Team:Harvard/Dailybook/Week4/Chemical and Light

From 2008.igem.org

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.

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
11 KB ladder
2-4CDF (~900 bp)
5-12mtrB 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+38SPAMP2 AFTER 48HRSPlate put in 4°C, not pickedno growth
63+59 FROM 7/9 LIGATIONKAN1 AFTER 48HRSno GFP, so plate tossedno growth
CDF XP (7/4)+P1XPKAN1 AFTER 48HRShas GFP- indicates wrong plasmid, tossed--
45XP(7/11 45)+39SPAMP 0no growth
45XP(7/11 45)+38SPAMP0no growth
77XP+38SPAMP0no growth
52XP+39SPAMP0no growth
77XP+39SPAMP0no growth
89XP+38SPAMP0no growth
45XP+39SPAMP0no growth
89XP+39SPAMP0no growth
51XP+39SPAMP0no growth
17XP+39SPAMP0no growth
P88KAN0--
63+58 FROM 7/9 LIGATIONKAN0no growth
58EX+63ESKAN0no growth
52XP+38SPAMP0no growth
89ES+45EXCARBTMTCThe 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)KAN68miniprepped--
17ES+45EXCARBTMTCThe 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+38SPAMP4DID NOT FLUORESCE- not minipreppedgrew, streaked for colonies- no GFP, tossed
51XP+38SPCARB52 picked colonies, neither grew- repicked 7/14, these did not grow eitherno growth
54ES+59EXKAN3DID NOT FLUORESCE- not miniprepped, no GFP, so plate tossedno growth
CDF XP(7/12)+P1XP(7/1)KANTMTCminiprepped--
CDF XP(7/12)+P1KAN232miniprepped--
77ES+59EXKANNOT PLATEDno growth
no DNAAMP0--
no DNAKAN0--
no DNACARB6 big + 10 small--
no cellsKAN0no growth
1μL pUC19CARB96 did grow in LB AMP medium (+ control)--
1μL pUC19AMP64--

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
DNA20 μL40 μL40 μL35 μL10 μL40 μL40 μL15 μL
10X NEB Bufferbuffer 3, 2.5 μLbuffer 2, 2.5 μL buffer 3, 2.5 μLbuffer 2, 2.5 μLbuffer 2, 2.5 μLbuffer 3, 2.5 μLbuffer 3, 2.5 μLbuffer 3, 2.5 μL
25X BSA1 μL1 μL1 μL1 μL1 μL1 μL1 μL1 μL
REs (1 μL each)XPSPXPSPSPXPXPXP
Water9.5 μL0 μL16.5 μL14.5 μL14.5 μL16.5 μL9.5 μL9.5 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
DNA20 μL20 μL20 μL10 μL10 μL4 μL20 μL4 μL
10X NEB Bufferbuffer 2, 2.5 μLbuffer 2, 2.5 μL buffer 2, 2.5 μLbuffer 2, 2.5 μLbuffer 2, 2.5 μLbuffer 3, 2.5 μLbuffer 3, 2.5 μLbuffer 3, 2.5 μL
25X BSA1 μL1 μL1 μL1 μL1 μL1 μL1 μL1 μL
REs (1 μL each)EXEXEXEXEXXPXPXP
Water0 μL0 μL0 μL9.5 μL9.5 μL15.5 μL0 μL15.5 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 XPKAN0
P90 SP with P49 XPKAN1 - set up liquid culture
P58 EX with P53 ESKAN0
P59 EX with P53 ESKAN0
NO DNAKAN0

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 P34AmpLawn, restreaked. Many orange colonies along streak. Can\'t detect fluor.Yes
S1 P59b cells + P12 vectorAmpLawn, restreaked. Fluorescent. ?Yes
S1 P13 cells + P59b vector (1)KanMany small colonies-- some fluoresce while others don\'t. Pinkish centers.Yes (both F and no F)
S1 P13 cells + P59b vector (2)KanMore 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)KanMany small colonies - fluorescent.Yes
S1 P27 cells + P59B vector (2)KanAlmost lawn of tiny colonies - fluorescent.Yes
S1 P59b Cells + P13 vectorSmMedium # 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 + P12Amp+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 + P12Kan+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
LB036.23
E1 + pET-Duet Vector0.361288.07797.9778393

Results:

Sample Temperature Measurement 1 to 1 dilution 1 to 2 dilution 1 to 4 dilution Undiluted
P8430YFP689.21643.31372.81308.37
OD0.8210.6980.3691.582
YFP/OD839.4762485921.64756451010.298103827.0353982
Corrected for autofluor.41.49840914123.6697251212.320263629.05755889
40YFP792.73607.11412.161326.16
OD0.8840.640.4051.665
YFP/OD896.7533937948.6093751017.679012796.4924925
Corrected for autofluor.98.77555433150.6315357219.701173-1.485346843
P8530YFP937.12665.39405.581341.37
OD1.1780.7390.4111.638
YFP/OD795.5178268900.3924222986.8126521818.9072039
Corrected for autofluor.-2.46001251102.4145829188.834812720.92936457
40YFP862.44600.56439.471364.59
OD1.0030.6330.441.711
YFP/OD859.8604187948.7519747998.7954545797.5394506
Corrected for autofluor.61.88257941150.7741354200.8176152-0.438388722

Putting Thermoinducible Lac and pLambda + GFP System on p15a Vector

RE Digestion 7/11

' P1 (vector) P1 (vector) P74 (insert) P84 (insert) P85 (insert)
DNA15 uL15 uL5 uL5 uL5 uL
10X Buffer (Volume & #)2.5 uL of 32.5 uL of 32.5 uL of 32.5 uL of 32.5 uL of 3
100X BSA0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL
Restriction Enzyme 11 uL XbaI1 uL XbaI1 uL XbaI1 uL XbaI1 uL XbaI
Restriction Enzyme 21 uL PstI1 uL PstI1 uL PstI1 uL PstI1 uL PstI
Water5.25 uL5.25 uL15.25 uL15.25 uL15.25 uL
Total Volume25 uL25 uL25 uL25 uL25 uL

Gel 7/14

File:7-11-08 Digestions.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
11 KB ladder
2P1, XP, 2750 bp
3P1, XP, 2750 bp
4P1 uncut
6P74, XP, ~925 bp
7P74, uncut
9P84, XP, 2314 bp
10P84, uncut
11P85, XP, 2314 bp
13P85, 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)
P8445 uL1 uL1 uL1 uL2 uL
P8545 uL1 uL1 uL1 uL2 uL
(-) ctrl, S1 P445 uL1 uL1 uL1 uL2 uL
(+) ctrl, E1, P345 uL1 uL1 uL1 uL2 uL

Gel from PCR 7/15

File:7-15-08 P84 PCR.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
11 kb Ladder
2P84, ~2314 bp
3Empty
4P85, ~2314 bp
5Empty
6(+) ctrl, E1 P3
7Empty
8(-) ctrl, S1 P4
9100 bp Ladder
10Empty

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)
DNA5 uL5 uL
10X Buffer (Volume & #)2.5 uL of 32.5 uL of 3
100X BSA0.25 uL0.25 uL
Restriction Enzyme 11 uL XbaI1 uL XbaI
Restriction Enzyme 21 uL PstI1 uL PstI
Water15.25 uL15.25 uL
Total Volume25 uL25 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
11 KB ladder
2P84, XbaI Single Cut
3P84, PstI Single Cut
4P84, XmnI Single Cut
5Empty
6P84, XPXmnI Triple Cut
7Empty
8100 bp Ladder
9P85, XbaI Single Cut
10P85, PstI Single Cut
11P85, XmnI Single Cut
12Empty
13P85, XPXmnI Triple Cut
14Empty
151 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
Vector15 uL
Dephos Buffer 10x2 uL
Alkaline Phosphatase1 uL
Water2 uL
Total20 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
Vector2 uL2 uL3 uL2 uL3 uL
Insert6 uL6 uL3 uL6 uL3 uL
DNA Dilution Buffer 5x2 uL2 uL2 uL2 uL2 uL
Water0 uL0 uL0 uL0 uL0 uL
(Added last:)
T4 DNA Ligation Buffer 2x10 uL10 uL10 uL10 uL10 uL
T4 DNA Ligase1 uL1 uL1 uL1 uL1 uL
Total21 uL21 uL21 uL21 uL21 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>100Small, white, fluorescent under microscope.
P92 (P1 + P84) 1:1Kan2 or 3Small, white
P92 (P1 + P84) 1:3Kan0
P93 (P1 + P85) 1:1Kan2Small, white
P93 (P1 + P85) 1:3Kan1Small, white
Kan ctrl (EB buffer)Kan0

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 P75a95.341.9071.952.2450
E1 P75b108.592.1721.982.2150
E1 P92a 1:1126.612.5322.011.6750
E1 P92b 1:1129.672.5931.982.0950
E1 P93a 1:1164.373.2871.941.9550
E1 P93b 1:1106.082.1221.922.1450
E1 P93 1:3142.492.852.032.1850

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)
Vector2 uL2 uL
Insert6 uL6 uL
DNA Dilution Buffer 5x2 uL2 uL
Water0 uL0 uL
(Added last:)
T4 DNA Ligation Buffer 2x10 uL10 uL
T4 DNA Ligase1 uL1 uL
Total21 uL21 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 PCRKan50+ small, white colonies.
P93 PCRKan50+ small white colonies
(+) ctrl pUC19Amp>100 small white colonies
(-) ctrlKan0 colonies

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

Plasmid/ Sample ng/uL A260 260/280 260/230 Constant
E1 P92a PCR111.172.2231.922.2950
E1 P92b PCR121.442.4291.982.2950
E1 P93a PCR99.131.9832.052.3950
E1 P93b PCR126.282.5261.932.1850
S1 P59b 1450.619.0121.92.3350
S1 P59b 2328.896.5781.922.3350
E1 P58a 1144.212.88422.3850
E1 P58a 291.061.8211.92.3850
E1 P80128.632.5732.042.2650

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)
P75a3 uLKan2 white colonies, ~1 mm diam. Fluorescent4 pink/ yellow colonies (4mm), ~20 white/cream colonies, 1mm
P75b3 uLKan2 white colonies, ~1 mm diam. Fluorescent.4 pink/ yellow colonies (4mm), ~30 white/cream colonies, 1mm
P93 1:32 uLKan~6 white, 1mm colonies, dimmish under scope*8 pink/yellow colonies (4mm), ~120 white/cream colonies, 1mm
P58b (miniprepped from S1)5 uLKan0 colonies~250 white/ cream colonies, 1mm
(+) ctrl (S1 P59b)3 uLKan>100 white, 1mm colonies120 pink/yellow colonies (4mm), ~10 white/ cream colonies, 1mm
(-) ctrlNone addedKan0 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 PCR3 uLKanN/A2 yellow/pink colonies (4mm), many white, 1mm colonies (=neg ctrl)
P92b PCR3 uLKanN/Amany white, 1mm colonies (= neg ctrl)
P93a PCR3 uLKanN/Amany white, 1mm colonies (= neg ctrl)
P93b PCR3 uLKanN/A2 yellow/pink colonies (4mm), many white, 1mm colonies (=neg ctrl)
P58a A (from E1)2 uLKanN/Amany white, 1mm colonies (> neg ctrl)
P84a B (from E1)3 uLKanN/Amany white, 1mm colonies (> neg ctrl)
P28 (TetR, GFP, ColE1)2 uLKanN/A12 yellow/pink colonies (4mm), many white, 1mm colonies (=neg ctrl)
P33 (TetR, Venus, p15a)5 uLChlorN/Ablank
(+) ctrl, p59B1 uLKanN/Amany white, 1mm colonies (TMTC)
(-) ctrl, EB buffer1 uLKanN/Amany 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 A0.420572.6601363.4761.150863.790751.1220.8181099.9801344.719593.597
p59 cells w/ p12 vector B0.427476.3001115.4571.011905.500895.6481.0121048.1501035.721140.073
p59 cells w/ p13 vector A0.47484.440178.1431.047177.100169.1501.026151.450147.612-21.538
p59 cells w/ p13 vector B0.33079.010239.4241.027171.320166.8160.930122.870132.118-34.698
p27 cells w/ p59b vector 1a0.36476.050208.9290.615180.420293.3660.775105.070135.574-157.792
p27 cells w/ p59b vector 1b0.29973.740246.6220.841189.910225.8150.824157.240190.825-34.989
p27 cells w/ p59b vector 2a0.30267.830224.6030.713170.020238.4570.716105.530147.388-91.069
p27 cells w/ p59b vector 2b0.47681.830171.9120.683178.370261.1570.647114.140176.414-84.742
p13 cells w/ p59b 1a0.371126.440340.8090.948186.400196.6240.852111.840131.268-65.357
p13 cells w/ p59b 1b0.53591.750171.4950.959167.660174.8280.941130.750138.948-35.880
p13 cells w/ p59b 1c0.431111.460258.6081.016184.350181.4470.937153.330163.639-17.808
p13 cells w/ p59b 2a0.58273.400126.1170.938183.240195.3520.924124.960135.238-60.114
p13 cells w/ p59b 2b0.543118.060217.4220.936189.170202.1051.054139.860132.694-69.410
p13 cells w/ p59b 2c0.523111.600213.3840.992192.720194.2741.160169.380146.017-48.257
p340.045191.1404247.5560.825413.930501.7331.006384.310382.018-119.715
p59b (glycerol) + cntl1.458131.24090.014#DIV/0!#DIV/0!#DIV/0!
p59b (plate) + cntl0.000288.750#DIV/0!1.025308.480300.9560.996261.480262.530-38.426
mtr A negative control0.097129.6201336.289#DIV/0!#DIV/0!#DIV/0!
mtrB negative control0.000109.910#DIV/0!#DIV/0!#DIV/0!#DIV/0!
LB106.800#DIV/0!0.000101.160#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)
LB0.000120.050#DIV/0!0.000110.640#DIV/0!#DIV/0!#DIV/0!
WT S10.232131.340566.1211.003136.600136.1910.700122.420174.88638.694
p59b0.038134.0803528.4210.299135.160452.0400.293137.400468.94216.902
p59b w/ p120.187175.520938.6100.621278.470448.4220.685312.750456.5698.147
p59b w/ p130.217115.500532.2580.655108.150165.1150.785126.850161.592-3.522
p13 w/ p59b 10.239101.130423.1380.751110.760147.4830.758109.220144.090-3.394
p13 w/ p59b 20.24590.400368.9800.708117.250165.6070.747126.030168.7153.108
p27 w/ p59b 10.141130.950928.7230.495156.170315.4950.493121.950247.363-68.132
p27 w/ p59b 20.171128.890753.7430.581127.040218.6570.487123.580253.75835.100

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)Amp10
E2/ TOP10 (new) + pUC19 (1 uL)Amp13
Amp (-) ctrlAmp0

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?67.451.3491.992.1350
E1 P39A 08 2362.897.2581.942.2650
E1 P39A 08 1417.468.3491.962.2650
E1 P38a 07 2351.017.021.972.0750
E1 P38a 07 1293.15.8621.962.1550
E1 P76 (17+45)534.1310.6831.92.2750
S1 P34 B amp941.8318.8371.92.2650
S1 P34 B amp994.719.8941.912.2650

7/18/08

Plasmid/ Sample ng/uL A260 260/280 260/230 Constant
S1 P59b 1450.619.0121.92.3350
S1 P59b 2328.896.5781.922.3350
E1 P58a 1144.212.88422.3850
E1 P58a 291.061.8211.92.3850
E1 P80128.632.5732.042.2650


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.