix &amp; suffix oligos)</span></li><li class="c1"><span>6.25 uL enzyme (Q5)</span></li><li class="c1"><span>0.3 uL of each template (0.5, 1, 2)</span></li></ul><ul class="c3 lst-kix_q28ky2oxurzk-0"><li class="c6 c10"><span>Ran PCR with iGEM protocol (previously saved)</span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 19, 2014 9:48:04 AM.jpg" src="images/image48.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>Some uneven loading in gel or at addition of template to tube step; either way it&rsquo;s fine. Pool with PCR cleanup protocol and then can use this for GA. 0.5 and 1.0 series look the most promising for this. </span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/19/14</span></p><p class="c6"><span class="c16">Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly yesterday</span></p><ul class="c3 lst-kix_l8zt1ty00aa9-0"><li class="c6 c10"><span>100s of colonies on bb+insert plates 2, 3, 4. None on insert-only plates or backbone-only. </span></li><li class="c6 c10"><span>Colony PCR with pSB1C3-up and -dn with 62C annealing temp (see 7/25/2014 entry, Farnitano figured this temp out for these oligos)</span></li><li class="c6 c10"><span>1ng pSB1C3-R0040, no template control, then 4 colonies from plate 2, 4 from 3, 6 from 4. </span></li><li class="c6 c10"><span>No amplification on anything. No picture</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c16">Objective: assemble decoy arrays. Insert putative arrays in to pSB1C3</span></p><ul class="c3 lst-kix_tzrfju21rmdv-0 start"><li class="c6 c10"><span>Delta pools reactions from Garima&rsquo;s PCR/gel produced yesterday in to three tubes. CBC sets up gibson assembly. 1 uL backbone and 0.3 insert as appropriate, H2O to 5 uL, vortexed briefly then added 2.5uL of this to 7.5 uL of master mix. Only enough master mix was in the freezer for 6 of these half-sized reactions so there is no insert-only control for reactions labeled 2 in Garima&rsquo;s gel.</span></li><li class="c6 c10"><span>Transforming all 10 uL in to 80uL of competent Z1s. </span></li><li class="c6 c10"><span>Next morning, lots of colonies on all the places there should be colonies. Huzzah</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/21/14</span></p><p class="c6"><span class="c16">Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </span></p><ul class="c3 lst-kix_sj5vnpizr8db-0 start"><li class="c6 c10"><span>Picked 18 colonies from gibson/transformation performed on 9/19/14 yesterday and miniprepped. EcoRI/PstI digest for 3h then run 15m on 0.8% gel</span></li></ul><ul class="c3 lst-kix_sj5vnpizr8db-1 start"><li class="c1"><span>Unadorned pSB1C3 is 41bp between EcoRI and PstI, 107 for minimal intended length of the four assembly oligos. It looks like there might be some incomplete digestion happening on these maybe, or just overloaded. </span></li><li class="c1"><span>taaggatgatttctgg aattcgcggccgcttctagagCCATCTAATTCAACAAGAATTGGtgatgttaatCCATCTAATTCAACAAGAATTGGtgatgttaattactagtagcggccgctgca gtccggcaaaaaagggc</span></li></ul><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 21, 2014 4:12:27 PM.jpg" src="images/image51.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>Left to right is 1-18. Two probably aberrant clones but they&rsquo;re getting sequenced anyway. Sent to bio sequencing core seqd from pSB1C3-up-1 and -dn-1. Clones 1-4, 15-18. </span></li></ul><p class="c6"><span class="c20">9/22/14</span></p><p class="c6"><span class="c16">Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </span></p><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>Mike picks 12 more colonies for miniprepping tomorrow</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/23/14</span></p><p class="c6"><span class="c16">Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </span></p><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>Mike starts miniprep for 12 new colonies, charlie takes over after N3 addition &amp; spin. Mike sets up EcoRI-HF/PstI-HF digest (cutsmart) for each clone. </span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 23, 2014 4:41:21 PM.jpg" src="images/image38.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>Garima loaded this 1.3% TAE gel. Lane 7 (ie clone 25) has one big band at ~600 but also looks to have a small band down at the 1x size. Contamination? Unstable repeats? Another gel 0.8% has the same thing, but smearier. </span></li><li class="c6 c10"><span>Sequencing results come back!! As expected and consistent with the gel above, almost all colonies have a single insert, but clone 17 has 5! It works, kind of!!! The chromatograms taper off in quality around the repeats which makes it difficult for seqman to put it assemble properly, but manually getting called bases and finding repeats makes it clear. Clone 2 from yesterday was trash. </span></li></ul><p class="c6 c85"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 806.50px; height: 48.18px;"><img alt="" src="images/image12.png" style="width: 806.50px; height: 48.18px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></p><p class="c4"><span></span></p><ul class="c3 lst-kix_rthy9filx2wx-0 start"><li class="c6 c10"><span>ctrl-F in text editor highlights each decoy site. To the left and right are prefix &amp; suffixes</span></li></ul><p class="c6"><span class="c16">Objective: transfer decoy arrays to pSB1A2</span></p><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>CBC sets up prep scale digest of EcoRI/PstI of clones 17 &amp; 18 &amp; pSB1A2-R0040-antitracr. PCR cleanup and CIP treatment of clones 17 &amp; 18 followed by a second PCR cleanup. Gel extraction of pSB1A2 backbone leaves only 22 ng/uL of dirty DNA by nanodrop. Tomorrow will try ligating, and also will set up another EcoRI/PstI digest of more pSB1A2-R0040-antitracr</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c16">Objective: optimize colony PCR conditions for testing pSB1C3-decoy arrays</span></p><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>Garima sets up master mix with 9x50uL rx. pSB1C3-up-1 and -dn-1 included at 0.25*9uL from 100uM stock. </span></li></ul><ul class="c3 lst-kix_sj5vnpizr8db-1 start"><li class="c1"><span>45 uL Taq buffer</span></li><li class="c1"><span>2.25 uL Taq polymerase</span></li><li class="c1"><span>9 uL dNTPs</span></li><li class="c1"><span>9 uL of each pSB1C3-up-1 and -dn-1</span></li><li class="c1"><span>375.75 uL dH2O</span></li><li class="c1"><span>9 uL template in positive tube/ 0 uL template in negative tubes</span></li></ul><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>Ran colony pcr using saved protocol:</span></li></ul><ul class="c3 lst-kix_sj5vnpizr8db-1 start"><li class="c1"><span>edited annealing step of protocol from 62 degrees to a gradient : 58-65 degrees</span></li><li class="c1"><span>extension time of 15s (was 30)</span></li><li class="c1"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 24, 2014 1:13:46 PM.jpg" src="images/image35.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c1"><span>Fairly crappy snapshot (crapshot?) but you can see that the primer dimers on the - gel are all at ~100bp and the product on +template are at 2-300 which is consistent with R0040-antitracr size. the best amplification took place at higher temps. New protocol: 65C with 15s extension for short pieces, next try with some actual colonies.</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/24/14</span></p><p class="c6"><span class="c16">Objective: transfer decoy arrays to pSB1A2</span></p><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>Setting up ligation reaction with EcoRI/PstI/CIP treated clone 17 and 18 from yesterday (5x and 1x decoys respectively) and EcoRI/PstI/gel-purified pSB1A2 bb. Also including controls below</span></li><li class="c6 c10"><span>used ligation calculator from dusseldorf </span></li><li class="c4 c10"><span></span></li></ul><a href="#" name="b2d32455b7b3438d6a59a365920ec8095fe8bb0e"></a><a href="#" name="8"></a><table cellpadding="0" cellspacing="0" class="c61 c19"><tbody><tr class="c33"><td class="c41" colspan="1" rowspan="1"><p class="c14 c4"><span class="c37"></span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">17 (5x)</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">18 (1x)</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1A2 bb</span></p></td></tr><tr class="c33"><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">length</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">~500</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">~100</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">2252</span></p></td></tr><tr class="c33"><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">conc</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">84.1</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">63.8</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">22.8</span></p></td></tr><tr class="c33"><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">ng needed</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">66</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">13</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">100</span></p></td></tr><tr class="c33"><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">using uL</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">0.5</span></p></td><td class="c41" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">5</span></p></td></tr></tbody></table><p class="c4"><span></span></p><a href="#" name="5465b9bebaea090720acd450041ece042a9665b5"></a><a href="#" name="9"></a><table cellpadding="0" cellspacing="0" class="c61 c19"><tbody><tr class="c33"><td class="c72 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">thing</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">bb+ins 17</span></p></td><td class="c70 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">insert only 17</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">bb+ins 17</span></p></td><td class="c64 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">insert only 17</span></p></td><td class="c40 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">bb only</span></p></td></tr><tr class="c33"><td class="c72 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">backbone</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">5uL</span></p></td><td class="c70 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">0</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">5</span></p></td><td class="c64 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">0</span></p></td><td class="c40 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">5</span></p></td></tr><tr class="c33"><td class="c72 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">insert</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1uL</span></p></td><td class="c70 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">0.5</span></p></td><td class="c64 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">0.5</span></p></td><td class="c40 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">0</span></p></td></tr><tr class="c33"><td class="c72 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">T4 buffer</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">2uL</span></p></td><td class="c62 c70" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">2</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">2</span></p></td><td class="c64 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">2</span></p></td><td class="c40 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">2</span></p></td></tr><tr class="c33"><td class="c62 c72" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">enzyme</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1uL</span></p></td><td class="c70 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1</span></p></td><td class="c62 c64" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1</span></p></td><td class="c40 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">1</span></p></td></tr><tr class="c33"><td class="c72 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">h2o</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">11uL</span></p></td><td class="c70 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">16</span></p></td><td class="c22" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">11.5</span></p></td><td class="c64 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">16.5</span></p></td><td class="c40 c62" colspan="1" rowspan="1"><p class="c14 c6"><span class="c37">12</span></p></td></tr></tbody></table><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>Mixed andl et sit for 3h at room temp on bench. Took 10uL and transformed with 80uL of frozen competent Z1s</span></li><li class="c6 c10"><span>next day: lots of colonies on bb+insert plates, none on bb-only or insert-only. huzzah</span></li></ul><p class="c6"><span class="c16">Objective: find more long decoy arrays</span></p><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>miniprepping 12 more colonies from plate 1.0, middle one from 9/18/14</span></li><li class="c6 c10"><span>EcoRI/PstI digest for 3h gives all small bands at 1x size. No picture taken, all tubes discarded. </span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/25/14</span></p><p class="c6"><span class="c16">Objective: find more long decoy arrays</span></p><ul class="c3 lst-kix_6peab1jnomrp-0 start"><li class="c6 c10"><span>colony PCR on 4 colonies from plate 1.0 next to no-template, 1ng of pSB1C3-R0040-antitracr, pSB1C3-5xGFP1 decoy, and pSB1C3-1xGFP1 decoy. 10s extension at 64C</span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 25, 2014 5:39:52 PM.jpg" src="images/image39.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span><span>&nbsp;No amplification on any of the colonies?</span></li></ul><ul class="c3 lst-kix_6peab1jnomrp-1 start"><li class="c1"><span>no template, pSB1C3-R0040-antitracr, 5x, 1x, then 4 colonies. At first I thought this was no amplification but actually I think it worked but all of the colonies are 1x or maybe a 2x for colony 4. Threw them all away in any case and before I had the realization that they amplified I already set up the next PCR with 8 colonies on a 58-65C gradient. </span></li></ul><ul class="c3 lst-kix_6peab1jnomrp-0"><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 25, 2014 6:04:08 PM.jpg" src="images/image22.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li></ul><ul class="c3 lst-kix_6peab1jnomrp-1 start"><li class="c1"><span>left to right is 65 to 58. My strong suspicion is that the long band in lane 1 is from a long template rather than 65. Picking colonies 1 and 3 for further investigation. Labeled 43 and 44 on the tubes, because that&rsquo;s where we are in the screening.</span></li></ul><p class="c6"><span class="c16">Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </span></p><ul class="c3 lst-kix_sj5vnpizr8db-0"><li class="c6 c10"><span>sequencing clone 25 with pSB1C3-up and -dn, dugsim job 1827</span></li></ul><p class="c4"><span></span></p><p class="c6"><span>It might be better to take the 5x repeat and then expand it with some other scheme. </span></p><ul class="c3 lst-kix_dd5gvcr9lwxi-0 start"><li class="c6 c10"><span>It is possible to do this by stepwise dig/lig/confirm cycles with XbaI/PstI dig&rsquo;d insert and SpeI/PstI dig&rsquo;d backbone (gel purified). </span></li><li class="c6 c10"><span>Another possible trick to speed up the expansion would be to cycle from 16C to 37C a couple of times with XbaI/SpeI dig&rsquo;d pSB1C3-5xGFP1 and also add XbaI/SpeI &amp; T4 ligase and a third enzyme that cuts somewhere else in the backbone. </span></li></ul><ul class="c3 lst-kix_dd5gvcr9lwxi-1 start"><li class="c1"><span>When XbaI and SpeI ligate it will destroy the recognition site. Any sites re-ligating to self will be cleaved in the next 37C cycle. The last step in the cycling reaction will be a long digest.</span></li><li class="c1"><span>Follow that with a ligation to XbaI/SpeI/CIP treated backbone. </span></li><li class="c1"><span>Circularization is possible with this scheme</span></li></ul><ul class="c3 lst-kix_dd5gvcr9lwxi-0"><li class="c6 c10"><span>Third possibility: cycle XbaI/PstI/CIPd insert with backbone, SpeI, PstI, ligase and cycle</span></li></ul><ul class="c3 lst-kix_dd5gvcr9lwxi-1 start"><li class="c1"><span>XbaI ligs to SpeI and destroys site. XbaI ligs to XbaI and gets re-cut, same with PstI. On re-cutting this would restore the phosphate removed by CIP so that doesn&rsquo;t help much. Hmm</span></li></ul><ul class="c3 lst-kix_dd5gvcr9lwxi-0"><li class="c6 c10"><span>&hellip;..or just the stepwise dig/lig/shock will work. I think the problems of circularization will be there no matter what I do with CIP &amp; including restriction enzymes the ligase.</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/26/14</span></p><p class="c6"><span class="c16">Objective: Confirm pSB1A2-GFP1 decoy arrays</span></p><ul class="c3 lst-kix_dbbueb2lh22a-0 start"><li class="c6 c10"><span>Yesterday picked 4 colonies from transformation of 5x and 1x repeat arrays and set up with EcoRI/PstI digest, alongside colonies 43 and 44 from yesterday&rsquo;s colony PCR and pSB1C3 5x and 1x parents. </span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 26, 2014 2:19:47 PM.jpg" src="images/image34.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>Slightly hard to read on the picture, but they&rsquo;re loaded in the order mentioned above. 43 might be longer than 5x, 44 looks like 1x so discarding. </span></li><li class="c6 c10"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 26, 2014 2:21:25 PM.jpg" src="images/image30.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></span></li><li class="c6 c10"><span>Zoom in on the 5x on 1A2 and Colony 1 might be slightly shorter than colony 4? Making bacteria with both of these just in case. Should probably sequence as well. pSB1C3-up-1 binds to 1A2, but not pSB1C3-dn</span></li><li class="c6 c10"><span>Shocking Z1 to make a couple of strains:</span></li></ul><ul class="c3 lst-kix_dbbueb2lh22a-1 start"><li class="c1"><span>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1</span></li><li class="c1"><span>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4</span></li><li class="c1"><span>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1</span></li><li class="c1"><span>pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1</span></li><li class="c1"><span>pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4</span></li><li class="c1"><span>pdCas9, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/27/14</span></p><p class="c6"><span class="c16">Objective: extend decoy arrays</span></p><ul class="c3 lst-kix_3e6a4wpaex86-0 start"><li class="c6 c10"><span>XbaI/PstI digest of 1C3-5xGFP1decoy followed by CIP, then SpeI/PstI of 1C3-5xGFP1 (same plasmid) with NO GEL PURIFICATION makes the backbone. I am hoping that since SpeI to PstI is only 18bp that the PCR cleanup kit gets rid of the fragment. Ligating with 3-fold molar excess of CIP-treated insert for 20m then shocking 10uL in to 70uL Z1. This is rushing the protocol; the other 10uL of the ligation mix will sit at RT for 90m in case the rushed version fails. </span></li></ul><ul class="c3 lst-kix_3e6a4wpaex86-1 start"><li class="c1"><span>Update: it looks like it did not fail; there are some colonies on bb-only and ins-only but ~10x as many on b+i.</span></li></ul><p class="c4"><span></span></p><p class="c6"><span class="c20">9/28/14</span></p><p class="c6"><span class="c16">Objective: test 1x and 5x decoy arrays</span></p><ul class="c3 lst-kix_3e6a4wpaex86-0"><li class="c6 c10"><span>4 colonies each picked from plates from</span></li></ul><ul class="c3 lst-kix_3e6a4wpaex86-1 start"><li class="c1"><span>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 </span><span class="c20">G51</span></li><li class="c1"><span>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 </span><span class="c20">G54</span></li><li class="c1"><span>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 </span><span class="c20">G11</span></li><li class="c1"><span>pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 </span><span class="c20">C51</span></li><li class="c1"><span>pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 </span><span class="c20">C54</span></li><li class="c1"><span>pdCas9, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 </span><span class="c20">C11</span></li></ul><ul class="c3 lst-kix_3e6a4wpaex86-0"><li class="c6 c10"><span>&hellip;.and grown overnight (started 9/27/14). 9:15am today diluted 1/200 in 3 mL fresh LB+AmpKanCm. 3h45m in 37C shaker. 1mL collected, spun down, and resuspended in PBS. Flowed 75000 events and got something that looks super promising. Means here are written down from stats view on MQ during acquisition and might change after full processing. There is a lot of variability in G51</span></li></ul><a href="#" name="225acd26b42536ecbf50f90518b34a9be550eb1e"></a><a href="#" name="10"></a><table cellpadding="0" cellspacing="0" class="c61 c66"><tbody><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">2014-09-28 flow data, not fully processed</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2 c32"><span class="c0"></span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">(constitutive GFP reporter)</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9 with or without GFP-targeting crRNA</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">decoy binding site count &amp; colony</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">expected GFP if working</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">tube label</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">unprocessed mean gfp</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">avg</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">std</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G51-1</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">42</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">69.25</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">28.14</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G51-2</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">50</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G51-3</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">83</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G51-4</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">102</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G54-1</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">64</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">68.33</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">5.13</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G54-2</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">67</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G54-3</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">nan</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">derepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G54-4</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">74</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">near repressed levels</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G11-1</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">16</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">16.00</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">0.82</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">near repressed levels</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G11-2</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">15</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">near repressed levels</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G11-3</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">17</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9-GFP1</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">near repressed levels</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">G11-4</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">16</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C51-1</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">131</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">141.75</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">7.46</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C51-2</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">143</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C51-3</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">145</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C51-4</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">148</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C54-1</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">153</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">149.00</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">3.16</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C54-2</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">150</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C54-3</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">146</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-5xGFP1decoy-4</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C54-4</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">147</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C11-1</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">150</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">153.25</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">3.95</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C11-2</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">152</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C11-3</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">159</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr><tr class="c33"><td class="c48" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB4K5-K608012</span></p></td><td class="c53" colspan="1" rowspan="1"><p class="c2"><span class="c0">pdCas9</span></p></td><td class="c50" colspan="1" rowspan="1"><p class="c2"><span class="c0">pSB1A2-1xGFP1decoy-1</span></p></td><td class="c11" colspan="1" rowspan="1"><p class="c2"><span class="c0">unrepressed</span></p></td><td class="c25" colspan="1" rowspan="1"><p class="c2"><span class="c0">C11-4</span></p></td><td class="c57" colspan="1" rowspan="1"><p class="c2"><span class="c0">152</span></p></td><td class="c30" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td><td class="c31" colspan="1" rowspan="1"><p class="c2"><span class="c0">&nbsp;</span></p></td></tr></tbody></table><p class="c4"><span></span></p><ul class="c3 lst-kix_3e6a4wpaex86-0"><li class="c6 c10"><span>Saved G51-1 through -4, then colony 1 for one of each different strain as glycerol stocks. Labeled very minimally with the bolded labels in first sub-bullet list. </span></li><li class="c6 c10"><span>Next steps:</span></li></ul><ul class="c3 lst-kix_3e6a4wpaex86-1 start"><li class="c1"><span>repeat</span></li><li class="c1"><span>try with more decoy site numbers: 10x is on the way, need to also make 0x</span></li></ul>
+/10/14</h2><p>Objective: assemble decoy binding site arrays</p><ul><li>Setting up two PCRs with same cycling program as last time: </li></ul><ul><li>one with 4x 50uL reactions using the same concs of everything as the 0.5 uL rx from 9/9/14 (blue labels)</li><li>one with equimolar oligo1 and oligo2 and varying amounts of reaction 0.5 from 9/9/14 (green labels)</li></ul><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 10, 2014 6:28:14 PM.jpg" src="images/image56.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>So something&rsquo;s happening. PCR cleanup&rsquo;d reactions 1-4 (green) and 5-8 (blue) in to one tube each labeled Decoy array (1-4 or 5-8) PCR1. Tomorrow will PCR with the end oligos and try gibson, dig/lig in to pSB1C3 and/or pSB1A2</li></ul><h2><a name="h.dmhnzg83ygzw"></a>9/11/14</h2><p>Objective: assemble decoy binding site arrays. Step 2: add prefix &amp; suffix to arrays</p><ul><li>Faw setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space&nbsp;as oligos on 0.5-2.0 ng of PCR cleanup&rsquo;d green and blue rx from 9/10/14 and running with the same cycling conditions.</li><li>CBC did not take a picture and doesn&rsquo;t remember why. PCR looked bad for some reason.</li></ul><h2><a name="h.knhldp48br8h"></a>9/12/14</h2><p>Objective: assemble decoy binding site arrays. Step 2: add prefix &amp; suffix to arrays</p><ul><li>Garima setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space&nbsp;as oligos on higher concs than yesterday ofPCR cleanup&rsquo;d green and blue rx from 9/10/14 and running with the same cycling conditions.</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 16, 2014 11:21:26 AM.jpg" src="images/image06.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>PCR cleanup to pool tubes 2-4 for both series and labeled Decoy Array PCR2</li></ul><h2><a name="h.qg2pv53zrivc"></a>9/13/14</h2><p>Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone</p><ul><li>CBC attempts a SLIC with 0.5 uL of the &ldquo;Super Clean&rdquo; XbaI/PstI pSB1C3 backbone. Note that puts 15 bp of nonhomology in the way of successful SLIC. I did this because sometimes it works despite mismatches and there was no EcoRI/PstI dig&rsquo;d pSB1C3 for me to work with. Green series looks like there is a bigger difference between no-template and +template tubes, so I will try that</li></ul><ul><li>set up tubes for backbone-only, backbone + 0.5, 1, 2, 3uL of insert from Green tube at 88.8 ng/uL, and 3uL insert-only.</li></ul><ul><li>Didn&rsquo;t work.</li></ul><p>Objective: observe fluorescence changes with antitracr time course over time</p><ul><li>Last night antitracr strains started in LB+AmpKanCm and let shake overnight. Diluted this morning in to +/- aTc and began collection at time indicated. Replaced LB with PBS before flowing</li><li>Isolated &ldquo;singlets&rdquo; which are probably not singlets (smalls) or left alone (bugs) and got mean GFPA or gfpa/fsca or /ssca to normalize roughly to size.</li><li></li></ul><a href="#" name="504882174bdbc3a066b549e955cdc3b34f759d86"></a><a href="#" name="7"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>time point</p></td><td><p>n/a</p></td><td><p>n/a</p></td><td><p>1</p></td><td><p>2</p></td><td><p>3</p></td><td><p>4</p></td><td><p>5</p></td></tr><tr><td><p>time (min)</p></td><td><p>n/a</p></td><td><p>n/a</p></td><td><p>120</p></td><td><p>180</p></td><td><p>240</p></td><td><p>300</p></td><td><p>360</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.52</p></td><td><p>2.74</p></td><td><p>3.17</p></td><td><p>3.16</p></td><td><p>2.81</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.39</p></td><td><p>2.8</p></td><td><p>3.18</p></td><td><p>3.42</p></td><td><p>2.88</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.45</p></td><td><p>2.92</p></td><td><p>3.31</p></td><td><p>3.53</p></td><td><p>2.88</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.53</p></td><td><p>3.13</p></td><td><p>3.72</p></td><td><p>3.6</p></td><td><p>2.89</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>45.6</p></td><td><p>56</p></td><td><p>68.4</p></td><td><p>76.8</p></td><td><p>74.9</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>46.9</p></td><td><p>54</p></td><td><p>67.8</p></td><td><p>73.8</p></td><td><p>74.7</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>48.4</p></td><td><p>53.1</p></td><td><p>65.8</p></td><td><p>73</p></td><td><p>76.2</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>45.6</p></td><td><p>54.2</p></td><td><p>68.2</p></td><td><p>74.3</p></td><td><p>78.4</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>0.109</p></td><td><p>0.16</p></td><td><p>0.185</p></td><td><p>0.139</p></td><td><p>0.143</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.43</p></td><td><p>3.08</p></td><td><p>3.65</p></td><td><p>3.67</p></td><td><p>3.07</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.32</p></td><td><p>3.17</p></td><td><p>3.67</p></td><td><p>3.88</p></td><td><p>3.07</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.73</p></td><td><p>3.3</p></td><td><p>4.15</p></td><td><p>3.99</p></td><td><p>3.18</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.57</p></td><td><p>3.47</p></td><td><p>4.16</p></td><td><p>3.83</p></td><td><p>3.29</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>41.7</p></td><td><p>53.4</p></td><td><p>67</p></td><td><p>75.8</p></td><td><p>75.6</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>41.4</p></td><td><p>51.3</p></td><td><p>67.4</p></td><td><p>74.3</p></td><td><p>71.7</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>39.4</p></td><td><p>52.4</p></td><td><p>66</p></td><td><p>73.9</p></td><td><p>60.8</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>40</p></td><td><p>52.7</p></td><td><p>67</p></td><td><p>76</p></td><td><p>73.2</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>0.0949</p></td><td><p>0.156</p></td><td><p>0.162</p></td><td><p>0.129</p></td><td><p>0.122</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>83.9</p></td><td><p>157</p></td><td><p>188</p></td><td><p>189</p></td><td><p>172</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>70.6</p></td><td><p>159</p></td><td><p>186</p></td><td><p>205</p></td><td><p>177</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>72.8</p></td><td><p>169</p></td><td><p>193</p></td><td><p>208</p></td><td><p>175</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>80.1</p></td><td><p>172</p></td><td><p>206</p></td><td><p>199</p></td><td><p>166</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2526</p></td><td><p>3227</p></td><td><p>3986</p></td><td><p>4592</p></td><td><p>4520</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2700</p></td><td><p>3078</p></td><td><p>4044</p></td><td><p>4486</p></td><td><p>4543</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2892</p></td><td><p>3028</p></td><td><p>3795</p></td><td><p>4291</p></td><td><p>4553</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2641</p></td><td><p>3131</p></td><td><p>4038</p></td><td><p>4531</p></td><td><p>4614</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>6.26</p></td><td><p>9.36</p></td><td><p>11.1</p></td><td><p>8.92</p></td><td><p>9.11</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>72.8</p></td><td><p>177</p></td><td><p>221</p></td><td><p>228</p></td><td><p>194</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>60.5</p></td><td><p>187</p></td><td><p>220</p></td><td><p>241</p></td><td><p>193</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>94.8</p></td><td><p>192</p></td><td><p>245</p></td><td><p>245</p></td><td><p>199</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>79.6</p></td><td><p>193</p></td><td><p>235</p></td><td><p>222</p></td><td><p>193</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2184</p></td><td><p>3100</p></td><td><p>4012</p></td><td><p>4663</p></td><td><p>4666</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2188</p></td><td><p>2957</p></td><td><p>4160</p></td><td><p>4554</p></td><td><p>4386</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2004</p></td><td><p>3047</p></td><td><p>4035</p></td><td><p>4565</p></td><td><p>3842</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2039</p></td><td><p>3051</p></td><td><p>4107</p></td><td><p>4630</p></td><td><p>4512</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>5.64</p></td><td><p>9.13</p></td><td><p>9.66</p></td><td><p>8.24</p></td><td><p>7.79</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>47.7</p></td><td><p>75.1</p></td><td><p>78.8</p></td><td><p>75.1</p></td><td><p>64.8</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>46.3</p></td><td><p>76.2</p></td><td><p>79.1</p></td><td><p>81</p></td><td><p>66</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>48</p></td><td><p>78.7</p></td><td><p>82.6</p></td><td><p>83.3</p></td><td><p>66.4</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>51.5</p></td><td><p>85.9</p></td><td><p>96.7</p></td><td><p>93.4</p></td><td><p>71.1</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1273</p></td><td><p>1469</p></td><td><p>1666</p></td><td><p>1779</p></td><td><p>1718</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1226</p></td><td><p>1413</p></td><td><p>1636</p></td><td><p>1719</p></td><td><p>1688</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1182</p></td><td><p>1382</p></td><td><p>1605</p></td><td><p>1704</p></td><td><p>1739</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1182</p></td><td><p>1391</p></td><td><p>1645</p></td><td><p>1714</p></td><td><p>1790</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>3.43</p></td><td><p>4.38</p></td><td><p>4.82</p></td><td><p>3.89</p></td><td><p>3.69</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>46.3</p></td><td><p>80.9</p></td><td><p>89.5</p></td><td><p>87.5</p></td><td><p>71</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>45.6</p></td><td><p>80.6</p></td><td><p>89.6</p></td><td><p>91.3</p></td><td><p>71.4</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>50.4</p></td><td><p>84.4</p></td><td><p>101</p></td><td><p>95</p></td><td><p>74</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>55.3</p></td><td><p>93.8</p></td><td><p>108</p></td><td><p>100</p></td><td><p>83.8</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1250</p></td><td><p>1375</p></td><td><p>1606</p></td><td><p>1758</p></td><td><p>1715</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1215</p></td><td><p>1320</p></td><td><p>1606</p></td><td><p>1734</p></td><td><p>1635</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1203</p></td><td><p>1319</p></td><td><p>1563</p></td><td><p>1718</p></td><td><p>1425</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1230</p></td><td><p>1338</p></td><td><p>1595</p></td><td><p>1754</p></td><td><p>1661</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>3.05</p></td><td><p>4.55</p></td><td><p>4.63</p></td><td><p>3.67</p></td><td><p>3.35</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.17</p></td><td><p>3.7</p></td><td><p>5.12</p></td><td><p>5.27</p></td><td><p>5.68</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.1</p></td><td><p>3.82</p></td><td><p>5.06</p></td><td><p>5.77</p></td><td><p>5.85</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.17</p></td><td><p>3.92</p></td><td><p>5.48</p></td><td><p>6.04</p></td><td><p>5.89</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.07</p></td><td><p>4.2</p></td><td><p>5.49</p></td><td><p>5.95</p></td><td><p>5.85</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>70.3</p></td><td><p>82.2</p></td><td><p>105</p></td><td><p>120</p></td><td><p>133</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>74.4</p></td><td><p>78.2</p></td><td><p>102</p></td><td><p>122</p></td><td><p>137</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>75.6</p></td><td><p>76.9</p></td><td><p>102</p></td><td><p>114</p></td><td><p>130</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>75.3</p></td><td><p>79.9</p></td><td><p>103</p></td><td><p>120</p></td><td><p>136</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>0.169</p></td><td><p>0.26</p></td><td><p>0.396</p></td><td><p>0.319</p></td><td><p>0.34</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.26</p></td><td><p>4.28</p></td><td><p>6.12</p></td><td><p>6.62</p></td><td><p>6.7</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.01</p></td><td><p>4.53</p></td><td><p>6.07</p></td><td><p>7.13</p></td><td><p>6.9</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.72</p></td><td><p>4.59</p></td><td><p>6.58</p></td><td><p>7.36</p></td><td><p>7.43</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.16</p></td><td><p>4.63</p></td><td><p>6.39</p></td><td><p>7.15</p></td><td><p>7.22</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>63.4</p></td><td><p>76.5</p></td><td><p>107</p></td><td><p>127</p></td><td><p>141</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>64.1</p></td><td><p>74.2</p></td><td><p>112</p></td><td><p>127</p></td><td><p>134</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>61.2</p></td><td><p>77.2</p></td><td><p>110</p></td><td><p>127</p></td><td><p>126</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>63.3</p></td><td><p>78.4</p></td><td><p>110</p></td><td><p>128</p></td><td><p>138</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>0.157</p></td><td><p>0.26</p></td><td><p>0.357</p></td><td><p>0.331</p></td><td><p>0.287</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>88.5</p></td><td><p>159</p></td><td><p>205</p></td><td><p>206</p></td><td><p>216</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>75.6</p></td><td><p>163</p></td><td><p>197</p></td><td><p>228</p></td><td><p>220</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>77.3</p></td><td><p>172</p></td><td><p>210</p></td><td><p>232</p></td><td><p>217</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>83.4</p></td><td><p>174</p></td><td><p>215</p></td><td><p>227</p></td><td><p>223</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2559</p></td><td><p>3229</p></td><td><p>4012</p></td><td><p>4818</p></td><td><p>5003</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2751</p></td><td><p>3078</p></td><td><p>4107</p></td><td><p>4768</p></td><td><p>5130</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>3006</p></td><td><p>3014</p></td><td><p>3787</p></td><td><p>4456</p></td><td><p>4927</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2695</p></td><td><p>3129</p></td><td><p>4076</p></td><td><p>4738</p></td><td><p>5032</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>6.33</p></td><td><p>10.1</p></td><td><p>15.8</p></td><td><p>12.9</p></td><td><p>14.1</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>78.4</p></td><td><p>182</p></td><td><p>242</p></td><td><p>263</p></td><td><p>257</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>64.6</p></td><td><p>194</p></td><td><p>236</p></td><td><p>278</p></td><td><p>259</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>101</p></td><td><p>195</p></td><td><p>263</p></td><td><p>287</p></td><td><p>277</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>82.5</p></td><td><p>195</p></td><td><p>250</p></td><td><p>270</p></td><td><p>274</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2213</p></td><td><p>3106</p></td><td><p>4099</p></td><td><p>5045</p></td><td><p>5411</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2212</p></td><td><p>2976</p></td><td><p>4289</p></td><td><p>5022</p></td><td><p>5084</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2022</p></td><td><p>3060</p></td><td><p>4164</p></td><td><p>4964</p></td><td><p>4574</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2061</p></td><td><p>3053</p></td><td><p>4158</p></td><td><p>4990</p></td><td><p>5205</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>5.59</p></td><td><p>10</p></td><td><p>14.4</p></td><td><p>13.3</p></td><td><p>12.3</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>49</p></td><td><p>73.4</p></td><td><p>74.8</p></td><td><p>71.9</p></td><td><p>69</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>48.4</p></td><td><p>74.5</p></td><td><p>74.4</p></td><td><p>77.2</p></td><td><p>68.9</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>49.8</p></td><td><p>77.1</p></td><td><p>79.2</p></td><td><p>80.5</p></td><td><p>69.6</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>53</p></td><td><p>84.8</p></td><td><p>90.9</p></td><td><p>88.6</p></td><td><p>74.1</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1267</p></td><td><p>1433</p></td><td><p>1538</p></td><td><p>1591</p></td><td><p>1583</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1210</p></td><td><p>1380</p></td><td><p>1489</p></td><td><p>1535</p></td><td><p>1541</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1170</p></td><td><p>1359</p></td><td><p>1497</p></td><td><p>1514</p></td><td><p>1583</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1174</p></td><td><p>1356</p></td><td><p>1512</p></td><td><p>1531</p></td><td><p>1634</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>3.25</p></td><td><p>4.27</p></td><td><p>5.57</p></td><td><p>4.17</p></td><td><p>4.13</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>48.5</p></td><td><p>77.5</p></td><td><p>84</p></td><td><p>84.1</p></td><td><p>77.8</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>47.8</p></td><td><p>77.4</p></td><td><p>83.9</p></td><td><p>88.1</p></td><td><p>76.9</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>51.9</p></td><td><p>80.9</p></td><td><p>94.2</p></td><td><p>92.9</p></td><td><p>82.8</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>56.8</p></td><td><p>90.5</p></td><td><p>101</p></td><td><p>94.2</p></td><td><p>90.5</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1246</p></td><td><p>1323</p></td><td><p>1446</p></td><td><p>1575</p></td><td><p>1613</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1208</p></td><td><p>1269</p></td><td><p>1413</p></td><td><p>1553</p></td><td><p>1529</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1196</p></td><td><p>1255</p></td><td><p>1395</p></td><td><p>1529</p></td><td><p>1366</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1231</p></td><td><p>1282</p></td><td><p>1420</p></td><td><p>1561</p></td><td><p>1553</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>2.82</p></td><td><p>4.23</p></td><td><p>4.92</p></td><td><p>4.26</p></td><td><p>3.62</p></td></tr></tbody></table><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 585.00px; height: 925.00px;"><img alt="" src="images/image49.jpg" style="width: 585.00px; height: 925.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>It looks like the decrease in fluor for pdCas9-GFP1 coli manifests fairly early and persists for 6 hours. Lutz &amp; Bujard&rsquo;s paper use slightly less aTc for full induction of pTet, but if the decrease we see here were due to aTc inhibiting translation then we would expect to see a decrease in +aTc cultures with pdCas9. If pTet expression of aTc was removing trasncription/translation resources away from GFP production then you would also expect that to manifest in pdCas9. However it might be possible that the pdCas9 strains are experiencing the same decrease from aTc or growht burden but the strong GFP expression is masking the effect.</li><li>The other possibility is that something scientifically interesting, but not useful for iGEM purposes, is going on.</li></ul><h2><a name="h.oz7brdgn00cg"></a>9/15/14</h2><p>Objective: assemble decoy binding site arrays. Step 2: add prefix &amp; suffix to arrays (retry)</p><ul><li>CBC sets up PCR with 0.5 ng/uL green or blue PCR1 template with 0, 0.4, 0.7, 1.0 uL in 50uL reactions with same cycling conditions and oligo concs etc as before. Maybe with less template the dominant species in the tubes will be that with prefix &amp; suffix added. Maybe</li><li>No difference between + template and notemplate tubes. No picture taken.</li><li></li></ul><h2><a name="h.bvksvj6lqq8i"></a>9/16/14</h2><p>Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone</p><ul><li>CBC prays, puts PCR2 products used on 9/13/14 in a digestion reaction with EcoRI/PstI and incubates 37C for 4h. PCR cleanup and CIP (buffer 2) for 20m then cleanup again. Both tubes have ~35 ng/uL and Dusseldorf ligation calculator calls for 12 ng of 100 (ish) bp insert &nbsp;for 86 ng 2000 bp bb. Assuming that the large majority of the inserts lack the prefix and suffix, I will try 1 and 2 uL of insert for 1 of the bb. Also bb only and insert only, of course. Ligating 90m at RT</li><li>Shocking in to Z1; 30m on ice with 10uL of the ligation mix, 80m recovery before plating on LB+Cm</li><li>next morning: no colonies on any plates. Cm works</li></ul><p>9/17/14</p><p>Objective: PCR to assemble decoy binding site array</p><ul><li>MZ does PCR on 4 tubes: 0 um, 0.5 um, 1 um, and 2 um of ~200 ng/uL prefix oligo respectively + 50 uL standard mix (Q5 buffer and polymerase, repeat-spacer-repeat and spacer-repeat-spacer oligos, dNTPS, H2O) </li><li>PCR for 75 min</li><li>Gel to confirm (picture)</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:40:25 PM.jpg" src="images/image03.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>From left to right, 0, 0.5, 1, 2 uL of prefix oligo. Shortening of schmear is consistent with intended effect but not conclusive evidence thereof.</li><li>CBC takes 0.3 uL out of wells 0.5, 1, 2 to serve as template for new PCR2 with prefix &amp; suffix oligos. MCF runs the gel</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:40:52 PM.jpg" src="images/image15.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><p>9/18/14</p><p>Objective: PCR to assemble decoy binding site array</p><ul><li>CBC sets up 3 new PCR series, each with 0.3, 0.6, 0.9, 1.2 uL of tubes 2, 3, 4 from previous reaction as template. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:44:02 PM.jpg" src="images/image14.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Left to right is series from reactions templated with tube 2, 3, and 4. Gels look more like small pieces are dominating (primer dimer??) but we will try gibson anyway</li></ul><p>Objective: PCR to assemble decoy binding site array</p><ul><li></li><li>MZ with CBC get Mert&rsquo;s homemade 1.33x gibson assembly mix and make reactions with backbone only, then backbone + insert</li><li>1uL of (conc) backbone (=EcoRI/PstI/CIP pSB1C3-R0040 made by CBC on [date] conc = [conc]) and 0.3uL of PCR products from series 2, 3, 4 at concs [conc conc conc] respectively.</li><li>Next morning: 100s of colonies.</li></ul><p>Objective: PCR to assemble decoy binding site array</p><ul><li>Master mix X13: &nbsp;(of a standard 50 uL reaction)</li></ul><ul><li>494 uL dH2O</li><li>130 uL buffer (Q5)</li><li>13 uL dNTPs</li><li>3.25 uL each oligo (prefix &amp; suffix oligos)</li><li>6.25 uL enzyme (Q5)</li><li>0.3 uL of each template (0.5, 1, 2)</li></ul><ul><li>Ran PCR with iGEM protocol (previously saved)</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 19, 2014 9:48:04 AM.jpg" src="images/image48.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Some uneven loading in gel or at addition of template to tube step; either way it&rsquo;s fine. Pool with PCR cleanup protocol and then can use this for GA. 0.5 and 1.0 series look the most promising for this. </li></ul><p>9/19/14</p><p>Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly yesterday</p><ul><li>100s of colonies on bb+insert plates 2, 3, 4. None on insert-only plates or backbone-only. </li><li>Colony PCR with pSB1C3-up and -dn with 62C annealing temp (see 7/25/2014 entry, Farnitano figured this temp out for these oligos)</li><li>1ng pSB1C3-R0040, no template control, then 4 colonies from plate 2, 4 from 3, 6 from 4. </li><li>No amplification on anything. No picture</li></ul><p>Objective: assemble decoy arrays. Insert putative arrays in to pSB1C3</p><ul><li>Delta pools reactions from Garima&rsquo;s PCR/gel produced yesterday in to three tubes. CBC sets up gibson assembly. 1 uL backbone and 0.3 insert as appropriate, H2O to 5 uL, vortexed briefly then added 2.5uL of this to 7.5 uL of master mix. Only enough master mix was in the freezer for 6 of these half-sized reactions so there is no insert-only control for reactions labeled 2 in Garima&rsquo;s gel.</li><li>Transforming all 10 uL in to 80uL of competent Z1s. </li><li>Next morning, lots of colonies on all the places there should be colonies. Huzzah</li></ul><p>9/21/14</p><p>Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </p><ul><li>Picked 18 colonies from gibson/transformation performed on 9/19/14 yesterday and miniprepped. EcoRI/PstI digest for 3h then run 15m on 0.8% gel</li></ul><ul><li>Unadorned pSB1C3 is 41bp between EcoRI and PstI, 107 for minimal intended length of the four assembly oligos. It looks like there might be some incomplete digestion happening on these maybe, or just overloaded. </li><li>taaggatgatttctgg aattcgcggccgcttctagagCCATCTAATTCAACAAGAATTGGtgatgttaatCCATCTAATTCAACAAGAATTGGtgatgttaattactagtagcggccgctgca gtccggcaaaaaagggc</li></ul><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 21, 2014 4:12:27 PM.jpg" src="images/image51.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Left to right is 1-18. Two probably aberrant clones but they&rsquo;re getting sequenced anyway. Sent to bio sequencing core seqd from pSB1C3-up-1 and -dn-1. Clones 1-4, 15-18. </li></ul><p>9/22/14</p><p>Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </p><ul><li>Mike picks 12 more colonies for miniprepping tomorrow</li></ul><p>9/23/14</p><p>Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </p><ul><li>Mike starts miniprep for 12 new colonies, charlie takes over after N3 addition &amp; spin. Mike sets up EcoRI-HF/PstI-HF digest (cutsmart) for each clone. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 23, 2014 4:41:21 PM.jpg" src="images/image38.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Garima loaded this 1.3% TAE gel. Lane 7 (ie clone 25) has one big band at ~600 but also looks to have a small band down at the 1x size. Contamination? Unstable repeats? Another gel 0.8% has the same thing, but smearier. </li><li>Sequencing results come back!! As expected and consistent with the gel above, almost all colonies have a single insert, but clone 17 has 5! It works, kind of!!! The chromatograms taper off in quality around the repeats which makes it difficult for seqman to put it assemble properly, but manually getting called bases and finding repeats makes it clear. Clone 2 from yesterday was trash. </li></ul><p><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 806.50px; height: 48.18px;"><img alt="" src="images/image12.png" style="width: 806.50px; height: 48.18px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></p><ul><li>ctrl-F in text editor highlights each decoy site. To the left and right are prefix &amp; suffixes</li></ul><p>Objective: transfer decoy arrays to pSB1A2</p><ul><li>CBC sets up prep scale digest of EcoRI/PstI of clones 17 &amp; 18 &amp; pSB1A2-R0040-antitracr. PCR cleanup and CIP treatment of clones 17 &amp; 18 followed by a second PCR cleanup. Gel extraction of pSB1A2 backbone leaves only 22 ng/uL of dirty DNA by nanodrop. Tomorrow will try ligating, and also will set up another EcoRI/PstI digest of more pSB1A2-R0040-antitracr</li></ul><p>Objective: optimize colony PCR conditions for testing pSB1C3-decoy arrays</p><ul><li>Garima sets up master mix with 9x50uL rx. pSB1C3-up-1 and -dn-1 included at 0.25*9uL from 100uM stock. </li></ul><ul><li>45 uL Taq buffer</li><li>2.25 uL Taq polymerase</li><li>9 uL dNTPs</li><li>9 uL of each pSB1C3-up-1 and -dn-1</li><li>375.75 uL dH2O</li><li>9 uL template in positive tube/ 0 uL template in negative tubes</li></ul><ul><li>Ran colony pcr using saved protocol:</li></ul><ul><li>edited annealing step of protocol from 62 degrees to a gradient : 58-65 degrees</li><li>extension time of 15s (was 30)</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 24, 2014 1:13:46 PM.jpg" src="images/image35.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Fairly crappy snapshot (crapshot?) but you can see that the primer dimers on the - gel are all at ~100bp and the product on +template are at 2-300 which is consistent with R0040-antitracr size. the best amplification took place at higher temps. New protocol: 65C with 15s extension for short pieces, next try with some actual colonies.</li></ul><p>9/24/14</p><p>Objective: transfer decoy arrays to pSB1A2</p><ul><li>Setting up ligation reaction with EcoRI/PstI/CIP treated clone 17 and 18 from yesterday (5x and 1x decoys respectively) and EcoRI/PstI/gel-purified pSB1A2 bb. Also including controls below</li><li>used ligation calculator from dusseldorf </li><li></li></ul><a href="#" name="b2d32455b7b3438d6a59a365920ec8095fe8bb0e"></a><a href="#" name="8"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td></td><td><p>17 (5x)</p></td><td><p>18 (1x)</p></td><td><p>1A2 bb</p></td></tr><tr><td><p>length</p></td><td><p>~500</p></td><td><p>~100</p></td><td><p>2252</p></td></tr><tr><td><p>conc</p></td><td><p>84.1</p></td><td><p>63.8</p></td><td><p>22.8</p></td></tr><tr><td><p>ng needed</p></td><td><p>66</p></td><td><p>13</p></td><td><p>100</p></td></tr><tr><td><p>using uL</p></td><td><p>1</p></td><td><p>0.5</p></td><td><p>5</p></td></tr></tbody></table><a href="#" name="5465b9bebaea090720acd450041ece042a9665b5"></a><a href="#" name="9"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>thing</p></td><td><p>bb+ins 17</p></td><td><p>insert only 17</p></td><td><p>bb+ins 17</p></td><td><p>insert only 17</p></td><td><p>bb only</p></td></tr><tr><td><p>backbone</p></td><td><p>5uL</p></td><td><p>0</p></td><td><p>5</p></td><td><p>0</p></td><td><p>5</p></td></tr><tr><td><p>insert</p></td><td><p>1uL</p></td><td><p>1</p></td><td><p>0.5</p></td><td><p>0.5</p></td><td><p>0</p></td></tr><tr><td><p>T4 buffer</p></td><td><p>2uL</p></td><td><p>2</p></td><td><p>2</p></td><td><p>2</p></td><td><p>2</p></td></tr><tr><td><p>enzyme</p></td><td><p>1uL</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td></tr><tr><td><p>h2o</p></td><td><p>11uL</p></td><td><p>16</p></td><td><p>11.5</p></td><td><p>16.5</p></td><td><p>12</p></td></tr></tbody></table><ul><li>Mixed andl et sit for 3h at room temp on bench. Took 10uL and transformed with 80uL of frozen competent Z1s</li><li>next day: lots of colonies on bb+insert plates, none on bb-only or insert-only. huzzah</li></ul><p>Objective: find more long decoy arrays</p><ul><li>miniprepping 12 more colonies from plate 1.0, middle one from 9/18/14</li><li>EcoRI/PstI digest for 3h gives all small bands at 1x size. No picture taken, all tubes discarded. </li></ul><p>9/25/14</p><p>Objective: find more long decoy arrays</p><ul><li>colony PCR on 4 colonies from plate 1.0 next to no-template, 1ng of pSB1C3-R0040-antitracr, pSB1C3-5xGFP1 decoy, and pSB1C3-1xGFP1 decoy. 10s extension at 64C</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 25, 2014 5:39:52 PM.jpg" src="images/image39.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title="">&nbsp;No amplification on any of the colonies?</li></ul><ul><li>no template, pSB1C3-R0040-antitracr, 5x, 1x, then 4 colonies. At first I thought this was no amplification but actually I think it worked but all of the colonies are 1x or maybe a 2x for colony 4. Threw them all away in any case and before I had the realization that they amplified I already set up the next PCR with 8 colonies on a 58-65C gradient. </li></ul><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 25, 2014 6:04:08 PM.jpg" src="images/image22.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><ul><li>left to right is 65 to 58. My strong suspicion is that the long band in lane 1 is from a long template rather than 65. Picking colonies 1 and 3 for further investigation. Labeled 43 and 44 on the tubes, because that&rsquo;s where we are in the screening.</li></ul><p>Objective: confirm insert presence &amp; size of decoy array inserts from gibson assembly </p><ul><li>sequencing clone 25 with pSB1C3-up and -dn, dugsim job 1827</li></ul><p>It might be better to take the 5x repeat and then expand it with some other scheme. </p><ul><li>It is possible to do this by stepwise dig/lig/confirm cycles with XbaI/PstI dig&rsquo;d insert and SpeI/PstI dig&rsquo;d backbone (gel purified). </li><li>Another possible trick to speed up the expansion would be to cycle from 16C to 37C a couple of times with XbaI/SpeI dig&rsquo;d pSB1C3-5xGFP1 and also add XbaI/SpeI &amp; T4 ligase and a third enzyme that cuts somewhere else in the backbone. </li></ul><ul><li>When XbaI and SpeI ligate it will destroy the recognition site. Any sites re-ligating to self will be cleaved in the next 37C cycle. The last step in the cycling reaction will be a long digest.</li><li>Follow that with a ligation to XbaI/SpeI/CIP treated backbone. </li><li>Circularization is possible with this scheme</li></ul><ul><li>Third possibility: cycle XbaI/PstI/CIPd insert with backbone, SpeI, PstI, ligase and cycle</li></ul><ul><li>XbaI ligs to SpeI and destroys site. XbaI ligs to XbaI and gets re-cut, same with PstI. On re-cutting this would restore the phosphate removed by CIP so that doesn&rsquo;t help much. Hmm</li></ul><ul><li>&hellip;..or just the stepwise dig/lig/shock will work. I think the problems of circularization will be there no matter what I do with CIP &amp; including restriction enzymes the ligase.</li></ul><p>9/26/14</p><p>Objective: Confirm pSB1A2-GFP1 decoy arrays</p><ul><li>Yesterday picked 4 colonies from transformation of 5x and 1x repeat arrays and set up with EcoRI/PstI digest, alongside colonies 43 and 44 from yesterday&rsquo;s colony PCR and pSB1C3 5x and 1x parents. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 26, 2014 2:19:47 PM.jpg" src="images/image34.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Slightly hard to read on the picture, but they&rsquo;re loaded in the order mentioned above. 43 might be longer than 5x, 44 looks like 1x so discarding. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 26, 2014 2:21:25 PM.jpg" src="images/image30.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Zoom in on the 5x on 1A2 and Colony 1 might be slightly shorter than colony 4? Making bacteria with both of these just in case. Should probably sequence as well. pSB1C3-up-1 binds to 1A2, but not pSB1C3-dn</li><li>Shocking Z1 to make a couple of strains:</li></ul><ul><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1</li><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4</li><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1</li></ul><p>9/27/14</p><p>Objective: extend decoy arrays</p><ul><li>XbaI/PstI digest of 1C3-5xGFP1decoy followed by CIP, then SpeI/PstI of 1C3-5xGFP1 (same plasmid) with NO GEL PURIFICATION makes the backbone. I am hoping that since SpeI to PstI is only 18bp that the PCR cleanup kit gets rid of the fragment. Ligating with 3-fold molar excess of CIP-treated insert for 20m then shocking 10uL in to 70uL Z1. This is rushing the protocol; the other 10uL of the ligation mix will sit at RT for 90m in case the rushed version fails. </li></ul><ul><li>Update: it looks like it did not fail; there are some colonies on bb-only and ins-only but ~10x as many on b+i.</li></ul><p>9/28/14</p><p>Objective: test 1x and 5x decoy arrays</p><ul><li>4 colonies each picked from plates from</li></ul><ul><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 G51</li><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 G54</li><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 G11</li><li>pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 C51</li><li>pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 C54</li><li>pdCas9, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 C11</li></ul><ul><li>&hellip;.and grown overnight (started 9/27/14). 9:15am today diluted 1/200 in 3 mL fresh LB+AmpKanCm. 3h45m in 37C shaker. 1mL collected, spun down, and resuspended in PBS. Flowed 75000 events and got something that looks super promising. Means here are written down from stats view on MQ during acquisition and might change after full processing. There is a lot of variability in G51</li></ul><a href="#" name="225acd26b42536ecbf50f90518b34a9be550eb1e"></a><a href="#" name="10"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>2014-09-28 flow data, not fully processed</p></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><p>(constitutive GFP reporter)</p></td><td><p>pdCas9 with or without GFP-targeting crRNA</p></td><td><p>decoy binding site count &amp; colony</p></td><td><p>expected GFP if working</p></td><td><p>tube label</p></td><td><p>unprocessed mean gfp</p></td><td><p>avg</p></td><td><p>std</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-1</p></td><td><p>42</p></td><td><p>69.25</p></td><td><p>28.14</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-2</p></td><td><p>50</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-3</p></td><td><p>83</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-4</p></td><td><p>102</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-1</p></td><td><p>64</p></td><td><p>68.33</p></td><td><p>5.13</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-2</p></td><td><p>67</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-3</p></td><td><p>nan</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-4</p></td><td><p>74</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-1</p></td><td><p>16</p></td><td><p>16.00</p></td><td><p>0.82</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-2</p></td><td><p>15</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-3</p></td><td><p>17</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-4</p></td><td><p>16</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-1</p></td><td><p>131</p></td><td><p>141.75</p></td><td><p>7.46</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-2</p></td><td><p>143</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-3</p></td><td><p>145</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-4</p></td><td><p>148</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-1</p></td><td><p>153</p></td><td><p>149.00</p></td><td><p>3.16</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-2</p></td><td><p>150</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-3</p></td><td><p>146</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-4</p></td><td><p>147</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-1</p></td><td><p>150</p></td><td><p>153.25</p></td><td><p>3.95</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-2</p></td><td><p>152</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-3</p></td><td><p>159</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-4</p></td><td><p>152</p></td><td><p>&nbsp;</p></td><td><p>&nbsp;</p></td></tr></tbody></table><ul><li>Saved G51-1 through -4, then colony 1 for one of each different strain as glycerol stocks. Labeled very minimally with the bolded labels in first sub-bullet list. </li><li>Next steps:</li></ul><ul><li>repeat</li><li>try with more decoy site numbers: 10x is on the way, need to also make 0x</li></ul>
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