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From 2008.igem.org

Contents 1 PCR/primers 1.1 HO+pcyA, P3 vector w/o GFP 1.2 mtrB 1.2.1 7/17: PCR 1.2.2 7/20: Retry (Gradient) 1.3 P38, P39 Oligos 1.4 P11 1.5 CDF 1.6 mtrB 2 Ligations 2.1 Results from 7/12 ligations (transformed into E1), 7/16 transformations 3 RE digests 07/15 4 RE digests 07/16 5 RE digests 07/17 6 RE digests 07/18 7 RE digests 07/19 8 Ligations 07/15 8.1 Results 7/16 9 Cross Transformations in S1 9.1 Transformations (7/11) and Picked Colonies (7/13) 9.2 Diluted and Re-grew in Media with Both Antibiotics 7/14 9.3 Restreaked in Plates with Double Selection Markers 7/15 9.4 Testing Cultures with IPTG 7/15 10 Thermoinducible Lac System 10.1 Testing Thermoinducible Cultures 7/14 10.2 Results from First Thermoinducible Test 7/14 11 Putting Thermoinducible Lac and pLambda + GFP System on p15a Vector 11.1 RE Digestion 7/11 11.1.1 Gel 7/14 11.2 Using BioBricks Primers to PCR Amplify P84-5 7/14 11.2.1 Gel from PCR 7/15 11.2.2 Digestion of PCR Products with XP 7/15 11.3 Single and Triple Digests of P84-5 7/14 11.3.1 Gel of Single and Triple Digests 7/15 11.4 Ligation of P74, Triple Digested P84-5, and P1 7/15 11.4.1 Dephosphorylation of P1, 7/16 11.4.2 Ligation 7/15 11.5 Transformation of P1/P74 and P1/P84-5 Triple Cuts 7/15 11.5.1 Picked Colonies for Liquid Culture 7/16 11.5.2 Minipreps of Liquid Culture 7/17 11.6 Ligation and Transformation of PCR Amplified P84-5 with P1 (7/16) 11.6.1 Ligation of P84-5 PCR with Dephos P1, 7/16 11.6.2 Transformation of P92-3 PCR into E1, 7/16 11.6.3 Liquid Cultures (7/17) and Minipreps (7/18) 11.7 Transformation of P75 and P92-3 (from 3x Cut) into S1 7/17 11.8 Transformation of P92-3 (from PCR) into S1 7/18 12 Test of IPTG Inducible System 13 Overnight cultures 07/18 14 Housekeeping! 14.1 Daily 14.2 Testing and Making Competent E. coli 14.2.1 Test Transformation of New Batches of Competent Cells 7/15 14.2.2 Making New Competent Cells 7/22 14.3 Keeping Up Stocks of Plasmids 14.3.1 7/15/08 Minipreps 14.3.2 7/18/08 14.4 Transformation of P86, P87, P97

PCR/primers

HO+pcyA, P3 vector w/o GFP

Primers were designed to PCR out the heme oxygenase and pcyA coding regions from the UT Austin plasmids. Due to the RE sites in these nonBB plasmids, their ends are given ApaLI and KpnI sites. The same is done for the P3 vector (the primers are such that we can PCR out the backbone, along with the RBS and terminator. The "P3-GFP" primers should also work for P1. However, I'm a little concerned, b/c the annotation of the P1 and P3 vectors disagrees with the sequence given for the RBS and terminator subparts.

mtrB

The same thing was done for mtrB (as for HO+pcyA). This gives us constitutive mtrB for testing the complement.

7/17: PCR

The primers arrived today, so PCR was performed using a colony of Shewanella Δ EnvZ (which should still have mtrB).
Rx mix: 90μL PCR supermix, 2μL mtrB-ApaLI-F primer (20μM), 2μL mtrB-KpnI-R (20μM), swirl of colony
Rx: 10min @ 94°C → 10x[45s @ 94°C → 45s @ 52°C → 2m30s @72°C] → 20x[45s @ 94°C → 45s @ 54°C → 4m45s @72°C]→ 5x[45s @ 94°C → 45s @ 56°C → 4m45s @72°C]→5min @ 72°C → ∞ @ 4°C

PCR failed- perhaps annealing temperature was too high or too many cells. Will retry.

7/20: Retry (Gradient)

Rx mix (split into 12 samples): 180μL PCR supermix, 4μL mtrB-ApaLI-F primer (20μM), 4μL mtrB-KpnI-R (20μM), swirl of WT Shewanella colony, 12μL water
Rx: 5min @ 94°C → 35x[45s @ 94°C → 45s @ {43.0, 43.2, 43.8, 44.5, 45.5, 46.9, 48.4, 49.7, 50.6, 51.3, 51.8, 52.0}°C → 2m38s @72°C] → 5min @ 72°C → ∞ @ 4°C

P38, P39 Oligos

5' phospho oligos were made to anneal into what is effectively P38 and P39 digested with ES.

P11

7/16 P11 (a piece of the filter paper) was PCRed: 2μL EB, 45μL PCR supermix, 1μL BBpfx primer (20μM), 1μL BBsfx (20μM) Rx: 5min @ 94°C → 15x[45s @ 94°C → 45s @ 54°C → 4m45s @72°C] → 20x[45s @ 94°C → 45s @ 56°C → 4m45s @72°C]→ 5min @ 72°C → ∞ @ 4°C
7/17

PCR failed- no 4kb band

CDF

8 100μL PCR reactions set up (standard mix): DNA template is S1 P13, 40 cycles, annealing temp is 58°C, extension time is 1:15

Run on gel; bands cut and frozen.

File:7-19 cdf, mtrb bb pcr mxh.jpg	1.2% agarose E-gel run for 30 min and visualized using EtBr/UV
	Lane	Contents
	1	[http://tools.invitrogen.com/content/sfs/manuals/15615016.pdf 1 KB ladder]
	2-4	CDF (~900 bp)
	5-12	mtrB w/ BB ends (~2.1kb)

mtrB

8 100μL PCR reactions set up (standard colony mix) w/ WT Shewanella

Rx: 5min @ 94°C → 10x[45s @ 94°C → 45s @ 52°C → 2m45s @72°C] → 25x[45s @ 94°C → 45s @ 55°C → 2m38s @72°C]→ 5min @ 72°C → ∞ @ 4°C

Run on gel; bands cut and frozen.

File:7-19 mtrb mxh.jpg

Ligations

Results from 7/12 ligations (transformed into E1), 7/16 transformations

DNA	Selection used on plate	# colonies	Notes	Liquid cultures of retransformed E1 (plating skipped) 7/16
17XP+38SP	AMP	2 AFTER 48HRS	Plate put in 4°C, not picked	no growth
63+59 FROM 7/9 LIGATION	KAN	1 AFTER 48HRS	no GFP, so plate tossed	no growth
CDF XP (7/4)+P1XP	KAN	1 AFTER 48HRS	has GFP- indicates wrong plasmid, tossed	--
45XP(7/11 45)+39SP	AMP	0		no growth
45XP(7/11 45)+38SP	AMP	0		no growth
77XP+38SP	AMP	0		no growth
52XP+39SP	AMP	0		no growth
77XP+39SP	AMP	0		no growth
89XP+38SP	AMP	0		no growth
45XP+39SP	AMP	0		no growth
89XP+39SP	AMP	0		no growth
51XP+39SP	AMP	0		no growth
17XP+39SP	AMP	0		no growth
P88	KAN	0		--
63+58 FROM 7/9 LIGATION	KAN	0		no growth
58EX+63ES	KAN	0		no growth
52XP+38SP	AMP	0		no growth
89ES+45EX	CARB	TMTC	The 45 EX used was not purified, so the miniprep culture was of religated P45 and did not glow. However, we picked fluorescent colonies from the plate and restreaked/set up liquid cultures of them. 7/15: Pelleted impure cultures were streaked for isolation. Individual fluorescent colonies picked for miniprep cultures (7/16: did not fluoresce, plates tosses).	--
CDF XP(7/4)+P1XP(7/1)	KAN	68	miniprepped	--
17ES+45EX	CARB	TMTC	The 45 EX used was not purified, so the miniprep cultures were of religated P45 and did not glow. However, we picked fluorescent colonies from the plate and restreaked/set up liquid cultures of them. 7/15: Miniprep done, glycerol stock made, pelleted impure cultures were streaked for isolation (7/16: did not fluoresce, plates tosses). Individual fluorescent colonies picked for miniprep cultures.	--
45XP+38SP	AMP	4	DID NOT FLUORESCE- not miniprepped	grew, streaked for colonies- no GFP, tossed
51XP+38SP	CARB	5	2 picked colonies, neither grew- repicked 7/14, these did not grow either	no growth
54ES+59EX	KAN	3	DID NOT FLUORESCE- not miniprepped, no GFP, so plate tossed	no growth
CDF XP(7/12)+P1XP(7/1)	KAN	TMTC	miniprepped	--
CDF XP(7/12)+P1	KAN	232	miniprepped	--
77ES+59EX	KAN	NOT PLATED		no growth
no DNA	AMP	0		--
no DNA	KAN	0		--
no DNA	CARB	6 big + 10 small		--
no cells	KAN	0		no growth
1μL pUC19	CARB	96	did grow in LB AMP medium (+ control)	--
1μL pUC19	AMP	64		--

We retransformed all of these on 07/15 using 5 μL DNA for 50 μL DH5α cells. We put the cells directly into liquid cultures after transforming them.

RE digests 07/15

We digested P53/54 with ES and P58/59 with EX. We intend to ligate these parts together. This will give us RBS + TetR/LacI + terminator + pTet/pLac + RBS + GFP + terminator.

We also digested P90 with SP and P48/P49 with XP. This will give us the CDF origin in a p15A vector with an Amp or Cm resistance cassette.

We followed the standard digest protocol under the "General Protocols" section.

Gel 1

File:7-15 gel 1 MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: P48 cut XP (769 bp)

Lane 3: P48 uncut (3477 bp)

Lane 4: P49A cut XP (943 bp)

Lane 5: P49A uncut (3651 bp)

Lane 6: P49B cut XP (943 bp)

Lane 7: P49B uncut (3651 bp)

Lane 8: P49C cut XP (943 bp)

Lane 9: P49C uncut (3651 bp)

Lane 10: P53 cut ES (840 bp)

Gel 2

File:7-15 gel 2 MXHTA.jpg

Lane 1: P53 uncut (2919 bp)

Lane 2: P54 cut ES (1308 bp)

Lane 3: P54 uncut (3387 bp)

Lane 4: P58 cut EX (~3016 bp)

Lane 5: P58 uncut (3016 bp)

Lane 6: P59A cut EX (~3201 bp)

Lane 7: P59A uncut (3201 bp)

Lane 8: P59B cut EX (~3201 bp)

Lane 9: 1 kb ladder

Gel 3

File:7-15 gel 3 MXHTA.jpg

Lane 1: P59B uncut (3201 bp)

Lane 2: P59C cut EX (~3201 bp)

Lane 3: P59C uncut (3201 bp)

Lane 4: P90A cut SP (~3650 bp)

Lane 5: P90A uncut (~3650 bp)

Lane 6: P90A cut SP (~3650 bp)

Lane 7: P90A uncut (~3650 bp)

Lane 8: P90A cut SP (~3650 bp)

Lane 9: P90A uncut (~3650 bp)

Lane 10: blank

Lane 11: 1 kb ladder

RE digests 07/16

We digested

• P63, P76 (A, B, C, D), P91 (A, B, C) with EX
• P90 (α, β) with SP
• P48, P49 (A, B, C) with XP

We used the standard digestion protocol.

We plan to ligate

• P90 with P48/P49. This will give us the CDF origin in the P1 vector with Amp and Cm resistance cassettes. We will also add P63, a terminator, and the high and low constitutive promoters (P38 and P39) later.
• P91/P76 with the high and low constitutive promoters. This should give us the entire Lac and Tet plasmids.

Gel 1

File:7-16 gel 1 MXHTA.jpg

• 1 kb ladder
• P48 cut XP
• P48 uncut
• P49A cut XP
• P49A uncut
• P49B cut XP
• P49B uncut
• P49C cut XP
• P49C uncut
• P63 cut EX
• P63 uncut
• P76A cut EX
• P76A uncut
• P76B cut EX
• P76B uncut

Gel 2

• 1 kb ladder
• P90α cut SP
• P90α uncut
• P90β cut SP
• P90β uncut
• P91A cut EX
• P91A uncut
• P91B cut EX
• P19B uncut

We extracted and purified P48, P49A, P49B, P63

RE digests 07/17

We digested

• P11 with XP
• P90α/β with SP (we plan to add the Amp/Cm resistance cassettes, P48 and P49)
• P91D/E with EX (we plan to add the high and low constitutive promoters, P38 and P39)
• P80 with ES (we think this is P90 with the Amp resistance marker; we plan to add terminators, P63)
• P76E/F with EX (we plan to add the high and low constitutive promoters, P38 and P39)

We digested more DNA (scaled everything up) to improve our yields from the gel extraction.

	P11	P90α	P90β	P91D	P91E	P80	P76E	P76F
DNA	20 μL	40 μL	40 μL	35 μL	10 μL	40 μL	40 μL	15 μL
10X NEB Buffer	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL
25X BSA	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL
REs (1 μL each)	XP	SP	XP	SP	SP	XP	XP	XP
Water	9.5 μL	0 μL	16.5 μL	14.5 μL	14.5 μL	16.5 μL	9.5 μL	9.5 μL

Gel 1

File:7-17 gel 1 MXHTA.jpg

• Lane 1: 1 kb ladder
• Lane 2: P76C cut EX (3857 bp)
• Lane 3: P76C uncut (~3857 bp)
• Lane 4: P76D cut EX (3857 bp)
• Lane 5: P76D uncut (~3857 bp)
• Lane 6: P76E cut EX (3857 bp)
• Lane 7: P76E uncut (~3857 bp)
• Lane 8: P76F cut EX (3857 bp)
• Lane 9: P76F uncut (~3857 bp)
• Lane 10: P80 cut ES (~1800 bp)

Gel 2

File:7-17 gel 2 MXHTA.jpg

• Lane 1: 1 kb ladder
• Lane 2: 80 uncut (~4500 bp)
• Lane 3: P90α cut SP (~3600 bp)
• Lane 4: P90α uncut (~3600 bp)
• Lane 5: P90β cut SP (~3600 bp)
• Lane 6: P90β uncut (~3600 bp)
• Lane 7: P91C cut EX (4327 bp)
• Lane 8: P91C uncut
• Lane 9: P91D cut EX (4327 bp)
• Lane 10: P91D uncut

Gel 3

File:7-17 gel 3 MXHTA.jpg

• Lane 1: 1 kb ladder
• Lane 2: P91E cut EX (4327 bp)
• Lane 3: P91E uncut
• Lane 4: P11 cut XP (4333 bp)

RE digests 07/18

Gel 1

File:7-18 gel 1 MXHTA.jpg

• Lane 1: 1 kb ladder
• Lane 2: P90α cut SP (~3600)
• Lane 3: P90α uncut
• Lane 4: P90β cut SP (~3600)
• Lane 5: P90β uncut
• Lane 6: P90γ cut SP (~3600)
• Lane 7: P90γ uncut
• Lane 8: P90δ cut SP (~3600)
• Lane 9: P90δ uncut
• Lane 10: ligated P76 cut EX (3857)
• Lane 11: ligated P76 uncut (3857)

Gel 2

File:7-18 gel 2.1 MXHTA.jpg

• Lane 1: 1 kb ladder
• Lane 2: P90ε cut SP (~3600)
• Lane 3: P90ε uncut
• Lane 4: P90ζ cut SP (~3600)
• Lane 5: P90ζ uncut
• Lane 6: Christina's plasmid uncut (?)
• Lane 7: Christina's plasmid cut EX (?)
• Lane 8: Christina's plasmid cut ES (~1300)
• Lane 9: Christina's plasmid cut XP (~1300)
• Lane 10: ligated P91 cut EX (4327)
• Lane 11: ligated P91 uncut (4327)
• Lane 12: mtrB PCR (2200)

File:7-18 gel 2.2 MXHTA.jpg

RE digests 07/19

We digested

• P17, P51, P52 (QPIs) with EX so we can add high/low constitutive promoters
• P63 (terminator) with EX
• P48, P49a, P49B (Amp, Cm resistance cassettes) with XP so we can add them to P90 (P1 + CDF)

	P17	P51	P52	P63A	P63B	P48	P49A	P49B
DNA	20 μL	20 μL	20 μL	10 μL	10 μL	4 μL	20 μL	4 μL
10X NEB Buffer	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL
25X BSA	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL
REs (1 μL each)	EX	EX	EX	EX	EX	XP	XP	XP
Water	0 μL	0 μL	0 μL	9.5 μL	9.5 μL	15.5 μL	0 μL	15.5 μL

File:7-19 mxh.jpg

Ligations 07/15

We ligated

• P90 SP with P48 XP
• P90 SP with P49 XP
• P58 EX with P53 ES
• P59 EX with P53 ES

These ligations were transformed into TOP10 cells that we made chemically competent.

Results 7/16

Transformed in E2:

'	Selection	# colonies
P90 SP with P48 XP	KAN	0
P90 SP with P49 XP	KAN	1 - set up liquid culture
P58 EX with P53 ES	KAN	0
P59 EX with P53 ES	KAN	0
NO DNA	KAN	0

Cross Transformations in S1

We transformed cells with repressor plasmid (P27), and duet vector (P12 and P13) with LacI with p59 (pLac + GFP) and vice versa in order to test the inducible system in S1.

Transformations (7/11) and Picked Colonies (7/13)

Plate	Marker	Description	Picked Colonies?
S1 P34	Amp	Lawn, restreaked. Many orange colonies along streak. Can\'t detect fluor.	Yes
S1 P59b cells + P12 vector	Amp	Lawn, restreaked. Fluorescent. ?	Yes
S1 P13 cells + P59b vector (1)	Kan	Many small colonies-- some fluoresce while others don\'t. Pinkish centers.	Yes (both F and no F)
S1 P13 cells + P59b vector (2)	Kan	More colonies than (1), but also some fluoresce while others don\'t. Pinkish centers.	Yes (both F and no F)
S1 P27 cells + P59B vector (1)	Kan	Many small colonies - fluorescent.	Yes
S1 P27 cells + P59B vector (2)	Kan	Almost lawn of tiny colonies - fluorescent.	Yes
S1 P59b Cells + P13 vector	Sm	Medium # of medium sized colonies, pink centers, no fluorescence.	Yes

Diluted and Re-grew in Media with Both Antibiotics 7/14

Sample	Media (5mL of each)	Grew?
S1 P59b + P12	Amp+Kan
S1 P13 + P59b (1)	Kan+Sm
S1 P13 + P59b (2)	Kan+Sm
S1 P27 + P59b (1)	Kan+Sm
S1 P27 + P59b (2)	Kan+Sm
S1 P59b + P12	Kan+Sm

Restreaked in Plates with Double Selection Markers 7/15

Sample	Plate (original w/ second antibiotic added on)	Grew?
S1 P59b + P12
S1 P13 + P59b (1)
S1 P13 + P59b (2)
S1 P27 + P59b (1)
S1 P27 + P59b (2)
S1 P59b + P12

Testing Cultures with IPTG 7/15

Thermoinducible Lac System

Grew up cultures of P84 (Lac mut265) and P85 (Lac mut 241) according to protocol derived from http://www3.interscience.wiley.com.ezp-prod1.hul.harvard.edu/journal/93519214/abstract?CRETRY=1&SRETRY=0 Chao et al. 2002 and http://sfx.hul.harvard.edu/sfx_local?id=doi:10.1016/0168-1656(94)00144-2 Yabuta et al. 1995

Testing Thermoinducible Cultures 7/14

1. Grow up 10mL culture overnight with antibiotic at 200 RPM, 37 degrees.
2. Dilute 2.5mL of overnight culture into 50mL of fresh LB.
3. Grow at 200 RPM, 30 degrees until OD660 = 0.2.
4. Split culture into two 25mL cultures, and put one in 30 degrees and the other in 40 degrees.
5. Grow for 4 hours at these separate temperatures at 200 RPM.
6. OD and measure YFP fluorescence (Ex: 514, Em: 527).

Results from First Thermoinducible Test 7/14

Controls:

Control	OD660	YFP (514/527)	YFP/OD
LB	0	36.23
E1 + pET-Duet Vector	0.361	288.07	797.9778393

Results:

Sample	Temperature	Measurement	1 to 1 dilution	1 to 2 dilution	1 to 4 dilution	Undiluted
P84	30	YFP	689.21	643.31	372.8	1308.37
		OD	0.821	0.698	0.369	1.582
		YFP/OD	839.4762485	921.6475645	1010.298103	827.0353982
		Corrected for autofluor.	41.49840914	123.6697251	212.3202636	29.05755889
	40	YFP	792.73	607.11	412.16	1326.16
		OD	0.884	0.64	0.405	1.665
		YFP/OD	896.7533937	948.609375	1017.679012	796.4924925
		Corrected for autofluor.	98.77555433	150.6315357	219.701173	-1.485346843
P85	30	YFP	937.12	665.39	405.58	1341.37
		OD	1.178	0.739	0.411	1.638
		YFP/OD	795.5178268	900.3924222	986.8126521	818.9072039
		Corrected for autofluor.	-2.46001251	102.4145829	188.8348127	20.92936457
	40	YFP	862.44	600.56	439.47	1364.59
		OD	1.003	0.633	0.44	1.711
		YFP/OD	859.8604187	948.7519747	998.7954545	797.5394506
		Corrected for autofluor.	61.88257941	150.7741354	200.8176152	-0.438388722

Putting Thermoinducible Lac and pLambda + GFP System on p15a Vector

RE Digestion 7/11

'	P1 (vector)	P1 (vector)	P74 (insert)	P84 (insert)	P85 (insert)
DNA	15 uL	15 uL	5 uL	5 uL	5 uL
10X Buffer (Volume & #)	2.5 uL of 3	2.5 uL of 3	2.5 uL of 3	2.5 uL of 3	2.5 uL of 3
100X BSA	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL
Restriction Enzyme 1	1 uL XbaI	1 uL XbaI	1 uL XbaI	1 uL XbaI	1 uL XbaI
Restriction Enzyme 2	1 uL PstI	1 uL PstI	1 uL PstI	1 uL PstI	1 uL PstI
Water	5.25 uL	5.25 uL	15.25 uL	15.25 uL	15.25 uL
Total Volume	25 uL	25 uL	25 uL	25 uL	25 uL

Gel 7/14

File:7-11-08 Digestions.jpg	1% Agarose, visualized using EtBr/UV
	Lane	Sample
	1	[http://tools.invitrogen.com/content/sfs/manuals/15615016.pdf 1 KB ladder]
	2	P1, XP, 2750 bp
	3	P1, XP, 2750 bp
	4	P1 uncut
	6	P74, XP, ~925 bp
	7	P74, uncut
	9	P84, XP, 2314 bp
	10	P84, uncut
	11	P85, XP, 2314 bp
	13	P85, uncut

P1 and P74 cut and uncut bands are in the right place, but P84 and P85 both have 2079 bp backbones, which does not account for the ~900 bp bands. We searched the sequence of the backbone in the registry for internal cut sites that could have produced the band, but found none.

Only P1 and P74 were extracted and gel purified.

Using BioBricks Primers to PCR Amplify P84-5 7/14

Sample	PCR Supermix	Template DNA	BBx Fwd	BBx Rev	Water (to 50 uL)
P84	45 uL	1 uL	1 uL	1 uL	2 uL
P85	45 uL	1 uL	1 uL	1 uL	2 uL
(-) ctrl, S1 P4	45 uL	1 uL	1 uL	1 uL	2 uL
(+) ctrl, E1, P3	45 uL	1 uL	1 uL	1 uL	2 uL

Gel from PCR 7/15

File:7-15-08 P84 PCR.jpg	1% Agarose, visualized using EtBr/UV
	Lane	Sample
	1	1 kb Ladder
	2	P84, ~2314 bp
	3	Empty
	4	P85, ~2314 bp
	5	Empty
	6	(+) ctrl, E1 P3
	7	Empty
	8	(-) ctrl, S1 P4
	9	100 bp Ladder
	10	Empty

P84 and P85 bands were extracted and gel purified so they could be digested with XP.

Digestion of PCR Products with XP 7/15

'	P84 (insert)	P85 (insert)
DNA	5 uL	5 uL
10X Buffer (Volume & #)	2.5 uL of 3	2.5 uL of 3
100X BSA	0.25 uL	0.25 uL
Restriction Enzyme 1	1 uL XbaI	1 uL XbaI
Restriction Enzyme 2	1 uL PstI	1 uL PstI
Water	15.25 uL	15.25 uL
Total Volume	25 uL	25 uL

After digestion, samples were PCR purified (7/16).

Single and Triple Digests of P84-5 7/14

P84 and P85 were found to have an internal XmnI cut site, so single cut digests were performed for both samples with XbaI, PstI, and XmnI. Triple digests were also done with all three enzymes. Digestions were incubated overnight at 37°C.

Gel of Single and Triple Digests 7/15

File:7-15-08 singletriple.jpg	1% Agarose, visualized using EtBr/UV
	Lane	Sample
	1	[http://tools.invitrogen.com/content/sfs/manuals/15615016.pdf 1 KB ladder]
	2	P84, XbaI Single Cut
	3	P84, PstI Single Cut
	4	P84, XmnI Single Cut
	5	Empty
	6	P84, XPXmnI Triple Cut
	7	Empty
	8	100 bp Ladder
	9	P85, XbaI Single Cut
	10	P85, PstI Single Cut
	11	P85, XmnI Single Cut
	12	Empty
	13	P85, XPXmnI Triple Cut
	14	Empty
	15	[http://tools.invitrogen.com/content/sfs/manuals/15615016.pdf 1 KB ladder]

The 2314 bp bands for the triple cuts of P84-5 were extracted and gel purified.

Ligation of P74, Triple Digested P84-5, and P1 7/15

Dephosphorylation of P1, 7/16

'	P1
Vector	15 uL
Dephos Buffer 10x	2 uL
Alkaline Phosphatase	1 uL
Water	2 uL
Total	20 uL

Ligation 7/15

The P84-5 inserts are roughly the same size as the vector (P1) so we tried different ratios of insert to vector.

'	P1/P74	P1/P84 1:3	P1/P84 1:1	P1/P85 1:3	P1/P85 1:1
Vector	2 uL	2 uL	3 uL	2 uL	3 uL
Insert	6 uL	6 uL	3 uL	6 uL	3 uL
DNA Dilution Buffer 5x	2 uL	2 uL	2 uL	2 uL	2 uL
Water	0 uL	0 uL	0 uL	0 uL	0 uL
(Added last:)
T4 DNA Ligation Buffer 2x	10 uL	10 uL	10 uL	10 uL	10 uL
T4 DNA Ligase	1 uL	1 uL	1 uL	1 uL	1 uL
Total	21 uL	21 uL	21 uL	21 uL	21 uL

Ligations were then transformed in DH5α cells.

Transformation of P1/P74 and P1/P84-5 Triple Cuts 7/15

5 uL Ligation in 50 uL chemically competent E1 cells (old batch). Plates were incubated overnight at 37°C.

Plate	Marker	# Colonies	Other Description
P75 (P1 + P74)	Kan	>100	Small, white, fluorescent under microscope.
P92 (P1 + P84) 1:1	Kan	2 or 3	Small, white
P92 (P1 + P84) 1:3	Kan	0
P93 (P1 + P85) 1:1	Kan	2	Small, white
P93 (P1 + P85) 1:3	Kan	1	Small, white
Kan ctrl (EB buffer)	Kan	0

For positive control plates, see the Competent Cell Test Plates under Housekeeping.

Picked Colonies for Liquid Culture 7/16

All cultures grew, except for (-) ctrl.

Minipreps of Liquid Culture 7/17

Plasmid	ng/uL	A260	260/280	260/230	Constant
E1 P75a	95.34	1.907	1.95	2.24	50
E1 P75b	108.59	2.172	1.98	2.21	50
E1 P92a 1:1	126.61	2.532	2.01	1.67	50
E1 P92b 1:1	129.67	2.593	1.98	2.09	50
E1 P93a 1:1	164.37	3.287	1.94	1.95	50
E1 P93b 1:1	106.08	2.122	1.92	2.14	50
E1 P93 1:3	142.49	2.85	2.03	2.18	50

Ligation and Transformation of PCR Amplified P84-5 with P1 (7/16)

Ligation of P84-5 PCR with Dephos P1, 7/16

P1 was dephosphorylated the day before, and used for this ligation reaction as well.

'	P1/P84 PCR (1:3)	P1/P85 PCR (1:3)
Vector	2 uL	2 uL
Insert	6 uL	6 uL
DNA Dilution Buffer 5x	2 uL	2 uL
Water	0 uL	0 uL
(Added last:)
T4 DNA Ligation Buffer 2x	10 uL	10 uL
T4 DNA Ligase	1 uL	1 uL
Total	21 uL	21 uL

Transformation of P92-3 PCR into E1, 7/16

5 uL Ligation in 50 uL chemically competent E1 cells (old batch). Plates were incubated overnight at 37°C.

Plate	Marker	# Colonies
P92 PCR	Kan	50+ small, white colonies.
P93 PCR	Kan	50+ small white colonies
(+) ctrl pUC19	Amp	>100 small white colonies
(-) ctrl	Kan	0 colonies

Liquid Cultures (7/17) and Minipreps (7/18)

Plasmid/ Sample	ng/uL	A260	260/280	260/230	Constant
E1 P92a PCR	111.17	2.223	1.92	2.29	50
E1 P92b PCR	121.44	2.429	1.98	2.29	50
E1 P93a PCR	99.13	1.983	2.05	2.39	50
E1 P93b PCR	126.28	2.526	1.93	2.18	50
S1 P59b 1	450.61	9.012	1.9	2.33	50
S1 P59b 2	328.89	6.578	1.92	2.33	50
E1 P58a 1	144.21	2.884	2	2.38	50
E1 P58a 2	91.06	1.821	1.9	2.38	50
E1 P80	128.63	2.573	2.04	2.26	50

Transformation of P75 and P92-3 (from 3x Cut) into S1 7/17

Used S1 cells frozen for electroporation.

Plasmid/ Sample	Vol DNA Added (250-300 ng DNA)	Marker	Plate After O/N incubation at 30 (7/18)	Plate Description after TWO days incubation at 30 (7/19)
P75a	3 uL	Kan	2 white colonies, ~1 mm diam. Fluorescent	4 pink/ yellow colonies (4mm), ~20 white/cream colonies, 1mm
P75b	3 uL	Kan	2 white colonies, ~1 mm diam. Fluorescent.	4 pink/ yellow colonies (4mm), ~30 white/cream colonies, 1mm
P93 1:3	2 uL	Kan	~6 white, 1mm colonies, dimmish under scope*	8 pink/yellow colonies (4mm), ~120 white/cream colonies, 1mm
P58b (miniprepped from S1)	5 uL	Kan	0 colonies	~250 white/ cream colonies, 1mm
(+) ctrl (S1 P59b)	3 uL	Kan	>100 white, 1mm colonies	120 pink/yellow colonies (4mm), ~10 white/ cream colonies, 1mm
(-) ctrl	None added	Kan	0 colonies	~20 white/ cream colonies, 1mm

1mm white/ cream colonies present on all plates after the 2 day incubation resemble the contamination we have observed in other E. coli plates. This is of interest because Kan is not as vulnerable to degradation as Carb/ Amp are.

P75a and P75b were both very bright in S1. Thermoinducible cI857 was ordered from http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=40166&Template=vectors ATCC.

The plates of P84-5 originally transformed in E. coli from the registry were used as a standard for fluorescence for the thermoinducible Lac colonies. They were left overnight at 37°C to stimulate thermosensitivity, which should lead to YFP expression. Although they were not very bright, they glowed. Compared to these plates, the P93 1:3 transformed in S1 was noticeably dimmer, though it had been incubated at 30°C O/N. Plate will be restreaked and grown at different temperatures after the weekend.

Transformation of P92-3 (from PCR) into S1 7/18

Plasmid/ Sample	Vol DNA Added (250-300 ng DNA)	Marker	Plate After O/N incubation at 30	Plate Description after TWO days incubation at 30
P92a PCR	3 uL	Kan	N/A	2 yellow/pink colonies (4mm), many white, 1mm colonies (=neg ctrl)
P92b PCR	3 uL	Kan	N/A	many white, 1mm colonies (= neg ctrl)
P93a PCR	3 uL	Kan	N/A	many white, 1mm colonies (= neg ctrl)
P93b PCR	3 uL	Kan	N/A	2 yellow/pink colonies (4mm), many white, 1mm colonies (=neg ctrl)
P58a A (from E1)	2 uL	Kan	N/A	many white, 1mm colonies (> neg ctrl)
P84a B (from E1)	3 uL	Kan	N/A	many white, 1mm colonies (> neg ctrl)
P28 (TetR, GFP, ColE1)	2 uL	Kan	N/A	12 yellow/pink colonies (4mm), many white, 1mm colonies (=neg ctrl)
P33 (TetR, Venus, p15a)	5 uL	Chlor	N/A	blank
(+) ctrl, p59B	1 uL	Kan	N/A	many white, 1mm colonies (TMTC)
(-) ctrl, EB buffer	1 uL	Kan	N/A	many white, 1mm colonies (TMTC)

Test of IPTG Inducible System

7/15/08 S1 containing 2 vectors one coding for LacI and one w/ a lac promoter controlling GFP expresssion were tested with IPTG

Strain	OD @ 0h	Fluor @ 0h	Fluor/OD	OD @ 2h no IPTG	Fluor @ 2h no IPTG	Fluor/OD2	OD @ 2h w/ IPTG	Fluor @ 2h w/ IPTG	Fluor/OD3	Difference in Levels of Fluor due to IPTG treatment (Fluor/OD3-Fluor/OD3)
p59 cells w/ p12 vector A	0.420	572.660	1363.476	1.150	863.790	751.122	0.818	1099.980	1344.719	593.597
p59 cells w/ p12 vector B	0.427	476.300	1115.457	1.011	905.500	895.648	1.012	1048.150	1035.721	140.073
p59 cells w/ p13 vector A	0.474	84.440	178.143	1.047	177.100	169.150	1.026	151.450	147.612	-21.538
p59 cells w/ p13 vector B	0.330	79.010	239.424	1.027	171.320	166.816	0.930	122.870	132.118	-34.698
p27 cells w/ p59b vector 1a	0.364	76.050	208.929	0.615	180.420	293.366	0.775	105.070	135.574	-157.792
p27 cells w/ p59b vector 1b	0.299	73.740	246.622	0.841	189.910	225.815	0.824	157.240	190.825	-34.989
p27 cells w/ p59b vector 2a	0.302	67.830	224.603	0.713	170.020	238.457	0.716	105.530	147.388	-91.069
p27 cells w/ p59b vector 2b	0.476	81.830	171.912	0.683	178.370	261.157	0.647	114.140	176.414	-84.742
p13 cells w/ p59b 1a	0.371	126.440	340.809	0.948	186.400	196.624	0.852	111.840	131.268	-65.357
p13 cells w/ p59b 1b	0.535	91.750	171.495	0.959	167.660	174.828	0.941	130.750	138.948	-35.880
p13 cells w/ p59b 1c	0.431	111.460	258.608	1.016	184.350	181.447	0.937	153.330	163.639	-17.808
p13 cells w/ p59b 2a	0.582	73.400	126.117	0.938	183.240	195.352	0.924	124.960	135.238	-60.114
p13 cells w/ p59b 2b	0.543	118.060	217.422	0.936	189.170	202.105	1.054	139.860	132.694	-69.410
p13 cells w/ p59b 2c	0.523	111.600	213.384	0.992	192.720	194.274	1.160	169.380	146.017	-48.257
p34	0.045	191.140	4247.556	0.825	413.930	501.733	1.006	384.310	382.018	-119.715
p59b (glycerol) + cntl	1.458	131.240	90.014			#DIV/0!			#DIV/0!	#DIV/0!
p59b (plate) + cntl	0.000	288.750	#DIV/0!	1.025	308.480	300.956	0.996	261.480	262.530	-38.426
mtr A negative control	0.097	129.620	1336.289			#DIV/0!			#DIV/0!	#DIV/0!
mtrB negative control	0.000	109.910	#DIV/0!			#DIV/0!			#DIV/0!	#DIV/0!
LB		106.800	#DIV/0!	0.000	101.160	#DIV/0!			#DIV/0!	#DIV/0!

7/17/08:

Strain	OD @ 0h	Fluor @ 0h	Fluor/OD	OD @ 2h no IPTG	Fluor @ 2h no IPTG	Fluor/OD2	OD @ 2h w/ IPTG	Fluor @ 2h w/ IPTG	Fluor/OD3	Difference in Levels of Fluor due to IPTG treatment (Fluor/OD3-Fluor/OD2)
LB	0.000	120.050	#DIV/0!	0.000	110.640	#DIV/0!			#DIV/0!	#DIV/0!
WT S1	0.232	131.340	566.121	1.003	136.600	136.191	0.700	122.420	174.886	38.694
p59b	0.038	134.080	3528.421	0.299	135.160	452.040	0.293	137.400	468.942	16.902
p59b w/ p12	0.187	175.520	938.610	0.621	278.470	448.422	0.685	312.750	456.569	8.147
p59b w/ p13	0.217	115.500	532.258	0.655	108.150	165.115	0.785	126.850	161.592	-3.522
p13 w/ p59b 1	0.239	101.130	423.138	0.751	110.760	147.483	0.758	109.220	144.090	-3.394
p13 w/ p59b 2	0.245	90.400	368.980	0.708	117.250	165.607	0.747	126.030	168.715	3.108
p27 w/ p59b 1	0.141	130.950	928.723	0.495	156.170	315.495	0.493	121.950	247.363	-68.132
p27 w/ p59b 2	0.171	128.890	753.743	0.581	127.040	218.657	0.487	123.580	253.758	35.100

Overnight cultures 07/18

We made overnight cultures of the parts we got from MIT

• P96
• P95
• P51
• P52
• P17

We also made cultures for plasmids we will need later

• P1 (A and B)
• P3 (A and B)
• P46 (A and B)
• P63 (A and B)
• P48
• P49 (a and B)
• P63 (A and B)

We are not completely sure if the LB Kan we used contains Kan or if it is just plain LB. So we also made a culture of P95, which is Amp resistant, in the this medium. So if it doesn't grow, then Kan was added to that bottle of LB.

07/19: All of these cultures grew except for P46A. The LB Kan really does have Kan.

Housekeeping!

Daily

• Pour LB-Amp plates.
• Ensure we have petri dishes, LB, LB agar, and agarose.
• Make LB-antibiotic medias as necessary.

Testing and Making Competent E. coli

Test Transformation of New Batches of Competent Cells 7/15

Plate	Marker	# Colonies
E1 (old) + pUC19 (1 uL)	Amp	>100
E1 (new) + pUC19 (1 uL)	Amp	10
E2/ TOP10 (new) + pUC19 (1 uL)	Amp	13
Amp (-) ctrl	Amp	0

Making New Competent Cells 7/22

As per Jason's lab's protocol.

Keeping Up Stocks of Plasmids

7/15/08 Minipreps

Plasmid/ Sample	ng/uL	A260	260/280	260/230	Constant
S1 P58b A?	67.45	1.349	1.99	2.13	50
E1 P39A 08 2	362.89	7.258	1.94	2.26	50
E1 P39A 08 1	417.46	8.349	1.96	2.26	50
E1 P38a 07 2	351.01	7.02	1.97	2.07	50
E1 P38a 07 1	293.1	5.862	1.96	2.15	50
E1 P76 (17+45)	534.13	10.683	1.9	2.27	50
S1 P34 B amp	941.83	18.837	1.9	2.26	50
S1 P34 B amp	994.7	19.894	1.91	2.26	50

7/18/08

Plasmid/ Sample	ng/uL	A260	260/280	260/230	Constant
S1 P59b 1	450.61	9.012	1.9	2.33	50
S1 P59b 2	328.89	6.578	1.92	2.33	50
E1 P58a 1	144.21	2.884	2	2.38	50
E1 P58a 2	91.06	1.821	1.9	2.38	50
E1 P80	128.63	2.573	2.04	2.26	50

Transformation of P86, P87, P97

We transformed each of the UTAustin plasmids (individually) and a new strong RBS into TOP 10 cells. The RBS has been miniprepped. The UTA plasmid transformants formed a lawn, so were restreaked first. Liquid cultures have been grown up. P87 requires mutagenesis to remove a PstI site ~100 bp into the coding region of the Cph/EnvZ fusion. We miniprepped P86A, P86B, P87A, P87B (all in E2) from liquid cultures. The amp and cm negative controls did not have any growth.

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