IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week4/Chemical and Light

From OpenWetWare
Jump to: navigation, search

Contents

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.

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 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.

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

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

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

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

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

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

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

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

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

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)

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

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 [Chao et al. 2002] and [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

7-11-08 Digestions.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
1 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

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

7-15-08 singletriple.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
1 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 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 [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.