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

From OpenWetWare
Jump to navigationJump to search
Line 407: Line 407:


'''Gel 1'''
'''Gel 1'''
[[Image:7-17_gel_1_MXHTA.jpg]]
[[Image:7-17_gel_1_MXHTA.jpg]]




'''Gel 2'''
'''Gel 2'''
[[Image:7-17_gel_2_MXHTA.jpg]]
[[Image:7-17_gel_2_MXHTA.jpg]]




'''Gel 3'''
'''Gel 3'''
[[Image:7-17_gel_3_MXHTA.jpg]]
[[Image:7-17_gel_3_MXHTA.jpg]]


Revision as of 09:54, 18 July 2008

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

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

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

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

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

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

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 und
E1 + pET-Duet Vector 0.361 288.07 798

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
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
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
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

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

  • 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


Gel 2


Gel 3

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 0.42 572.66 1363.47619 1.15 863.79 751.1217391 0.818 1099.98 1344.718826 593.5970873
p59 cells w/ p12 vector B 0.427 476.3 1115.456674 1.011 905.5 895.6478734 1.012 1048.15 1035.721344 140.0734705
p59 cells w/ p13 vector A 0.474 84.44 178.1434599 1.047 177.1 169.1499522 1.026 151.45 147.6120858 -21.53786647
p59 cells w/ p13 vector B 0.33 79.01 239.4242424 1.027 171.32 166.8159688 0.93 122.87 132.1182796 -34.69768927
p27 cells w/ p59b vector 1a 0.364 76.05 208.9285714 0.615 180.42 293.3658537 0.775 105.07 135.5741935 -157.7916601
p27 cells w/ p59b vector 1b 0.299 73.74 246.6220736 0.841 189.91 225.8145065 0.824 157.24 190.8252427 -34.98926382
p27 cells w/ p59b vector 2a 0.302 67.83 224.602649 0.713 170.02 238.457223 0.716 105.53 147.3882682 -91.06895484
p27 cells w/ p59b vector 2b 0.476 81.83 171.9117647 0.683 178.37 261.1566618 0.647 114.14 176.4142195 -84.74244231
p13 cells w/ p59b 1a 0.371 126.44 340.8086253 0.948 186.4 196.6244726 0.852 111.84 131.2676056 -65.35686694
p13 cells w/ p59b 1b 0.535 91.75 171.4953271 0.959 167.66 174.8279458 0.941 130.75 138.9479277 -35.88001804
p13 cells w/ p59b 1c 0.431 111.46 258.6078886 1.016 184.35 181.4468504 0.937 153.33 163.6392743 -17.80757611
p13 cells w/ p59b 2a 0.582 73.4 126.1168385 0.938 183.24 195.3518124 0.924 124.96 135.2380952 -60.11371713
p13 cells w/ p59b 2b 0.543 118.06 217.4217311 0.936 189.17 202.1047009 1.054 139.86 132.6944972 -69.4102037
p13 cells w/ p59b 2c 0.523 111.6 213.3843212 0.992 192.72 194.2741935 1.16 169.38 146.0172414 -48.25695217
p34 0.045 191.14 4247.555556 0.825 413.93 501.7333333 1.006 384.31 382.0178926 -119.7154407
p59b (from glycerol stock) postive control 1.458 131.24 90.01371742 #DIV/0! #DIV/0! #DIV/0!
p59b (plate) positive control 0 288.75 #DIV/0! 1.025 308.48 300.9560976 0.996 261.48 262.5301205 -38.42597708
mtr A negative control 0.097 129.62 1336.28866 #DIV/0! #DIV/0! #DIV/0!
mtrB negative control 0 109.91 #DIV/0! #DIV/0! #DIV/0! #DIV/0!
LB 106.8 #DIV/0! 0 101.16 #DIV/0! #DIV/0! #DIV/0!
Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:Harvard/2008/Lab_Notebooks/DailyBook/Week4/Chemical_and_Light&oldid=222075"