IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week4/Chemical and Light: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Difference in Levels of Fluor due to IPTG treatment (Fluor/OD3-Fluor/OD3)''' | | align="center" style="background:#f0f0f0;"|'''Difference in Levels of Fluor due to IPTG treatment (Fluor/OD3-Fluor/OD3)''' | ||
|- | |- | ||
| p59 cells w/ p12 vector A||0. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | 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. | | p34||0.045||191.140||4247.556||0.825||413.930||501.733||1.006||384.310||382.018||-119.715 | ||
|- | |- | ||
| p59b ( | | p59b (glycerol) + cntl||1.458||131.240||90.014||||||#DIV/0!||||||#DIV/0!||#DIV/0! | ||
|- | |- | ||
| p59b (plate) | | 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. | | mtr A negative control||0.097||129.620||1336.289||||||#DIV/0!||||||#DIV/0!||#DIV/0! | ||
|- | |- | ||
| mtrB negative control||0||109. | | mtrB negative control||0.000||109.910||#DIV/0!||||||#DIV/0!||||||#DIV/0!||#DIV/0! | ||
|- | |- | ||
| LB||||106. | | LB||||106.800||#DIV/0!||0.000||101.160||#DIV/0!||||||#DIV/0!||#DIV/0! | ||
|} | |} | ||
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| align="center" style="background:#f0f0f0;"|'''Fluor @ 2h w/ IPTG''' | | align="center" style="background:#f0f0f0;"|'''Fluor @ 2h w/ IPTG''' | ||
| align="center" style="background:#f0f0f0;"|'''Fluor/OD3''' | | align="center" style="background:#f0f0f0;"|'''Fluor/OD3''' | ||
| align="center" style="background:#f0f0f0;"|'''Difference in Levels of Fluor due to IPTG treatment (Fluor/OD3-Fluor/ | | align="center" style="background:#f0f0f0;"|'''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! | | LB||0.000||120.050||#DIV/0!||0.000||110.640||#DIV/0!||||||#DIV/0!||#DIV/0! |
Revision as of 13:54, 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
- Grow up 10mL culture overnight with antibiotic at 200 RPM, 37 degrees.
- Dilute 2.5mL of overnight culture into 50mL of fresh LB.
- Grow at 200 RPM, 30 degrees until OD660 = 0.2.
- Split culture into two 25mL cultures, and put one in 30 degrees and the other in 40 degrees.
- Grow for 4 hours at these separate temperatures at 200 RPM.
- 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
- 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
- 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
- Lane 1: 1 kb ladder
- Lane 2: P91E cut EX (4327 bp)
- Lane 3: P91E uncut
- Lane 4: P11 cut XP (4333 bp)
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.