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| Line 1: |
Line 1: |
| - | {{:Team:Kyoto/Header}}
| |
| - | ==Wednesday, August 11 <span class="by">By: Wataru</span>==
| |
| - | ===Overnight culture===
| |
| - | {| class="experiments"
| |
| - | !Name||Strain||Parts||Medium||Inducer(IPTG)||Incubation
| |
| - | |-
| |
| - | |ML-||KRX||Lac promoter with GFP (<partinfo>K358000</partinfo>)||LB (Kan+), 3ml||0mmol/l||rowspan="5"|At 37℃ 20:20- (Overnight)
| |
| - | |-
| |
| - | |ML+||KRX||Lac promoter with GFP (<partinfo>K358001</partinfo>)||LB (Kan+), 3ml||1mmol/l
| |
| - | |-
| |
| - | |MS-||KRX||Measurement Kit Test of <partinfo>J23101</partinfo> (<partinfo>I20260</partinfo>)||LB (Kan+), 3ml||0mmol/l
| |
| - | |-
| |
| - | |MS+||KRX||Measurement Kit Test of <partinfo>J23101</partinfo> (<partinfo>I20260</partinof>)||LB (Kan+), 3ml||1mmol/l
| |
| - | |-
| |
| - | |KRX||KRX||-||SOC, 3ml||0mmol/l
| |
| - | |}
| |
| | | | |
| - |
| |
| - | ==Thursday, August 12 <span class="by">By: Tomonori, Takuya O.</span>==
| |
| - | ===Culture===
| |
| - | We stopped overnight cultures at 11:10 and took out 30µl culture from each test tube, and added it to 3ml fresh medium which is same for overnight culture. They are grown at 37℃ from 11:10-14:10. After 3-hours culture, OD600 of each culture were measured. Because we found that the 3-hours culture of the sample KRX had not grown, we use the overnight culture of the sample KRX in later procedures. We took out 1ml medium from test tubes and put them into 1.5ml tubes and freeze them. The data is shown in table below.
| |
| - | ===Measure===
| |
| - | We melt samples and centrifuge them at 14,000rpm, at 4℃, for 2min. Discard the supernatant and 150µl cel lysis reagent to each pellet. These pellets were dissolved by vortex for 2min and centrifuged for 2min. From each tubes, 100µl of the supernatant was took out and added to each well of the plate for the plate reader. 100µl cell lysis reagent was also added to them. We measured GFP fluorescence in each sample (Ex/Em = 485/535nm, 1sec). The data is shown in table below.
| |
| - | We calculated the promoter activity and the results are shown in table below.
| |
| - | {| class="experiments"
| |
| - | !Name||Optical Density (600nm)||Fluorescence (485/535nm)||Calculation 1||Calculation 2||Calculation 3||Promoter Activity
| |
| - | |-
| |
| - | |ML-||0.531||4881||4697||8.85 × 10<sup>3</sup>||6.40 × 10<sup>3</sup>||8.65 × 10<sup>-2</sup>
| |
| - | |-
| |
| - | |ML+||0.546||91549||91365||1.67 × 10<sup>5</sup>||1.65 × 10<sup>5</sup>||2.04
| |
| - | |-
| |
| - | |MS-||0.645||49469||49285||7.64 × 10<sup>4</sup>||7.40 × 10<sup>4</sup>||1 (0mmol/l IPTG)
| |
| - | |-
| |
| - | |MS+||0.623||52017||51833||8.32 × 10<sup>4</sup>||8.08 × 10<sup>4</sup>||1 (1mmol/l IPTG)
| |
| - | |-
| |
| - | |KRX||1.413||3644||3460||2.449 × 10<sup>3</sup>||-||-
| |
| - | |-
| |
| - | |Cell Lysis Reagent||-||184||-||-||-||-
| |
| - | |}
| |
| - | * Calculation 1: Fluorescence - Fluorescence<sub>Cell Lysis Reagent</sub>
| |
| - | * Calculation 2: Calculation 1 / Optical Density (600nm)
| |
| - | * Calculation 3: Calculation 2 - Calculation 2<sub>KRX</sub>
| |
| - | * Promoter Activity: Calculation 3<sub>ML-</sub> / Calculation 3<sub>MS-</sub>, Calculation 3<sub>ML+</sub> / Calculation 3<sub>MS+</sub>
| |
| - |
| |
| - |
| |
| - | ==Friday, August 20 <span class="by">By: Tomonori</span>==
| |
| - | ===Make M9 Medium===
| |
| - | M9 salts were added to 1l MilliQ and sterilized by autocrave. M9 medium was stored in a fridge.
| |
| - |
| |
| - |
| |
| - | ==Monday, August 23 <span class="by">By: Tasuku, Takuya O.</span>==
| |
| - | ===Make Supplemented M9 Medium===
| |
| - | We tried to dissolve CaCl<sub>2</sub> 0.1mol/l (final concentration) in 1l M9 medium, but we failed because CaCl<sub>2</sub> was too much.
| |
| - |
| |
| - |
| |
| - | ==Tuesday, August 24 <span class="by">By: Tasuku, Takuya O.</span>==
| |
| - | ===Make Supplemented M9 Medium===
| |
| - | M9 salts were added to 1l MilliQ and sterilized by autocrave. We changed amount of CaCl<sub>2</sub> from 0.1mol/l (final concentration) to 0.01mmol/l (final concentration), and we could make supplemented M9 medium.
| |
| - | ===Overnight culture===
| |
| - | The ML, i.e., E.coli KRX transformed with <partinfo>K358000</partinfo> was grown in the supplemented M9 medium including 1mmol/l IPTG for a night (16h).
| |
| - |
| |
| - |
| |
| - | ==Wednesday, August 25 <span class="by">By: Tasuku, Takuya O.</span>==
| |
| - | ===Culture===
| |
| - | OD600 of the overnight culture was measured. The overnight culture was diluted in fresh supplemented M9 medium including 7 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected (To know more detail procedures, see [[Team:Kyoto/Protocols#Measurement|Protocols]]).
| |
| - | {| class="experiments"
| |
| - | !||0.00mmol/l||0.01||0.03||0.05||0.07||0.10||1.00
| |
| - | |-
| |
| - | |0h||-0.003||0.014||0.000||-0.008||-||-||-
| |
| - | |-
| |
| - | |4h||0.001||0.100||0.033||0.062||-||-||-
| |
| - | |-
| |
| - | |6h||-||-||-||-||0.118||0.187||0.178
| |
| - | |-
| |
| - | |8h||0.227||0.402||0.363||0.346||0.360||0.479||0.517
| |
| - | |-
| |
| - | |16||1.117||1.142||1.131||1.237||1.211||1.202||1.156
| |
| - | |-
| |
| - | |20h||1.107||1.118||1.084||1.178||1.177||1.178||1.131
| |
| - | |}
| |
| - | * The OD600 of medium including 0.07mmol/l, 0.10mmol/l, and 1mmol/l IPTG were not measured at 4h, but at 6h.
| |
| - | * Measurement at 16h and 20h was conducted the day after.
| |
| - | [[Image:KyotoGrp100825-1.png]]
| |
| - | We found that the number of the cell was in steady state at 16h and after.
| |
| - | ===Overnight Culture===
| |
| - | The MS, i.e., E.coli KRX transformed with <partinfo>I20260</partinfo> was grown in supplemented M9 medium including 1mmol/l IPTG for a night for 16 hours.
| |
| - |
| |
| - |
| |
| - | ==Thursday, August 26 <span class="by">By: Tasuku, Takuya O.</span>==
| |
| - | ===Culture===
| |
| - | OD600 of the overnight culture was measured. The overnight culture was diluted in fresh supplemented M9 medium including 7 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected (To know more detail procedures, see [[Team:Kyoto/Protocols#Measurements|Protocols]]).
| |
| - | {| class="experiments"
| |
| - | !Time||0.00mmol/l||0.01||0.03||0.05||0.07||0.10||1.00
| |
| - | |-
| |
| - | |0h||-0.016||-0.015||0.000||-0.016||-0.018||0.001||-0.007
| |
| - | |-
| |
| - | |4||0.108||0.112||0.116||0.094||0.127||0.116||0.160
| |
| - | |-
| |
| - | |8||0.626||0.585||0.591||0.522||0.516||0.562||0.553
| |
| - | |-
| |
| - | 16||1.351||1.425||1.442||1.483||1.346||1.420||1.497
| |
| - | |-
| |
| - | |20||1.363||1.405||1.403||1.442||1.383||1.361||1.434
| |
| - | |-
| |
| - | |24||-||1.297||-||-||-||-||1.352
| |
| - | |}
| |
| - | * Measurement at 16h, 20h, and 24h was conducted the day after.
| |
| - | [[Image:KyotoGrp100826-1.png]]
| |
| - | ===Measure OD600 of the culture of ML and collect samples===
| |
| - |
| |
| - |
| |
| - | ==Friday, August 27 <span class="by">By: Tasuku, Takuya O.</span>==
| |
| - | ===Measure OD600 of the culture of ML and collect samples===
| |
| - |
| |
| - |
| |
| - | ==Make the plate of E.coli C2===
| |
| - | The E.coli competent cell, C2, was diluted to three different concentration, that is 10<sup>-3</sup>, 10<sup>-5</sup>, 10<sup-6</sup>. The 200µl of each culture was plated without antibiotics.
| |
| - |
| |
| - |
| |
| - | ==Monday, August 30 <span class="by">By: Tomonori, Tasuku</span>==
| |
| - | ===Overnight culture===
| |
| - | A colony of each plate made on Friday was picked out and put into the test tube including 5ml supplemented M9 medium without antibiotics. They were grown at 37℃ for a night (16 hours).
| |
| - |
| |
| - |
| |
| - | ==Tuesday, August 31 <span class="by">By: Tomonori, Tasuku, Kazuya</span>==
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | OD600 of the overnight culture was measured. We found that E.coli C2 had not grown.
| |
| - | {| class="experiments"
| |
| - | !Name||OD600
| |
| - | |-
| |
| - | |C2 (diluted to 10<sup>-3</sup>)||0.034
| |
| - | |-
| |
| - | |C3 (diluted to 10<sup>-5</sup>)||0.062
| |
| - | |-
| |
| - | |C4 (diluted to 10<sup>-6</sup>)||0.040
| |
| - | |}
| |
| - | Because we noticed that the color of the three overnight cultures were strange, we suspected that other bacterium had grwon in these culture.
| |
| - | ===Culture E.coli C2 wighout antibiotics again===
| |
| - | A colony of paltes were picked out and put into the test tube including 5ml supplemented M9 medium wighout antibiotics. They were grown at 37℃ for 11 hours. OD600 of the culture was measured. As the previous experiment, C2 did not grow.
| |
| - | {| class="experiments"
| |
| - | !Name||OD600
| |
| - | |-
| |
| - | |C2 (diluted to 10<sup>-3</sup>)||0.031
| |
| - | |-
| |
| - | |C3 (diluted to 10<sup>-5</sup>)||0.042
| |
| - | |-
| |
| - | |C4 (diluted to 10<sup>-6</sup>)||0.091
| |
| - | |}
| |
| - | ===Overnight culture E.coli C2 in new medium===
| |
| - | The supllemented M9 medium wighout antibiotics was made again. E.coli C2 (diluted to 10<sup>-3</sup>) was grown for a night (16 hours).
| |
| - | ===Measure the fluorescence of GFP of the ML and the MS===
| |
| - | The fluorescence of GFP of the ML and the MS was measured (Ex/Em = 485/535 nm, 1sec).
| |
| - | {| class="experiments"
| |
| - | |+ ML
| |
| - | !Time||0.00mmol/l||0.01||0.03||0.05||0.07||0.10||1.00
| |
| - | |-
| |
| - | 0h||51||329||57||62||68||40||5
| |
| - | |-
| |
| - | 4||1261||6121||3046||4317||2606||13677||12120
| |
| - | |-
| |
| - | 8||36181||74292||67032||65566||42850||78761||68736
| |
| - | |-
| |
| - | 16||5573||7274||4759||3526||6131||2844||3091
| |
| - | |-
| |
| - | 20||1229||2990||1512||3189||3579||7919||2437
| |
| - | |}
| |
| - | * Back corrected.
| |
| - | {| class="experiments"
| |
| - | |+ MS
| |
| - | !Time||0.00mmol/l||0.01||0.03||0.05||0.07||0.10||1.00
| |
| - | |-
| |
| - | |0h||3||-13||-11||16||56||32||-14
| |
| - | |-
| |
| - | |4||17638||7790||13413||8056||19632||8090||8800
| |
| - | |-
| |
| - | 8||66508||85100||236319||83459||126515||177323||84395
| |
| - | |-
| |
| - | |16||6504||7556||6159||7650||4228||4240||10662
| |
| - | |-
| |
| - | |20||7835||11638||9070||15135||15383||8681||18276
| |
| - | |-
| |
| - | |24||-||22526||-||-||-||-||34230
| |
| - | |}
| |
| - | * Back corrected.
| |
| - | Because amounts of samples collected at 0h are very little and the data indicates that is not enough to measure OD600 and fluorescence of GFP correctoly, we decide to measure OD600 after 4 hours only.
| |
| - | We calculated the promoter activity of the lactose promoter.
| |
| - | {| class="experiments"
| |
| - | !Time||0.00mmol/l||0.01||0.03||0.05||0.07||0.10||1.00
| |
| - | |-
| |
| - | |0h||-||-||-||-||-||-||-
| |
| - | |-
| |
| - | |4||0.000000||0.880041||0.798267||0.812454||0.142867||1.048718||1.237998
| |
| - | |-
| |
| - | |8||1.500221||1.270405||0.461811||1.185223||0.485463||0.521131||0.871168
| |
| - | |-
| |
| - | |16||1.036360||1.201241||0.985163||0.552576||1.611748||0.792406||0.375426
| |
| - | |-
| |
| - | |20||0.193135||0.322870||0.215761||0.257924||0.273380||1.053934||0.169068
| |
| - | |}
| |
| - | * The autofluorescence of E.coli KRX is not corrected.
| |
| - | [[Image:KyotoGrp100831-1.png]]
| |
| - | [[Image:KyotoGrp100831-2.png]]
| |
| - | Though we drew the graph, we couldn't find any tendency. We decide to conduct experiment with more samples and measure the autofluorescence of E.coli.
| |
| - |
| |
| - |
| |
| - | ==Wednesday, September 1 <span class="by">By: Tomonori, Tasuku<span>==
| |
| - | ===Measure OD600 of the overnight culture of C2===
| |
| - | OD600 of the overnight culture of C2 was measured. In this experiment, C2 grew.
| |
| - | {| class="experitments"
| |
| - | !Name||OD600
| |
| - | |-
| |
| - | |C2 (diluted to 10<sup>-3</sup>)||1.172
| |
| - | |-
| |
| - | |C3 (diluted to 10<sup>-5</sup>)||1.774
| |
| - | |-
| |
| - | |C4 (diluted to 10<sup>-6</sup>)||1.765
| |
| - | |}
| |
| - | We decide not to culture of C2 for measurement of promoter activity because other bacterium can grow without antibiotics.
| |
| - | ===[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] for to making a low copy plasmid without insert===
| |
| - | {| class="experiments"
| |
| - | !Name||Sample||10xBuffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
| |
| - | |-
| |
| - | |pSB4K5||2.0 µl||5.0||0.5||XbaI||0.2||SpeI||0.2||42.1||50.0||At 37℃ for 2h
| |
| - | |}
| |
| - | * The concentration of the sample is 56ng/µl.
| |
| - | ===[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Phosphorylation|Phosphorylation]]===
| |
| - | After PCR purification, the sample was ligated.
| |
| - | {| class="experiments"
| |
| - | !Name||Sample||Ligation High||Total
| |
| - | |-
| |
| - | |pSB4K5||1.0 µl||1.0||2.0
| |
| - | |}
| |
| - | ===[[Team:Kyoto/Protocols#Transformation|Transformation]]===
| |
| - | {| class="experiments"
| |
| - | !Name||Sample||Competent Cell||Total||Plate||Incubation||Result
| |
| - | |-
| |
| - | |pSB4K5 without insert||2.0 µl||50.0||52.0||LB (Kan+)||09/01 - 09/02
| |
| - | |}
| |
| - | ===Overnight culture===
| |
| - | The ML and the MS were grown at 37℃ for a night (16 hours).
| |
| - | ===Overnight culture for the makeup test of 08/11, 08/12===
| |
| - | {| class="experiments"
| |
| - | !Name||Medium||IPTG
| |
| - | |-
| |
| - | |ML||rowspan="4"|Supplemented M9 (Kan+)||0.00 mmol/l
| |
| - | |-
| |
| - | |ML||1.00
| |
| - | |-
| |
| - | |MS||0.00
| |
| - | |-
| |
| - | |MS||1.00
| |
| - | |}
| |
| - | These samples were grown at 37℃ for 16 hours.
| |
| - |
| |
| - |
| |
| - | ==Thursday, September 2 <span class="by">By: Tomonori</span>==
| |
| - | ===Culture===
| |
| - | OD600 of the overnight culture was measured.
| |
| - | {|class="experiments"
| |
| - | !sample name||OD600
| |
| - | |-
| |
| - | |ML||1.580
| |
| - | |-
| |
| - | |MS||1.582
| |
| - | |}
| |
| - | The overnight culture of ML was diluted in fresh supplemented M9 medium including 3 different concentration of IPTG and that of MS was diluted without IPTG. OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.
| |
| - | {| class="experiments"
| |
| - | !ML 0mM||ML 0.01mM||ML 0.1mM||MS1||MS2||MS3
| |
| - | |-
| |
| - | |4h||0.119||0.120||0.124||0.130||0.125||0.111
| |
| - | |-
| |
| - | |8h||0.974||1.018||0.958||0.848||0.816||0.792
| |
| - | |-
| |
| - | |12h||1.127||1.180||1.242||1.262||1.264||1.189
| |
| - | |-
| |
| - | |16h||1.027||1.090||1.137||1.172||1.180||1.159
| |
| - | |-
| |
| - | |20h||1.103||1.110||1.151||1.233||1.169||1.225
| |
| - | |}
| |
| - | *Measurement at 12h, 16h and 20h was conducted the day after.
| |
| - | [[image:KyotoGrp100902-1.png]]
| |
| - | We found that the number of the cell had been in steady state at 12h and after.
| |
| - | ===Culture for the makeup test of 8/11, 8/12===
| |
| - | The overnight culture was diluted to 10-2 in the fresh culture and grown for 3hours. OD600 of the culture was measured and samples for measurement of GFP fluorescence were collected.
| |
| - | {|class="experiments"
| |
| - | !sample name||OD600
| |
| - | |-
| |
| - | |ML(IPTG 1mM)||0.326
| |
| - | |-
| |
| - | |ML(IPTG 0mM)||0.329
| |
| - | |-
| |
| - | |MS(IPTG 1mM)||0.328
| |
| - | |-
| |
| - | |MS(IPTG 0mM)||0.314
| |
| - | |}
| |
| - | ===Transformation of pSB4K5 without insertion===
| |
| - | Colonies were seen on the plate.
| |
| - |
| |
| - |
| |
| - | ==Friday, 3 September <span class="by"> By:Tomonori, Tasuku </span>==
| |
| - | ===Measure OD600 of the culture of MS and collect samples.===
| |
| - | ===Measure the fluorescence of GFP of the ML and the MS===
| |
| - | {|class="experiments"
| |
| - | !||ML<br>IPTG 0mM</br>||ML<br>IPTG 0.01mM</br>||ML<br>IPTG 0.1mM</br>||MS1||MS2||MS3
| |
| - | |-
| |
| - | |4h||155||392||1836||6526||6572||8274
| |
| - | |-
| |
| - | |8h||1046||1667||92602||145341||149358||119017
| |
| - | |-
| |
| - | |12h||918||2733||119459||180575||137615||152878
| |
| - | |-
| |
| - | |16h||883||2543||80473||138505||159007||235987
| |
| - | |-
| |
| - | |20h||868||3229||157578||400829||152590||157942
| |
| - | |}
| |
| - | *Back corrected
| |
| - | We calculate the promoter activity of the lactose promoter
| |
| - | {|class="experiments"
| |
| - | !||0mM||0.01mM||0.1mM
| |
| - | |-
| |
| - | |4h||0.022||0.055||0.251
| |
| - | |-
| |
| - | |8h||0.006||0.010||0.575
| |
| - | |-
| |
| - | |12h||0.006||0.018||0.758
| |
| - | |-
| |
| - | |16h||0.006||0.015||0.465
| |
| - | |-
| |
| - | |20h||0.004||0.015||0.703
| |
| - | |}
| |
| - | *the autofluorescence of E.coli KRX is not corrected.
| |
| - | [[image:KyotoExp100903-1.png]]
| |
| - | We could not find the relationship between promoter activity and incubation time.
| |
| - | [[image:KyotoExp100903-2.png]]
| |
| - | This graph indicates that the higher IPTG concentration is, the higher the activity of the lactose promoter is.
| |
| - |
| |
| - |
| |
| - | ==Sunday, 5 September <span class="by"> By Kazuya </span>==
| |
| - | ===Colony PCR of pSB4K5 without insertion===
| |
| - |
| |
| - |
| |
| - | ==Monday, 6 September <span class="by"> By Kazuya </span>==
| |
| - | ===Electrophoresis of pSB4K5 without insertion===
| |
| - | {|class="experiments"
| |
| - | |Marker||1||2||3||4||5||6||7||8||9||10||11||12||Marker
| |
| - | |-
| |
| - | |lambda||colspan="12"|pSB4K5 without insertion||100bp
| |
| - | |}
| |
| - | The pSB4K5 must be about 2300bp. The result of the electrophoresis indicates that the transformation was faile.
| |
| - | M 1 2 3 4 5 6 7 8 9 10 11 12 M
| |
| - | [[image:KyotoExp100906-1.png]]
| |
| - | 500bp <これだけ離れていてどこにつければいいか分かりません>
| |
| - | ===Overnight culture for measurement the promoter===
| |
| - | The ML and the MS were grown at 37℃ for a night (16hours).
| |
| - |
| |
| - |
| |
| - | ==Tuesday, 7 September <span class="by"> By: Kazuya, Tomo, Wataru, Fumitaka</span>==
| |
| - | ===Digest pSB4K5 by XbaI, SpeI to make low copy plasmid without insert again===
| |
| - | {|class="experiments"
| |
| - | !Sample||10xBuffer||BSA||Enzyme1||Enzyme2||MilliQ||Total||Incubation
| |
| - | |-
| |
| - | |pSB4K5 2µL||5µL||0.5µL||XbaI 0.2µL||SpeI 0.2µL||42.1µL||50µL||At 37℃2h
| |
| - | |}
| |
| - | *the concentration of the sample, pSB4K5, is 56ng/µL
| |
| - | ===Ligation and pospholylation===
| |
| - | After PCR purification, the sample was ligated.
| |
| - | {|class="experiments"
| |
| - | |Sample||Ligation High||Total
| |
| - | |-
| |
| - | |pSB4K5 1µL||1µL||2µL
| |
| - | |}
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | We found that the overnight culture had not grown. We think this is because we added too much antibiotics to the culture.
| |
| - |
| |
| - |
| |
| - | ==Wednesday, 8 September <span class="by"> By: Tasuku, Wataru </span>==
| |
| - | ===Transformation of pSB4K5 without insert===
| |
| - | {|class="experiments"
| |
| - | !Sample||Conc(/µL)||Sample Volume(µL)||Competent Cell(µL)||Total||Plate||Incubation
| |
| - | |-
| |
| - | |pSB4K5 without insert||-||2||50||52||LB Kan||9/8‾9/9
| |
| - | |}
| |
| - | ===Overnight culture for measurement the promoter===
| |
| - | The ML was grown at 37℃ for a night (18hours).
| |
| - |
| |
| - |
| |
| - | ==Thursday, 9 September <span class="by"> By:Tasuku </span>==
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | We measure the OD600 of overnight culture. The OD600 is 0.376.
| |
| - | ===Culture for measurement of Promoter activity===
| |
| - | The overnight culture was diluted in fresh supplemented M9 medium including 5 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.
| |
| - | {|class="experiments"
| |
| - | !ML||0mM||0.01mM||0.03mM||0.1mM||1mM
| |
| - | |-
| |
| - | |4h||0.373||0.325||0.326||0.359||0.341
| |
| - | |-
| |
| - | |8h||1.328||1.355||1.481||1.405||1.457
| |
| - | |}
| |
| - | We stopped incubation at 8h because strange floating matters were seen in the culture.
| |
| - | ===Overnight culture for measurement the promoter===
| |
| - | The ML was grown at 37℃ for a night (16hours).
| |
| - | ===Transformation of pSB4K5 without insert===
| |
| - | Colonies were seen on the plate.
| |
| - |
| |
| - |
| |
| - | ==Friday, 10 September <span class="by"> By: Tasuku </span>==
| |
| - | The overnight culture was diluted in fresh supplemented M9 medium including 5 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.
| |
| - | {|class="experiments"
| |
| - | |ML||0mM||0.01mM||0.03mM||0.1mM||1mM
| |
| - | |-
| |
| - | |4h||0.103||0.109||0.117||0.11||0.055
| |
| - | |-
| |
| - | |8h||0.196||0.256||0.285||0.377||0.174
| |
| - | |}
| |
| - | We stopped incubation at 8h because the cell growth rate was too bad.
| |
| - |
| |
| - |
| |
| - | ==Sunday, 12 September <span class="by"> By: Wataru</span>==
| |
| - | ===Overnight culture of MB===
| |
| - | MB (Measurement ? Back), that is, E.coli KRX transformed with pSB4K5 without insert was grown in the supplemented M9 medium for a night (16h).
| |
| - |
| |
| - |
| |
| - | ==Monday, 13 September <span class="by"> By: Takuya Okada, Ken </span>==
| |
| - | ===Culture for measurement of the promoter activity===
| |
| - | OD600 of MB was measured.
| |
| - | {|class="experiments"
| |
| - | |4h||8h||12h||16h||20h
| |
| - | |-
| |
| - | |0.148||1.738||1.952||1.8||2.178
| |
| - | |}
| |
| - | *Measurements at 12h, 16h and 20h were conducted the day after.
| |
| - |
| |
| - |
| |
| - | ==Tuesday, 14 September <span class="by"> By: Kazuya, Tomonori </span>==
| |
| - | ===MeasureOD600 of MB culture and collect samples===
| |
| - | ===Measure the fluorescence of the samples===
| |
| - | {|class="experiments"
| |
| - | |||colspan="5"|9/9||colspan="5"|9/10||MB
| |
| - | |-
| |
| - | |||0mM||0.01mM||0.03mM||0.1mM||1mM||0mM||0.01mM||0.03mM||0.1mM||1mM
| |
| - | |-
| |
| - | |4h||2487||3322||4728||23302||69101||225||264||389||3391||5500||77
| |
| - | |-
| |
| - | |8h||4472||11295||52260||147947||241965||1761||2139||43379||138454||187563||635
| |
| - | |-
| |
| - | |12h||||||||||||||||||||||459
| |
| - | |-
| |
| - | |16h||||||||||||||||||||||382
| |
| - | |-
| |
| - | |20h||||||||||||||||||||||751
| |
| - | |}
| |
| - | *Back corrected
| |
| - | We measure GFP fluorescence of the samples, but we did not these data because GFP fluorescence of ML samples might have grown abnormally.
| |
| - | ===Transformation of pSB4K5 without insert to E.coli BL21 for measurement of lactose degradation===
| |
| - | {|class="experiments"
| |
| - | |Sample Conc(/µL)||Sample Volume(µL)||Competent Cell(µL)||Total||Plate||Incubation
| |
| - | |-
| |
| - | |pSB4K5 without insert||-||2||50||52||LB Kan||9/14‾9/15
| |
| - | |}
| |
| - | ===Overnight culture of ML for measurement promoter activity===
| |
| - | ML in three test tubes was grown for 13hours.
| |
| - |
| |
| - |
| |
| - | ==Wednesday, 15 September <span class="by"> By: Tomonori </span>==
| |
| - | ===Make the standard curve for measurement lactose concentration===
| |
| - | [[image:KyotoExp100915-1.png]]
| |
| - | We also measure the concentration of lactose in the supplemented M9 medium and found that it contains approximately 0.01mM lactose.
| |
| - |
| |
| - |
| |
| - | ==Thursday, 16 September <span class="by"> By: Tomonori </span>==
| |
| - | ===Overnight culture for measurement of RPU of the lactose promoter===
| |
| - | We changed the way of measurement of promoter activity. (to know more detail, see protocol.)
| |
| - | Three falcon tubes of ML, three falcon tubes of MS and three falcon tubes of MB were grown with 1mM IPTG at 37℃ for 12hours.
| |
| - | ===Transformation of pSB4K5 without insert to E.coli BL21 for measurement of lactose degradation===
| |
| - | Any colony was not seen on the plate.
| |
| - |
| |
| - |
| |
| - | ==Friday, 17 September <span class="by"> By: Tomonori, Hitoshi, Takuya Okada </span>==
| |
| - | ===Measure OD600 of overnight culture===
| |
| - | {|class="experiments"
| |
| - | |sample name||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||1.900||1.745||1.937||1.905||1.938||1.488||3.276||3.300||3.202
| |
| - | |}
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | Overnight culture was diluted in the fresh medium including 1mM IPTG and OD600 was measured at 1h, 2h, 3h, 3.5h, 4h.
| |
| - | {|class="experiments"
| |
| - | !OD600||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |1h||0.017||0.103||0.118||0.057||0.073||0.044||-0.028||0.101||0.098
| |
| - | |-
| |
| - | |2h||0.270||0.295||0.245||0.167||0.241||0.149||0.208||0.179||0.294
| |
| - | |-
| |
| - | |3h||0.33||0.33||0.35||0.36||0.42||0.31||0.66||0.70||0.66
| |
| - | |-
| |
| - | |3.5h||0.47||0.37||0.46||0.49||0.55||0.40||0.98||0.98||0.96
| |
| - | |-
| |
| - | |4h||0.675||0.595||0.665||0.684||0.793||0.607||1.358||1.362||1.360
| |
| - | |}
| |
| - | [[image:KyotoExp100917-1.png]]
| |
| - | This graph indicates that all samples were in the exponential growth from 2h to 4h.
| |
| - | The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.
| |
| - | {|class="experiments"
| |
| - | |GFP||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |3h||58603||49991||50845||53416||55463||40321||204||167||152
| |
| - | |-
| |
| - | |3.5h||89428||76573||85433||67457||83329||59463||218||271||246
| |
| - | |}
| |
| - | Back corrected
| |
| - | We calculate the RPU of the lactose promoter with 1mM IPTG is 1.65.
| |
| - | {|class="experiments"
| |
| - | |||ML-A||ML-B||ML-C||average||stdev||CV
| |
| - | |-
| |
| - | |RPU||1.60||1.58||1.77||1.65||0.106||0.0642
| |
| - | |}
| |
| - |
| |
| - | Overnight culture of the ML was also diluted in the fresh medium including 0mM IPTG, 0.01mM IPTG, 0.03mM IPTG, 0.1mM IPTG and 1mM IPTG and the samples for measurement of GFP fluorescence were collected at 3h and 3.5h.
| |
| - | {|class="experiments"
| |
| - | |IPTG 0mM||ML-A||ML-B||ML-C
| |
| - | |-
| |
| - | |3h||1906||2138||2190
| |
| - | |-
| |
| - | |3.5h||1781||1141||980
| |
| - | |-
| |
| - | |IPTG 0.01mM||ML-A||ML-B||ML-C
| |
| - | |-
| |
| - | |3h||5037||2588||2189
| |
| - | |-
| |
| - | |3.5h||1191||1700||1511
| |
| - | |-
| |
| - | |IPTG 0.03mM||ML-A||ML-B||ML-C
| |
| - | |-
| |
| - | |3h||2525||2410||3065
| |
| - | |-
| |
| - | |3.5h||1982||3330||3723
| |
| - | |-
| |
| - | |IPTG 0.1mM||ML-A||ML-B||ML-C
| |
| - | |-
| |
| - | |3h||14191||9154||9856
| |
| - | |-
| |
| - | |3.5h||6897||5427||7084
| |
| - | |}
| |
| - | *Back corrected
| |
| - | We did not calculate RPU from these data because GFP fluorescence at 3.5h was lower than that at 3h in most of the samples, and we couldn't calculate GFP synthesis rate.
| |
| - | We think this is because GFP synthesized in overnight culture with IPTG 1mM had stayed more at 3h than 3.5h.
| |
| - | ===Make a new plate of the MB===
| |
| - |
| |
| - |
| |
| - | ==Monday, 20 September <span class="by"> By: Tomonori </span>==
| |
| - | ===Make the supplemented M9 medium without casamino acids===
| |
| - | We think that lactose in the supplemented M9 medium come from casamino acids, so we decide to measure RPU of the lactose promoter without casamino acids because lactose in medium induces the lactose promoter.
| |
| - | ===Overnight culture for measurement of RPU of the lactose promoter with IPTG 0.1mM===
| |
| - | Three falcon tubes of the ML, three falcon tubes of the MS and three falcon tubes of the MB were grown in the supplemented M9 medium without casamino acids including 0.1mM IPTG at 37℃ for 23 hours.
| |
| - |
| |
| - |
| |
| - | ==Tuesday, 21 September <span class="by"> By: Hitoshi </span>==
| |
| - | ===Measure OD600 of overnight culture===
| |
| - | {|class="experiments"
| |
| - | |sample name||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||2.140||2.080||2.103||1.975||1.964||1.993||0.041||0.062||0.025
| |
| - | |}
| |
| - | We found that MB had not grown.
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | ML and MS were cultured and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h in the medium without casamino acids. The samples were collected at 3h and 3,5h.
| |
| - | {|class="experiments"
| |
| - | !OD600<br>IPTG 0.1mM</br>||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |2h||-0.056||-0.002||0.004||0.027||-0.001||0.024
| |
| - | |-
| |
| - | |2.5h||-0.023||0.000||0.014||-0.050||0.003||0.0025
| |
| - | |-
| |
| - | |3h||-0.050||0.003||0.0025||0.0036||0.009||0.040
| |
| - | |-
| |
| - | |3.5h||-0.036||0.002||0.041||0.061||0.061||0.039
| |
| - | |-
| |
| - | |4h||-0.030||0.045||0.04||0.0175||0.074||0.104
| |
| - | |}
| |
| - | We found that ML and MS had grown hardly.
| |
| - | ===Overnight culture for measurement of RPU of the lactose promoter with IPTG 0mM===
| |
| - | Three falcon tubes of ML, three falcon tubes of MS and three falcon tubes of MB were grown in the supplemented M9 medium without casamino acids including 0mM IPTG and 0.01mM ITPG at 37℃ for 23 hours.
| |
| - |
| |
| - |
| |
| - | ==Wednesday, 22 September <span class="by"> By: Hitoshi, Takuya Okada </span>==
| |
| - | ===Measure OD600 of overnight culture===
| |
| - | {|class="experiments"
| |
| - | |IPTG 0mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||2.009||1.948||1.965||2.168||1.951||1.719||0.042||0.052||0.032
| |
| - | |-
| |
| - | |IPTG 0.01mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||2.091||1.414||2.082||1.460||1.959||2.099||0.035||0.050||0.042
| |
| - | |}
| |
| - | We found that MB had not grown.
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | ML and MS were cultured and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h in the medium without casamino acids. The samples were collected at 3h and 3,5h.
| |
| - | {|class="experiments"
| |
| - | |OD600<br>IPTG 0mM</br>||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |2h||0.099||0.102||0.119||0.080||0.082||0.068
| |
| - | |-
| |
| - | |2.5h||0.115||0.141||0.136||0.092||0.068||0.110
| |
| - | |-
| |
| - | |3h||0.176||0.173||0.193||0.130||0.093||0.124
| |
| - | |-
| |
| - | |3.5h||0.236||0.237||0.248||0.201||0.129||0.169
| |
| - | |-
| |
| - | |4h||0.320||0.249||0.256||0.243||0.156||0.232
| |
| - | |-
| |
| - | |OD600<br>IPTG 0.01mM</br>||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |2h||0.135||0.070||0.116||0.060||0.075||0.092
| |
| - | |-
| |
| - | |2.5h||0.155||0.091||0.142||0.071||0.103||0.127
| |
| - | |-
| |
| - | |3h||0.167||0.092||0.196||0.080||0.148||0.168
| |
| - | |-
| |
| - | |3.5h||0.227||0.180||0.331||0.151||0.230||0.271
| |
| - | |}
| |
| - | [[image:KyotoExp100922-1.png]]
| |
| - | [[image:KyotoExp100922-2.png]]
| |
| - | We found that cell growth rate was low without casamino acids and that OD600 of the culture at 3h was low, therefore we decide to measure OD600 at 4h, 4.5h, 5h, 5.5h and 6h, and to collect samples at 5h and 5.5h in later experiment without casamino acids.
| |
| - | ===Overnight culture for the measurement of lactose promoter===
| |
| - | We cultivated ML and MS in the supplemented M9 medium without casamino acids including 0.03mM IPTG and 0.1mM IPTG.
| |
| - |
| |
| - |
| |
| - | ==Thursday, 23 September <span class="experiments"> By: Hitoshi, Kazuya </span>==
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | {|class="experoments"
| |
| - | |0.03mM||ML||MS
| |
| - | |-
| |
| - | |OD600||1.417||1.483
| |
| - | |-
| |
| - | |0.1mM||ML||MS
| |
| - | |-
| |
| - | |OD600||2.126||2.131
| |
| - | |}
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | ML and MS were cultured and OD600 was measured at 4h, 4.5h, 5h, 5.5h and 6h in the medium without casamino acids. The samples were collected at 5h and 5.5h.
| |
| - | {|class="experiments"
| |
| - | |0.03mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |4h||0.266||0.256||0.273||0.208||0.188||0.267
| |
| - | |-
| |
| - | |4.5h||0.345||0.316||0.360||0.280||0.232||0.213
| |
| - | |-
| |
| - | |5h||0.506||0.456||0.531||0.337||0.309||0.270
| |
| - | |-
| |
| - | |5.5h||0.580||0.569||0.623||0.380||0.330||0.334
| |
| - | |-
| |
| - | |6h||0.764||0.699||0.775||0.415||0.396||0.395
| |
| - | |}
| |
| - | [[image:KyotoExp100923-1.png]]
| |
| - | {|class="experiments"
| |
| - | |0.1mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |4h||0.237||0.259||0.26||0.279||0.282||0.286
| |
| - | |-
| |
| - | |4.5h||0.325||0.339||0.348||0.429||0.423||0.419
| |
| - | |-
| |
| - | |5h||0.442||0.455||0.485||0.563||0.541||0.572
| |
| - | |-
| |
| - | |5.5h||0.602||0.599||0.615||0.749||0.675||0.682
| |
| - | |-
| |
| - | |6h||0.719||0.678||0.741||0.987||0.815||0.900
| |
| - | |}
| |
| - | [[image:KyotoExp100923-2.png]]
| |
| - | ===Overnight culture for the measurement of lactose promoter===
| |
| - | We cultivated ML and MS in the supplemented M9 medium without casamino acids including 1mM IPTG.
| |
| - |
| |
| - |
| |
| - | ==Friday, 24 September <span class="by"> By: Tomonori, Hitoshi, Tasuku, Takuya Okada, Ken, Kazuya </span>==
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | {|class="experiments"
| |
| - | |1mM||ML||MS
| |
| - | |-
| |
| - | |OD600||1.187||1.427
| |
| - | |}
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | ML and MS were cultured and OD600 was measured at 4h, 4.5h, 5h, 5.5h and 6h in the medium without casamino acids. The samples were collected at 5h and 5.5h.
| |
| - | {|class="experiments"
| |
| - | |1mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |4h||0.127||0.11||0.107||0.168||0.169||0.208
| |
| - | |-
| |
| - | |4.5h||0.161||0.153||0.153||0.255||0.254||0.275
| |
| - | |-
| |
| - | |5h||0.220||0.206||0.209||0.366||0.353||0.383
| |
| - | |-
| |
| - | |5.5h||0.349||0.324||0.293||0.479||0.483||0.501
| |
| - | |-
| |
| - | |6h||0.439||0.431||0.384||0.628||0.651||0.645
| |
| - | |}
| |
| - | [[image:KyotoExp100924-1.png]]
| |
| - | ===Measure the fluorescence of GFP of the ML and the MS===
| |
| - | {|class="experiments"
| |
| - | |0mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |3h||'''292'''||'''263'''||'''88'''||'''6086'''||'''3007'''||'''6443'''
| |
| - | |-
| |
| - | |3.5h||'''131'''||'''93'''||'''-45'''||'''1700'''||'''1676'''||'''1652'''
| |
| - | |-
| |
| - | |0.01mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |3h||124||'''42'''||378||3425||6640||5990
| |
| - | |-
| |
| - | |3.5h||330||'''-41'''||632||5830||10515||13601
| |
| - | |-
| |
| - | |0.03mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |5h||48848||23900||51150||'''38837'''||13416||33454
| |
| - | |-
| |
| - | |5.5h||54980||54980||54980||'''35758'''||34595||34443
| |
| - | |-
| |
| - | |0.1mM<br>20 September</br>||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |3h||'''31'''||'''3327'''||'''2219'''||'''3331'''||'''2298'''||'''3766'''
| |
| - | |-
| |
| - | |3.5h||'''61'''||'''5341'''||'''3489'''||'''4840'''||'''3955'''||'''4512'''
| |
| - | |-
| |
| - | |0.1mM<br>23 September</br>||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C
| |
| - | |-
| |
| - | |5h||72902||68382||78054||56365||42563||57917
| |
| - | |-
| |
| - | |5.5h||98103||84737||81525||89239||91295||68302
| |
| - | |}
| |
| - | Back corrected
| |
| - | We can't use the data of IPTG 0mM to calculate RPU because GFP fluorescence at 3.5h is lower than that at 3h. Also we can't use the data of ML-B 0.01mM IPTG and the data of MS-A of 0.03mM. Although GFP fluorescence of 0.1mM (20 September) at 3.5h is higher than that at 3h, OD600 is so low. Accordingly, We can't use the data of 0.1mM (20 September).
| |
| - | ===Overnight culture for the measurement of lactose promoter===
| |
| - | Three tubes of the ML, three tubes of the MS and three tubes of the MB were grown with casamino acids including 0.1mM ITPG and 0.01mM IPTG for 16hours.
| |
| - |
| |
| - |
| |
| - | ==Saturday, 25 September <span class="by"> By: Hitoshi, Tomonori </span>==
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | {|class="experiments"
| |
| - | |0.1mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||1.961||1.823||2.078||2.241||2.383||1.857||2.674||2.674||2.632
| |
| - | |-
| |
| - | |0.01mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||1.754||1.868||1.702||1.885||2.009||1.940||2.693||2.631||2.622
| |
| - | |}
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | Overnight culture was diluted in the fresh medium and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h. The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.
| |
| - | {|class="experiments"
| |
| - | |0.1mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |2h||0.186||0.184||0.217||0.191||0.208||0.204||0.347||0.298||0.311
| |
| - | |-
| |
| - | |2.5h||0.289||0.302||0.334||0.289||0.313||0.315||0.624||0.542||0.567
| |
| - | |-
| |
| - | |3h||0.408||0.44||0.441||0.432||0.426||0.406||0.947||0.843||0.858
| |
| - | |-
| |
| - | |3.5h||0.519||0.584||0.602||0.558||0.6||0.539||1.33||1.21||1.25
| |
| - | |-
| |
| - | |4h||0.784||0.805||0.893||0.777||0.848||0.743||1.655||1.561||1.582
| |
| - | |}
| |
| - | [[image:KyotoExp1000925-1.png]]
| |
| - | ===Overnight culture for the measurement of lactose promoter===
| |
| - | Three tubes of the ML, three tubes of the MS and three tubes of the MB were grown with casamino acids including 0mM ITPG.
| |
| - |
| |
| - |
| |
| - | ==Sunday, 26 September <span class="by"> By: Tomonori </span>==
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | {|class="experiments"
| |
| - | |0mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||1.646||1.576||1.643||1.702||1.832||1.826||2.272||2.090||1.985
| |
| - | |}
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | Overnight culture was diluted in the fresh medium and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h. The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.
| |
| - | {|class="experiments"
| |
| - | |0mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |2h||0.289||0.195||0.247||0.213||0.237||0.243||0.289||0.265||0.229
| |
| - | |-
| |
| - | |2.5h||0.365||0.317||0.307||0.278||0.316||0.316||0.387||0.358||0.428
| |
| - | |-
| |
| - | |3h||0.385||0.295||0.31||0.344||0.349||0.332||0.651||0.578||0.642
| |
| - | |-
| |
| - | |3.5h||0.456||0.519||0.493||0.453||0.464||0.49||0.859||0.885||0.883
| |
| - | |-
| |
| - | |4h||0.65||0.659||0.645||0.567||0.565||0.642||1.215||1.189||1.199
| |
| - | |}
| |
| - | [[image:KyotoExp1000926-1.png]]
| |
| - | ===Overnight culture for the measurement of lactose promoter===
| |
| - | Three tubes of the ML, three tubes of the MS and three tubes of the MB were grown with casamino acids including 0.03mM ITPG.
| |
| - |
| |
| - |
| |
| - | ==Monday, 27 September <span class="by"> By: Tomonori, Tasuku, Ken </span>==
| |
| - | ===Measure OD600 of the overnight culture===
| |
| - | {|class="exepriments"
| |
| - | |0.03mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |OD600||1.646||1.576||1.643||1.702||1.832||1.826||2.272||2.090||1.985
| |
| - | |}
| |
| - | ===Culture for measurement of RPU of the lactose promoter===
| |
| - | Overnight culture was diluted in the fresh medium and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h. The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.
| |
| - | {|class="experiments"
| |
| - | |0.03mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |2h||0.214||0.239||0.218||0.238||0.232||0.217||0.376||0.383||0.315
| |
| - | |-
| |
| - | |2.5h||0.343||0.307||0.329||0.318||0.258||0.316||0.583||0.541||0.554
| |
| - | |-
| |
| - | |3h||0.43||0.373||0.389||0.39||0.358||0.406||0.773||0.803||0.746
| |
| - | |-
| |
| - | |3.5h||0.509||0.446||0.538||0.46||0.488||0.548||1.148||1.114||1.054
| |
| - | |-
| |
| - | |4h||0.637||0.635||0.709||0.626||0.616||0.74||1.524||1.479||1.385
| |
| - | |}
| |
| - | [[image:KyotoExp100927-1.png]]
| |
| - | ===Measure the GFP fluorescence===
| |
| - | {|class="experiments"
| |
| - | |0mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |3h||589||508||417||43120||40563||50343||'''-368'''||230||255
| |
| - | |-
| |
| - | |3.5h||704||938||868||58066||66566||65910||'''346'''||765||508
| |
| - | |-
| |
| - | |0.01mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |3h||1187||1316||1329||59806||69376||62702||360||396||439
| |
| - | |-
| |
| - | |3.5h||1883||1707||1507||96634||93095||88262||583||559||588
| |
| - | |-
| |
| - | |0.03mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |3h||2341||884||'''2575'''||'''34995'''||'''33056'''||'''14529'''||273||171||'''268'''
| |
| - | |-
| |
| - | |3.5h||3219||3124||'''1243'''||'''18855'''||'''20536'''||'''14040'''||416||230||'''209'''
| |
| - | |-
| |
| - | |0.1mM||ML-A||ML-B||ML-C||MS-A||MS-B||MS-C||MB-A||MB-B||MB-C
| |
| - | |-
| |
| - | |3h||17202||20387||25090||61144||'''13667'''||54117||313||'''401'''||388
| |
| - | |-
| |
| - | |3.5h||22040||25372||32237||87691||'''89368'''||76786||613||'''338'''||476
| |
| - | |}
| |
| - | Back corrected
| |
| - | We did not use the data in grey cells of the above table.
| |
| - | {|class="experiments"
| |
| - | |RPU||ML-A||ML-B||ML-C||average||stdev||CV
| |
| - | |-
| |
| - | |0mM||'''-0.00062'''||0.0122||0.0147||0.0134||0.00821||0.611
| |
| - | |-
| |
| - | |0.01mM||0.0191||0.00963||0.00244||0.0144||0.00838||0.583
| |
| - | |-
| |
| - | |0.1mM||0.205||0.191||0.269||0.221||0.0415||0.188
| |
| - | |}
| |
| - | We did not use the data in grey cells of the above table.
| |
| - | CV of RPU of 0mM IPTG and that of 0.01mM IPTG is high. In addition, the supplemented M9 medium used in the experiment includes 0.01mM lactose. The RPU of the lactose promoter induced by IPTG only must be lower than the data resulted from this experiment.
| |
| - | ----
| |
"
