Team:Kyoto/Notebook1

From 2010.igem.org

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(→=Restriction Digestion of LTC)
(→Ligation and Transformation of LTCS)
 
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Line 1,495: Line 1,495:
====Tuesday, September 7 <span class="by">By: Wataru, Ken</span>====
====Tuesday, September 7 <span class="by">By: Wataru, Ken</span>====
=====Insert Check=====
=====Insert Check=====
 +
[[Image:KyotoExp100907-1.png|300px|right]]
We did colony PCR, and three colonies were inserted rrS<sub>&Delta;TMD1</sub>-E0840. So we succeeded in making J23105 (RPU0.3)-rrS<sub>&Delta;TMD1</sub>-E0840.
We did colony PCR, and three colonies were inserted rrS<sub>&Delta;TMD1</sub>-E0840. So we succeeded in making J23105 (RPU0.3)-rrS<sub>&Delta;TMD1</sub>-E0840.
Line 1,658: Line 1,659:
The propagation wasn't observed on some samples on CTLS cultured with M9-Kan +IPTG. It meant that the construction of CTLS was in success.
The propagation wasn't observed on some samples on CTLS cultured with M9-Kan +IPTG. It meant that the construction of CTLS was in success.
However, the growth was observed on all of samples of LTCS. So, we decided to retry the construction of LTCS.
However, the growth was observed on all of samples of LTCS. So, we decided to retry the construction of LTCS.
-
=====Restriction Digestion of LTC=====
 
-
After the restriction digestion, we did the gel extraction.
 
-
=====Ligation and Transformaiton of LTCS=====
 
=====Miniprep=====
=====Miniprep=====
{| class="experiments"
{| class="experiments"
Line 1,675: Line 1,673:
====Thursday, October 7 <span class="by">By: Ken</span>====
====Thursday, October 7 <span class="by">By: Ken</span>====
-
===== =====
+
=====Miniprep=====
 +
{| class="experiments"
 +
!Name||Concentration
 +
|-
 +
|S<sub>&Delta;TMD1</sub>||84.1 ng/&micro;L
 +
|-
 +
|lac-SRRz||172.5
 +
|}
 +
=====Restriction Digestion=====
 +
{| class="experiments"
 +
!Name||Template||10xbuffer||100xbuffer||EcoRI||PstI||Water||Total
 +
|-
 +
|LT||4||4||0.4||0.4||0.4||30.8||40
 +
|-
 +
|CT||6||4||0.4||0.4||0.4||28.8||40
 +
|-
 +
|CTL||11||4||0.4||0.4||0.4||23.8||40
 +
|-
 +
|LTC||4||4||0.4||0.4||0.4||30.8||40
 +
|-
 +
|SRRz||7||4||0.4||0.4||0.4||27.8||40
 +
|-
 +
|S<sub>&Delta;TMD1</sub>||17||4||0.4||0.4||0.4||17.8||40
 +
|}
 +
=====Concentration after purification=====
 +
{| class="experiments"
 +
!Name||Concentration
 +
|-
 +
|LT||107.9 ng/&micro;L
 +
|-
 +
|CT||56.9
 +
|-
 +
|CTL||37.1
 +
|-
 +
|LTC||101.9
 +
|-
 +
|SRRz||55.7
 +
|-
 +
|S<sub>&Delta;TMD1</sub>||102.9
 +
|}
====Friday, October 8 <span class="by">By: Ken</span>====
====Friday, October 8 <span class="by">By: Ken</span>====
=====Screening=====
=====Screening=====
Screened lac-SRRz 1-7(LB-Tc with IPTG 1.5mM). Except for sample 4, the propagation could be observed.  So, we decided to use sample 4.
Screened lac-SRRz 1-7(LB-Tc with IPTG 1.5mM). Except for sample 4, the propagation could be observed.  So, we decided to use sample 4.
 +
=====Restriction Digestion of pSB1C3=====
 +
{| class="experiments"
 +
!Template||10xbuffer||100xbuffer||EcoRI||PstI||Water||Total
 +
|-
 +
|5||4||0.4||0.4||0.4||28.4
 +
|}
 +
=====Concentration after purification=====
 +
{| class="experiments"
 +
!Name||Concentration
 +
|-
 +
|pSB1C3(E-P)||16.1 ng/&micro;L
 +
|}
=====Ligation and Transformation=====
=====Ligation and Transformation=====
Inserted LTC, CTL, T, SRRz, LT or CT to pSB1C3.
Inserted LTC, CTL, T, SRRz, LT or CT to pSB1C3.
Line 1,697: Line 1,746:
|CT||Some colonies
|CT||Some colonies
|}
|}
-
 
====Saturday, October 9 <span class="by">By: Ken</span>====
====Saturday, October 9 <span class="by">By: Ken</span>====
=====Making culture=====
=====Making culture=====
Cultured LTC, T, SRRz, LT, and CT with LB (Cp/Amp+).
Cultured LTC, T, SRRz, LT, and CT with LB (Cp/Amp+).
-
 
+
=====Restriction digestion of LTC and SRRz-low=====
 +
{| class="experiments"
 +
!Name||Template||10xbuffer||100xbuffer||EcoRI||SpeI||Water||Total
 +
|-
 +
|LTC||3.5||4||0.4||0.6||0.6||30.9||40
 +
|-
 +
|SRRz-low||4||4||0.4||0.6||0.6||30.4||40
 +
|}
 +
=====Ligation and Transformation of LTCS=====
 +
{| class="experiments"
 +
!Name||Colony
 +
|-
 +
|LTCS||Many colonies
 +
|-
 +
|control||few colonies
 +
|}
 +
Used Top 10.
====Sunday, October 10 <span class="by">By: Ken</span>====
====Sunday, October 10 <span class="by">By: Ken</span>====
Line 1,732: Line 1,796:
|}
|}
=====Electrophoreis=====
=====Electrophoreis=====
 +
[[Image:KyotoExp101010-1.png|300px|right]]
* Lane 1, 8: Lambda, 100bp Marker
* Lane 1, 8: Lambda, 100bp Marker
* Lane 2-7: LTC, T, SRRz, LT, CT, CTLS
* Lane 2-7: LTC, T, SRRz, LT, CT, CTLS
Line 1,762: Line 1,827:
# ctctagtattattgatttctaccatcttctactcc
# ctctagtattattgatttctaccatcttctactcc
-
=====Isolation of R0011-SRRz (4)=====
+
 
====Monday, October 11 <span class="by">By: Ken</span>====
====Monday, October 11 <span class="by">By: Ken</span>====
Line 1,772: Line 1,837:
|}
|}
-
{| class="experiments"
+
[[Image:KyotoExp101011-1.png|300px|right]]
 +
{| class="electrophoresis"
!Lane||Name
!Lane||Name
|-
|-
Line 1,793: Line 1,859:
|}
|}
 +
{| class="experiments"
 +
!Name||Colony
 +
|-
 +
|S<sub>&Delta;TMD1</sub>-dT||Many colonies
 +
|-
 +
|S<sub>&Delta;TMD1</sub>||Many colonies
 +
|}
*Competent Cell: TOP10
*Competent Cell: TOP10
Line 1,829: Line 1,902:
====Tuesday, October 12 <span class="by">By: Ken</span>====
====Tuesday, October 12 <span class="by">By: Ken</span>====
-
===== =====
+
=====Screening of CTL=====
-
 
+
The propagation could not be observed with LB-Amp and could be observed with LB-Cp. It meant that the part, CTL, was correctly inserted to the plasmid, pSB1C3.
 +
=====Making culture=====
 +
Cultured two samples on each part: S<sub>&Delta;TMD1</sub>-dT and S<sub>&Delta;TMD1</sub>. Named T1,3,4 and 6.
====Wednesday, October 13 <span class="by">By: Ken</span>====
====Wednesday, October 13 <span class="by">By: Ken</span>====
Line 1,837: Line 1,912:
====Thursday, October 14 <span class="by">By: Ken</span>====
====Thursday, October 14 <span class="by">By: Ken</span>====
-
===== =====
+
=====Retry of the construction of CTLS=====
-
 
+
====Friday, October 15 <span class="by">By: Ken</span>====
====Friday, October 15 <span class="by">By: Ken</span>====
Line 1,847: Line 1,921:
We did the sequencing on ML, MS, CTLS, T4 and CTL. The sequencing of T4 and CTL were in success and the others were failed. The results of CTL was desirable, but that of T4 was bad sequence and there was a mutation on PstI site. Including the last time sequencing, we could say that the deletion of GFP-terminator from S<sub>&Delta;TMD1</sub>-GFP was failed. So, we decided to retry the deletion PCR.
We did the sequencing on ML, MS, CTLS, T4 and CTL. The sequencing of T4 and CTL were in success and the others were failed. The results of CTL was desirable, but that of T4 was bad sequence and there was a mutation on PstI site. Including the last time sequencing, we could say that the deletion of GFP-terminator from S<sub>&Delta;TMD1</sub>-GFP was failed. So, we decided to retry the deletion PCR.
-
====Saturday, October 16 <span class="by">By: Ken</span>====
 
-
===== =====
 
Line 1,871: Line 1,943:
=====Electrophoresis=====
=====Electrophoresis=====
 +
[[Image:KyotoExp101017-1.png|300px|right]]
*Lane 1,2,5,6: Lambda, 100bp Maker
*Lane 1,2,5,6: Lambda, 100bp Maker
*Lane 3,4 PCR products[T'1,T'2]
*Lane 3,4 PCR products[T'1,T'2]
Line 1,939: Line 2,012:
=====Electrophoresis=====
=====Electrophoresis=====
 +
[[Image:KyotoExp101023-1.png|300px|right]]
*Lane 1,8,2,7: Lambda, 100bp Maker
*Lane 1,8,2,7: Lambda, 100bp Maker
*Lane3,4: lac-S<sub>&Delta;TMD1</sub>RRz (1,2)
*Lane3,4: lac-S<sub>&Delta;TMD1</sub>RRz (1,2)
Line 2,004: Line 2,078:
=====Electrophoresis=====
=====Electrophoresis=====
 +
[[Image:KyotoExp101025-1.png|300px|right]]
*Lane 1,8,2,7: Lambda, 100bp Maker
*Lane 1,8,2,7: Lambda, 100bp Maker
*Lane 3,4: C'TLS(1,2)
*Lane 3,4: C'TLS(1,2)

Latest revision as of 02:17, 28 October 2010

Contents

Notebook1

Construction

Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

Transformation
NameWellSampleCompetent CellsTotalPlateIncubationColony
J231001-18-C1 µL2021LB (Amp+)At 37℃, 7/20 20:50 - 7/21 17:00○
J231051-18-M12021○
J231161-20-M12021○
R00111-6-G12021○
E08401-12-O12021○
J067022-8-E12021○
pSB4K51-5-G12021×
B00151-23-L12021LB (Kan+)×

A vector of pSB4K5 is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture B0015 despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of pSB4K5 and B0015.


Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.

Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate
Transformation
NameWellSampleCompetent CellsTotalPlateIncubationColony
pSB4K51-5-G1 µL2021LB (Kan+)At 37℃, 7/21 20:50 - 7/22 16:30○
B00151-23-L12021○
PCR for SRRz and S
No.WaterMgSO4dNTPs10xBufferTemplate DNAPrimer Fwd.Primer Rev. (SRRz)Primer Rev. (S)KOD Plus ver.2Total
128 µL35551.51.5-150
22835551.51.5-150
32835551.5-1.5150
42835551.5-1.5150
52835551.51.5-150
62835551.51.5-150
72835551.5-1.5150
82835551.5-1.5150
94℃2min
98℃10s30 cycles
55℃30s
68℃4min
4℃forever


Thursday, July 22 By: Wataru

Electrophoresis (40min) of the PCR Products
KyotoExp100722-1.png
  • Sample: 1, 2, 5, 6 = SRRz, 3, 4, 7, 8 = S
  • Marker: 100bp, 1kb, 1kb, 100bp.
Miniprep
NameConcentration
J2310018.5 (ng/µL)
J2310512.5
J2311614.6
R00118.6
E084012.1
J0670214.7

The concentration of all samples was very week. Probably our shaking incubation was week.

Culture from 07/22 17:00 to 07/23 10:00 and Making Master Plates of pSB4K5 and B0015

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep
NameConcentration
pSB4K579.2 (ng/µL)
B0015-

We lost B0015 by our mistake. The concentration of pSB4K5 is high, so this condition of shaking incubation is moderate.

PCR Purification
No.NameConcentrationNew Name
1SRRz18.6 ng/µL-
3S77.6SSam7(1)
5SRRz33.6-
7S65.4SSam7(2)

The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.

PCR for SRRz
No.WaterMgSO4dNTPs10xBufferTemplate DNAPrimer Fwd. (SRRz)Primer Rev. (SRRz)KOD plus ver.2Total
128 µL35551.51.5150
22835551.51.5150
326.54.55551.51.5150
426.54.55551.51.5150
52565551.51.5150
62565551.51.5150
94℃2min
98℃10s30 cycles
55℃30s
68℃4min
4℃forever
Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme
No.NameSample10xBufferBSAEnzymeMilliQTotalIncubation
1J067025 µL10.1EcoRI0.13.610At 37℃ 7/23 18:00 - 7/23 18:30
2J06702510.1XbaI0.13.610
3J06702510.1SpeI0.13.610
4J06702510.1PstI0.13.610
5J06702510.1-3.710
KyotoExp100723-1.png

Marker: 1kb. Comparison to No. 5 (control, circular DNA), the bands of No. 1, 2, 3, and 4 was shifted. The DNA of them was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

Restriction Digestion and Ligation to insert S gene to E0840
NameSample10xBufferEnzyme 1Enzyme 2MilliQTotalIncubation
SSam7(1)11 µL5EcoRI0.2SpeI0.233.650At 37℃ for 2h
SSam7(2)115EcoRI0.2SpeI0.233.650
E0840455EcoRI0.2XbaI0.2050

After PCR Purification, evaporated them and diluted 3µL.

NameVectorInsertLigation HighTotal
SSam7(1)-E0840E08400.5µLSSam7(1)0.512
SSam7(2)-E0840E08400.5SSam7(2)0.512


Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products
KyotoExp100726-1.png
No.NameLength(bp)Result
1SRRz1386
2SRRz1386
3SRRz1386
4SRRz1386
5SRRz1386
6SRRz1386

Marker: 1kb. At the condition 4 (4.5µL MgSO4) and 6 (6µL MgSO4), SRRz is amplified very much. So we decided to use them.

PCR Purification
No.NameConcentrationNew Name
4SRRZ51.6 ng/µLSRRzSam7(1)
5SRRZ59.3
6SRRZ59.6SRRzSam7(2)
Transformation
NameWellSampleCompetent CellTotalPlateIncubationColony
E02401-12-M1 µL2021LB (Amp+)At 37℃ 7/26 - 7/27×
I202602-17-F12021LB (Kan+)×
J044501-5-E12021×
Culture of pSB4K5, E0840, and B0015

Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi

Colony PCR of SSam7-E0840 (Electrophoresis for 35min)
KyotoExp100727-1.png
No.NameLengthResult
1SSam7(1)-E08401522○
2SSam7(1)-E08401522×
3SSam7(1)-E08401522○
4SSam7(1)-E08401522×
5SSam7(1)-E08401522○
6SSam7(1)-E08401522◎ (Use as SSam7(1)-E0840)
7SSam7(2)-E08401522×
8SSam7(2)-E08401522×
9SSam7(2)-E08401522×
10SSam7(2)-E08401522×
11SSam7(2)-E08401522◎ (Use as SSam7(2)-E0840)
12SSam7(2)-E08401522○
13SSam7(2)-E08401522○
+E08401116
-None

Marker: 1kb, 100bp

Miniprep
NameConcentration
R001126.9 ng/µL
B0015120.0
E0840120.1
Restriction Digestion
NameSample2 bufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
B001530 µL50.5EcoRI0.4XbaI0.313.750At 37℃ 16:45 - 18:00
SRRzSam7(1)4050.5EcoRI0.4SpeI0.43.850
SRRzSam7(2)4050.5EcoRI0.4SpeI0.43.850
Ligation
Transformation
NameSampleCompetent CellsTotalPlateIncubationColony
SRRzSam7(1)-B0015 ○
SRRzSam7(2)-B0015 ○


Wednesday, July 28 By:

Miniprep
NameConcentration
SSam7(1)-E084095.5 ng/µL
SSam7(2)-E084098.6

Diluted SSam7(1)-E0840 and SSam7(2)-E0840 20 times with water, and used as template DNA.

Deletion PCR to delete a functional domain of S gene
WaterMgSO4dNTPs10xBufferPrimer Fwd.Primer Rev.Template (1)Template (2)KOD Plus ver.2Total
SSam7,ΔTMD1(1)-E0840 (1)28 µL3551.51.55-150
SSam7,ΔTMD1(1)-E0840 (2)283551.51.55-150
SSam7,ΔTMD1(2)-E0840 (1)283551.51.5-5150
SSam7,ΔTMD1(2)-E0840 (2)283551.51.5-5150
94℃2min
98℃10s35 cycles
55℃30s
68℃4min
4℃forever
Restriction Digestion to check the function of DpnI
NameSamplefast digestion bufferDpnIMilliQTotal
SSam7(1)-E08403 µL10.15.810
SSam7(2)-E0840310.15.810
Electrophoresis for 35min
KyotoExp100728-1.png
No.NameLengthResult
1Not digested SSam7(1)-E08403363bp
2Not digested SSam7(2)-E08403363
3Digested SSam7(1)-E08401021, 933, 402, 341, 258, 105, ...
4Digested SSam7(2)-E08401021, 933, 402, 341, 258, 105, ...

Marker: 1kb, 100bp DpnI works correctly.


Thursday, July 29 By:

Restriction Digestion
NameSample volumeFastdigestion BufferEnzyme 1MilliQTotalIncubation
SSam7,ΔTMD1(1)-E0840 (1)50 µL6DpnI0.23.86007/29 09:40 - 07/29 11:00
SSam7,ΔTMD1(2)-E0840 (1)506DpnI0.23.860
Ligation and Phosphorylation
NameSampleMilliQLigation HighT4 KinaseTotalIncubation
SSam7,ΔTMD1(1)-E0840 (1)2 µL7511507/29 11:30 ~ 07/29 13:00
SSam7,ΔTMD1(2)-E0840 (1)275115
Transformation
NameSample VolumeCompetent CellTotalPlateIncubationColony
SSam7,ΔTMD1(1)-E0840 (1)3 µL3033LB (Amp+)07/29 ~ 07/30○
SSam7,ΔTMD1(2)-E0840 (1)33033○


Monday, August 2 By: Wataru, Ken

Miniprep
NameConcentration
SSam7,ΔTMD1-E0840 (1)52.7 ng/µL
SSam7,ΔTMD1-E0840 (2)54.4
SSam7,ΔTMD1-E0840 (3)89.5
pSB4K550.7
R001118.6
PCR of E0240

E0240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.

NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate E0240KOD Pllus ver.2Total
E0240(1)28 µL3551.51.55150
E0240(2)283551.51.55150
94℃2min
98℃10s35 cycles
55℃30s
68℃4min
4℃forever
Electrophoresis
PCR Purification
NameConcentration
E0240(1)42.6 ng/µL
E0240(2)55.3
Restriction Digestion for inserting E0240 to pSB4K5 by 3A assembly
NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
E0240(1) [XP]30 µL50.5XbaI0.2PstI0.214.150
E0240(2) [XP]3050.5XbaI0.2PstI0.214.150
PCR Purification
NameConcentrationVolume
E0240(1) [XP]21.8 ng/µL40 µL
E0240(2) [XP]32.445

Stored at -20℃.

Error PCR
NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate (1)Template (2)Template (3)KOD Pllus ver.2Total
SSam7,ΔTMD1-E0840 (1)32 µL3551.51.51--150
SSam7,ΔTMD1-E0840 (2)323551.51.5-1-150
SSam7,ΔTMD1-E0840 (3)323551.51.5--1150
94℃2min
98℃10s20 cycles
68℃4min
4℃forever
Restriction Digestion of SSam7,ΔTMD1-E0840 by DpnI
Transformation
NameSampleCompetent CellsTotalPlateIncubationColony
SSam7,ΔTMD1-E0840 (1)2 µL2022--○
SSam7,ΔTMD1-E0840 (2)22022×
SSam7,ΔTMD1-E0840 (3)22022○

Tuesday, August 3 By:

Culture

Picked two colonies from SSam7,ΔTMD1-E0840 (1), and SSam7,ΔTMD1-E0840 (3), and cultured at 37℃ from 08/03 to 08/04.

Miniprep
NameConcentration
pSB4K560.7 ng/µL
R001126.8
Restriction Digestion
NameSample2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
R0011 [ES]50 µL60.6EcoRI0.2SpeI0.2360
pSB4K5 [EP]5060.6EcoRI0.2PstI0.2360
E0240(1) [XP]5060.6XbaI0.2PstI0.2360
E0240(2) [XP]5060.6XbaI0.2PstI0.2360
PCR Purification
NameConcentration
pSB4K5 [EP]39.5 ng/µL
E0240(1) [XP]21.8
E0240(2) [XP]32.4

pSB4K5 [EP] is concentrated 10µL and E0240(1) [XP], E0240(2) [XP] are concentrated 1µL.

Ethanol Precipitation

After ethanol precipitation, we diluted pSB4K5 by 2µL MilliQ

Ligation
NameVectorInsert 1Insert 2Ligation HighT4 KinaseTotalIncubation
ML (1)pSB4K5 [EP]1R0011 [ES]1E0240(1) [XP]131517:30 - 20:20
ML (2)pSB4K5 [EP]1R0011 [ES]1E0240(2) [XP]1315
PCR of I20260
NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate I20260KOD plus ver.2Total
I20260 (1)32µL3551.51.51150
I20260 (2)323551.51.5-150
94℃2min
98℃10s30 cycles
55℃30s
68℃4min
4℃forever
PCR Purification
NameConcentration
I2026040.6 ng/µL
Restriction Digestion
NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
I20260 [EP]45 µL60.6EcoRI0.2PstI0.2860
PCR Purification
NameConcentrationVolume
I20260 [EP]74.1 ng/µL30

I20260 [EP] is concentrated at 7µL

Ligation
VectorInsertLigation HighTotalIncubation
MSpSB4K5 [EP]1I20260 [EP]12420:00-20:30
Transformation
NameSampleCompetent CellTotalPlateIncubationColony
ML (1)1 µL2021LB (Kan+)08/03-08/04○
ML (2)12021○
MS12021○


Thursday, August 5 By:

Culture and Master Plates

pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red. So, white colony is correctly inserted parts.

However, white colonies and green colonies are observed in ML (1) and ML (2) plate. We cultured both white and green colonies.

In MS, Many of colonies are red, but green colonies are observed. We cultured green colonies.

NameColorIncubation
ML (1-1)Green Colony8/5-8/6
ML (1-2)Green Colony
ML (1-3)White Colony
ML (1-4)White Colony
ML (2-1)Green Colony
ML (2-2)White Colony
ML (2-3)White Colony
ML (2-4)White Colony
MS (1)Green Colony
MS (2)Green Colony
MS (3)Green Colony
Sequence
NameConcentration
SΔTMD1-E0840(1) A28.9 ng/µL
SΔTMD1-E0840(1) B25.3
SΔTMD1-E0840(3) A26.6
SΔTMD1-E0840(3) B24.0

As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.


Friday, August 6

Miniprep
Name
ML (1-1)
ML (1-2)
ML (1-3)
ML (1-4)
ML (2-1)
ML (2-2)
ML (2-3)
ML (2-4)
MS (1)
MS (2)
MS (3)
Restriction Digestion
NameSample2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
ML (1-1) [EP]50 µL60.6EcoRI0.3PstI0.32.860
ML (1-2) [EP]5060.6EcoRI0.3PstI0.32.860
ML (1-3) [EP]5060.6EcoRI0.3PstI0.32.860
ML (1-4) [EP]5060.6EcoRI0.3PstI0.32.860
ML (2-1) [EP]5060.6EcoRI0.3PstI0.32.860
ML (2-2) [EP]5060.6EcoRI0.3PstI0.32.860
ML (2-3) [EP]5060.6EcoRI0.3PstI0.32.860
ML (2-4) [EP]5060.6EcoRI0.3PstI0.32.860
MS (1) [EP]5060.6EcoRI0.3PstI0.32.860
MS (2) [EP]5060.6EcoRI0.3PstI0.32.860
MS (3) [EP]5060.6EcoRI0.3PstI0.32.860
Electrophoresis
KyotoExp100806-1.png
No.NameLengthResults
1MS (1) [EP]960, 4339
2MS (2) [EP]960, 4339
3MS (3) [EP]960, 4339
4ML (1-1) [EP]980 3378○
5ML (1-2) [EP]980 3378○
6ML (1-3) [EP]980 3378×
7ML (1-4) [EP]980 3378×
8ML (2-1) [EP]980 3378○
9ML (2-2) [EP]980 3378×
10ML (2-3) [EP]980 3378×
11ML (2-4) [EP]980 3378×
12MS (1) [EP]960, 4339○
13MS (2) [EP]960, 4339○

White colonies are not inserted R0011 but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of lacI gene.

Error PCR (Retry)
NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate SSam7,ΔTMD1-E0840 failed (50ng/µL)KOD plus ver.2Total
SSam7,ΔTMD1-E0840 (1)323551.51.51150
SSam7,ΔTMD1-E0840 (2)323551.51.51150
94℃2min
98℃10s25 cycles
68℃4min
Add DpnI 2µL
Incubate1h
4℃forever
Transformation
NameWellSampleCompetent CellTotalPlateIncubationColony
SSam7,ΔTMD1-E0840 (1)-4 µL5054LB (Kan+)08/06-08/09○
SSam7,ΔTMD1-E0840 (2)-45054○
I202602-17-F25052○
2-I-525052LB (Amp+)○


Monday, August 9 By: Wataru, Tomonori, Ken, Takuya

Miniprep
Nameconcentration
MS116.2 ng/µL
ML146.6
Transfotrmation
SampleSampleCompetent CellTotalPlateIncuvationResults
MS2 µLKRX5052LB (Kan+)08/09 18:00-08/10 12:00○
ML2KRX5052○
Restriction Eigestion and Ethanol Precipitation

To use R0011 for next ligation, we digested it by EcoRI and PstI

NameSample10x BufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
R0011 [EP]5060.6EcoRI0.5PstI0.52.460At 37℃ 08/09 16:20-18:20

After restriction enzyme digestion, we did ethanol precipitation.

Ligation and Transformation
NameSampleCompetent cellTotalPlateIncuvationColony
R0011 [pSB4K5, KRX]2 µLKRX5052LB (Kan+)08/09 20:00-08/10 09:00○
R0011 [pSB4K5</partrinfo>, C2]2C25052○


Tuesday, August 10 By: Wataru, Tomonori, Ken, Fumitaka

Culture

Cultured I20260 [pSB4K5, ML, R0011 [pSB4K5, KRX], and R0011 [pSB4K5</partrinfo>, C2].

Minprep
NameConcentration
SSam7,ΔTMD1-E0840 (1-1)9.9 ng/µL
SSam7,ΔTMD1-E0840 (1-2)27.3
SSam7,ΔTMD1-E0840 (2-1)43.2
SSam7,ΔTMD1-E0840 (2-2)34.7
Culture and Master Plate

At 37℃ 08/09 18:00-08/10 9:00


Wednesday, August 11 By: Wataru, Naoi, Ken, Takuya

No.MediumCloudIncubation
1Kanamycin○At 37℃, 08/10 20:00-08/11 9:00
Ampicillin×
2Kanamycin○
Ampicillin○
3Kanamycin○
Ampicillin×
4Kanamycin○
Ampicillin×
5Kanamycin○
Ampicillin×
6Kanamycin○
Ampicillin○
7Kanamycin○
Ampicillin×

Discussion: About sample 1, 3, 4, 5 and 7, lac promoter was correctly inserted in low copy plasmid. About sample 2 and 6, low copy plasmid and vector derived from lac promoter were ligated. We decided to use sample 1 or 3.

Miniprep of R0011 [pSB4K5, C2], SRRz 1', 3'
NameConcentration
R0011 [pSB4K5, C2] (1)31.2 ng/µL
R0011 [pSB4K5, C2] (3)29.9
Restriction Digestion and electrophoresis of R0011 [pSB4K5, C2]
NameEcoRIPstI
10.2-
2-0.2
30.20.2
N--
KyotoExp100811-1.png
No.NameLengthResults
1R0011 [pSB4K5, C2] (1-1)
2R0011 [pSB4K5, C2] (1-2)
3R0011 [pSB4K5, C2] (1-3)
4R0011 [pSB4K5, C2] (1-N)
5R0011 [pSB4K5, C2] (2-1)
6R0011 [pSB4K5, C2] (2-2)
7R0011 [pSB4K5, C2] (2-3)
8R0011 [pSB4K5, C2] (2-N)

Each enzyme correctly cut samples.

Screening PCR of SRRz
KyotoExp100811-2.png
KyotoExp100811-3.png
No.NameResults
1None
2Control B0015
3Control J06702
4Control B0015
5-24SRRz-B0015×

Marker: Lambda Marker

Discussion: All of the sample were self-ligation of DT. SRRz weren't inserted.

Thursday, August 12 By: Wataru, Ken

Restriction Digestion and electrophoresis of B0015
NameTemplate10xbuffer100xbufferEcoRIXbaI 1XbaI 2SpeIPstI 1PstI 2WaterTotal
1310.10.2-----5.710
2310.1-0.2----5.710
3310.1--0.2---5.710
4310.1---0.2--5.710
5310.1----0.2-5.710
6310.1-----0.25.710
N310.1------5.910

Maker: Lambda, 100bp

KyotoExp100812-1.png

Discussion: Each enzyme correctly cut each sample and was active.


Thursday, August 19 By: Wataru, Tomo, Ken

Miniprep of SSam7,ΔTMD1-E0840
NameConcentration
SSam7,ΔTMD1-E084029.6 ng/µL
Point mutation PCR of SSam7,ΔTMD1-E0840
NameTemplate10xbufferdNTPsMgSO4Primer Fwd.Primer Rev.MilliQKOD plus ver.2Total
SΔTMD1-E0840 (1)1.55531.51.531.5150
SΔTMD1-E0840 (2)1.55531.51.531.5150
Control1.55531.51.532.5-50
94℃2min
98℃10s30cycles
55℃30s
68℃3.5min
4℃forever
Restriction Digestion by DpnI from 17:50 to 18:50
Electrophoresis
KyotoExp100819-1.png
Name
SΔTMD1-E0840 (1)
SΔTMD1-E0840 (2)
Control

Marker: Lambda, 100bp

Ligation and Transformation
NameColony
SΔTMD1-E0840 (1)○
SΔTMD1-E0840 (2)○
Control×


Friday, August 20 By: Wataru, Ken

Making Culture and Master Plate of SΔTMD1-E0840
Miniprep
NameConcentration
B001541.1 ng/µL
PCR of SRRz
Name10xBufferMgS04dNTPTemplatePrimer Fwd.Primer Rev.MilliQKOD plus ver.2Total
SRRzSam7 (1)5 µL355F11.51.528150
SRRzSam7 (2)5355F21.51.528150
SRRzSam7 (3)5355F11.51.528150
SRRzSam7 (4)5355F21.51.528150
SRRzSam7 (5)5355F11.51.528150
SRRzSam7 (6)5355F21.51.528150
94℃2min
98℃10s30cycles
55℃30s
68℃2min
4℃forever
Electrophoresis
KyotoExp100820-1.png
Name
SRRzSam7 (1)
SRRzSam7 (3)
SRRz Sam7(5)
SRRzSam7 (2)
SRRzSam7 (4)
SRRzSam7 (6)

Discussion: Primer F1 might be better than F2, because the bands of 1, 2 and 3 were clearer. We decided to use sample 1 and 3. Their bands were clearer in the three.

PCR Purification
NameConcentration
SRRzSam7 (1)134.0 ng/µL
SRRzSam7 (3)69.0
Restriction Digestion
NameSample10xBuffer100xBufferEcoRIXbaISpeIMilliQTotalIncubation
B0015 [EX]50 µL60.60.40.4-2.66017:45-18:45
SRRzSam7 (1) [EP]5060.60.4-0.42.660
SRRzSam7 (3) [EP]5060.60.4-0.42.660
Purification
NameConcentration
SRRzSam7 (1) [EP]109.0 ng/µL
SRRzSam7 (2) [EP]110.0
B001525.5
Ligation and Transformation

Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku

Miniprep
Sample numberConcentration
SΔTMD1-E0840 (1-1)58.9 ng/µL
SΔTMD1-E0840 (2-2)49.9
Sequence

Sample: SΔTMD1-E0840 (1-1). SΔTMD1-E0840 (2-2), MS Discussion: The sequencing was in success and the results were desirable. It meant the point mutation was succeeded and sequence of MS was confirmed. We decided to use SΔTMD1-E0840.

Screening PCR of SRRzSam7-B0015
90℃10min
94℃30s35cycles
50℃30s
72℃1.5min
72℃4min
4℃hold
KyotoExp100823-1.png
No.Name
1-13SRRzSam7-B0015
CControl: B0015
NNone

Marker: Lambda, 100bp

Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.

Deletion PCR of SΔTMD1-E0840 (2-2)
NameSample10xBufferdNTPsPrimer Fwd.Primer Rev.TemplateWaterKOD-plus-Total
rrSΔTMD1-E0840 (1)2 µL551.51.5135150
rrSΔTMD1-E0840 (2)2551.51.5135150
rrSΔTMD1-E0840 (Control)2551.51.5135-50
94℃2min
94℃10s35cycles
56℃30s
68℃3.5min
4℃forever
Restriction Digestion (DpnI)
SampleDpnITotalIncubation
25 µL12619:00-20:10
Ligation
NameSampleWaterLigation highT4 KinaseTotalIncubation
rrSΔTMD1-E0840 (1)3 µL6511520:15-21:15
rrSΔTMD1-E0840 (2)365115
rrSΔTMD1-E0840 (Control)365115
Transformation

Tuesday, August 24 By: Ken, Tomo, Tasuku, Takuya

Retry of deletion PCR of SΔTMD1-E0840
NameSample10xBufferdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-Total
rrSΔTMD1-E0840 (1)2 µL5531.51.5132150
rrSΔTMD1-E0840 (2)25531.51.5132150
Control25531.51.5132150
94℃2min
94℃10s35cycles
58℃30s
68℃3.5min
4℃hold
Restriction Digestion (DpnI)

14:15-15:15

Electrophoreis
KyotoExp100824-1.png
LaneName
1rrSΔTMD1-E0840 (1)
2rrSΔTMD1-E0840 (3)
CrrSΔTMD1-E0840 (Control)

Marker: 100bp, Lambda.

We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and Restriction Digestion were completed successfully.

Ligation
Point mutation of SRRz
Name10xdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-total
SRRzSam7-B0015 (1)5531.51.5132150
SRRzSam7-B0015 (2)5531.51.5132150
SRRzSam7-B0015 (Control)5531.51.5132150
94℃2min
98℃10s30cycles
55℃30s
68℃4min
4℃hold
Restriction Digestion (DpnI), Electrophoresis and Ligation
KyotoExp100824-2.png

We could find point mutation PCR and restriction enzyme of DpnI was done.

PCR of E0240
Sample10xBufferdNTPsMgSO4VF2VRTemplateWaterKOD-plus-Total
E0240 (1)5531.51.5131.5150
E0240 (2)5531.51.5131.5150
PCR Purification
NameConcentration
E0240 (1)5.5 x 50 ng/µL
E0240 (2)5.2 x 50
Restriction Digestion (EcoRI, PstI) and Gel Extraction
NameConcentration
E0240 (1) 28.8 ng/µL
E0240 (2) 26.4
Transformation

Wednesday, August 25 By:Ken, Tomo, Kazuya, Tasuku, Takuya

Making culture and Master plate
NameColony
rrSΔTMD1-E0840 (1)○
rrSΔTMD1-E0840 (2)○
rrSΔTMD1-E0840 (Control)×
SRRzSam7-B0015 (1)○
SRRzSam7-B0015 (2)○
SRRzSam7-B0015 (Control)×
Miniprep
NameConcentration
pSB4K529.0 ng/µL
Restriction Digestion
Sample nameTemplate10xbuffer100xbufferEcoRISpeIPstIWaterTotal
pSB4K55060.60.40.4-2.660
R0011 [pSB4K5]1040.4-0.30.32540
Purification
Sample NameConcentration
pSB4K518.4 ng/µL
R0011 [pSB4K5]8.6
Ligation of E0240 and pSB4K5, Transformation

Thursday, August 26 By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka

Miniprep
Sample nameConcentration
J23116 (RPU0.7)44.5 ng/µL
Restriction Digestion
NameTemplate10xbuffer100xbufferSpeIPstIWaterTotal
J23116 (RPU0.7)2540.40.30.31040
Purification of
NameConcentration
J23116 (RPU0.7)49.8 ng/µL


Friday, August 27 By: Ken, Tomo, Kazuya, Fumitaka

Making master plate of E0240 [pSB4K5]
Sample NameConcentration
rrSΔTMD1-E0840 (1-2)20.9 ng/µL
SRRz-B0015 (1-1)16.4
Restriction Digestion
NameTemplate10xbuffer100xbufferXbaIPstIWaterTotalIncubation
rrSΔTMD1-E0840 (1-2)45 µL60.60.30.37.86013:20-14:20
SRRz-B0015 (1-1)4560.60.30.37.860
Purification
rrSΔTMD1-E0840 (1-2)44.7 ng/µL
SRRz-B0015 (1-1)56.1
Ligation and Transformation
Name
R0011-rrSΔTMD1-E0840 (1-2)
J23116 (RPU0.7)- rrSΔTMD1-E0840 (1-2)
R0011-SRRz-B0015 (1-1) [pSB4K5]

Monday, August 30 By: Tomonori, Kazuya, Tasuku, Ken

Making culture and Master plate
NameColony
R0011-rrSΔTMD1-E0840Many colonies
R0011-rrSΔTMD1-E0840 (Control)Some colonies
J23116 (RPU0.7)- rrSΔTMD1-E0840Many colonies
J23116 (RPU0.7)- rrSΔTMD1-E0840 (Control)Many colonies
R0011-SRRz-B0015 (1-1) [pSB4K5]No colony
R0011-SRRz-B0015 (1-1) [pSB4K5] (Control)No colony

Discussion: There ware some colonies, which emitted green light, on the plate 1. So, we cultured those colonies on master plate. On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead. However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.


Tuesday, August 31 By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken

Miniprep
NameConcentration
J23105 (RPU0.3)48.5 ng/µL
R0011-rrSΔTMD1-E0840107.3
Restriction Digestion
Gel Extraction of R0011-rrSΔTMD1-E0840 (Electrophoresis for 45min)

Discussion: There were two band at the bottom of the gel. It was too long -45min-, and insert and vector might be contaminated. But we went on next operation.

Purification of J23105 (RPU0.3) and R0011-rrSΔTMD1-E0840
NameConcentration
J23105 (RPU0.3)5.8 ng/µL
R0011-rrSΔTMD1-E08407.8
Ligation and Transformation
InsertVector
R0011-rrSΔTMD1-E0840J23105 (RPU0.3)

Wednesday, September 1 By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken

Making culture and Master plate
NameColony
R0011-rrSΔTMD1-E0840-J23105 (RPU0.3)Many colonies
R0011-rrSΔTMD1-E0840-J23105 (RPU0.3) (Control)Many colonies
Screenig PCR of R0011-rrSΔTMD1-E0840-J23105 (RPU0.3)
  • Sample: 1-13
  • Control: Positive (B0015)
  • Maker: lambda, 100

Discussion: All of the sample except sample 10 might be self-ligation products of J23105 (RPU0.3).

Miniprep
SRRz-B0015 (1-1)33.8 ng/µL
pSB4K556.0
Restriction Digestion of SRRz and pSB4K5
NameTemplate10xbuffer100xbufferEcoRIPstIWaterTotalIncubation
SRRz-B0015 (1-1)20 µL40.40.30.3154013:25-14:30
pSB4K52040.40.30.31540
Purification
SRRz-B0015 (1-1)6.5 ng/µL
pSB4K516.8
Ligation and transformation
  • Insert: SRRz-B0015 (1-1)
  • Vector: pSB4K5

Thursday, September 2 By: Tomonori, Tomo, Takuya, Ken

Making culture and Master plate
SRRz-B0015 (1-1) [pSB4K5]13 colonies
SRRz-B0015 (1-1) [pSB4K5] (Control)13 colonies
Screening PCR of rSRRz low

Sample: (1-13) SRRz-B0015 (1-1) [pSB4K5] Maker: Lambda, 100 Control: Positive (B0015), Neganive Discussion: From sample 1, two vectors might be ligated. Sample 3 and 4, SRRz-B0015 might be inserted in low copy plasmid correctly. Sample 11, it might be the self-ligation product of low copy plasmid. Anyway, we decided to culture those 4 colonies on master plate.


Friday, September 3 By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken

Making culture
  • R0011-rrSΔTMD1-E0840 (1)
  • R0011-rrSΔTMD1-E0840 (3)
  • rrSΔTMD1-E0840 (1-1)
  • rrSΔTMD1-E0840 (1-2)
  • SRRz-B0015 (1-1) [pSB4K5]
  • SRRz-B0015 (1-2) [pSB4K5]
  • ML


Monday, September 6 By: Wataru, Tomo, Kazuya, Ken

Sequence Analysis
  • R0011-rrSΔTMD1-E0840 (A)
  • R0011-rrSΔTMD1-E0840 (B)
  • SRRz-B0015 [pSB4K5] (A)
  • SRRz-B0015 [pSB4K5] (B)
  • rrSΔTMD1-E0840 (1-1)
  • rrSΔTMD1-E0840 (1-2)

R0011-rrSΔTMD1-E0840 (A), SRRz-B0015 [pSB4K5] (A) is correct.

Miniprep
NameConcentration
SRRz29.6 ng/µL
R0011-rrSΔTMD1-E084070.2
J23105 (RPU0.3)75.3
rrSΔTMD1-E0840 (1-1)30.3
Restriction Digestion
  • J23105 (RPU0.3): SpeI, PstI
  • rrSΔTMD1-E0840: XbaI, PstI
Gel Extraction
NameConcentration
J23105 (RPU0.3) [SP]20ng / 10µL
rrSΔTMD1-E0840100ng / 1µL
Ligation
Vector Insert Ligation High Total Incubation
J23105 (RPU0.3) [SP]1 µLrrSΔTMD1-E0840 [XP]12430min
Transformation
NameCompetent CellProduct of Ligation
J23105 (RPU0.3)-rrSΔTMD1-E084050 µL4


Tuesday, September 7 By: Wataru, Ken

Insert Check
KyotoExp100907-1.png

We did colony PCR, and three colonies were inserted rrSΔTMD1-E0840. So we succeeded in making J23105 (RPU0.3)-rrSΔTMD1-E0840.

Thirsday, September 9 By: Wataru, Ken

Culture

Culture pSB4K5 and SRRzSam7


Friday, September 10 By: Wataru, Ken

Miniprep
NameConcentration
pSB4K548.8
SRRzSam733.4
Mutagenesis

We lost SRRz-B0015, so we decided to retry point mutation.

NameMgS04dNTP10xBufferTemplateKODMilliQTotal
SRRz (1)3551.513450
SRRz (2)3551.513450
Control3551.513450
94℃1min
94℃5s25 cycles
55℃30s
68℃3min 40s
4℃Forever

After digestion by DpnI, Ligation and Transformation.

Sunday, September 12 By: Wataru

Culture

Culture SRRz (1), (2), (3) from original plate.


Monday, September 13 By: Wataru, Ken

Miniprep
NameConcentration
SRRz (1)61.3 ng/µL
SRRz (2)59.3
SRRz (3)69.3
Sequence Analysis
  • SRRz (1), (2), (3)

SRRz (1), (2), (3) are correct.

Restriction Digestion
  • J23105 (RPU0.3)-rrSΔTMD1-E0840: EcoRI, SpeI
  • R0011: EcoRI, XbaI
Ligation

J23105 (RPU0.3)-rrSΔTMD1-E0840 [ES] + R0011 [EX]

Transformation

Tuesday, September 14 By: Ken, Wataru

Colony PCR

We did Colony PCR, and we succeeded in making J23105 (RPU0.3)-rrSΔTMD1-E0840-R0011

Sequence Analysis

From the product of Colony PCR.

  • J23105 (RPU0.3)-rrSΔTMD1-E0840-R0011

J23105 (RPU0.3)-rrSΔTMD1-E0840-R0011 is correct.


Tuesday, September 30 By: Ken

Miniprep
NameConcentration
LTC (1)84.6 ng/µL
LTC (2)97.6
LTC (3)127.4
LTC (4)85.4
LTC (5)70.0


Friday, October 1 By: Ken

Sequence Analysis

Analyzed LTC 1-5. The sequencing on each sample was in success. The results of sequencing with VF2 were desirable, but, on the other, only one of those with VR was desirable, the others were found bad sequencing. We decided to use LTC 2.


Saturday, October 2 By: Ken

Miniprep
NameConcentration
SRRz-low162.3 ng/µL
CTL60.8 ng/µL
Restriction Digestion
  • LTC (EcoRI-SpeI)
  • CTL (EcoRI-SpeI)
  • SRRz low (EcoRI-XbaI)

Sunday, October 3 By: Ken

Miniprep
NameConcentration
SRRz-low237.3 ng/µL
CTL138.8
LTC441.2
CT252.1
Restriction Digestion
NameTemplate10xbuffer100xbufferEcoRIXbaISpeICIAPWaterTotal
SRRz-low740.40.30.3-12740
CTL1040.40.3-0.3-2540
LTC440.40.3-0.3-3140
Concentration(after purification)
NameConcentration
SRRz-low(E-X)36.9 ng/µL
CTL(E-S)25.0
LTC(E-S)64.3
Ligation and Transformation

Ligation and transformation of CTL-SRRz(CTLS) and LTC-SRRz(LTCS).

NameColony
CTLSSome colonies
LTCSSome colonies

Monday, October 4 By: Ken

Making culture

Cultured CTLS and LTCS with LB-Kan(+/- IPTG).

Tuesday, October 5 By: Ken

Results of Screening

The propagation wasn't observed on some samples on CTLS cultured with M9-Kan +IPTG. It meant that the construction of CTLS was in success. However, the growth was observed on all of samples of LTCS. So, we decided to retry the construction of LTCS.

Miniprep
NameConcentration
LT387.1 ng/µL
Making culture

Cultured CTLS.

Wednesday, October 6 By: Ken

Thursday, October 7 By: Ken

Miniprep
NameConcentration
SΔTMD184.1 ng/µL
lac-SRRz172.5
Restriction Digestion
NameTemplate10xbuffer100xbufferEcoRIPstIWaterTotal
LT440.40.40.430.840
CT640.40.40.428.840
CTL1140.40.40.423.840
LTC440.40.40.430.840
SRRz740.40.40.427.840
SΔTMD11740.40.40.417.840
Concentration after purification
NameConcentration
LT107.9 ng/µL
CT56.9
CTL37.1
LTC101.9
SRRz55.7
SΔTMD1102.9

Friday, October 8 By: Ken

Screening

Screened lac-SRRz 1-7(LB-Tc with IPTG 1.5mM). Except for sample 4, the propagation could be observed. So, we decided to use sample 4.

Restriction Digestion of pSB1C3
Template10xbuffer100xbufferEcoRIPstIWaterTotal
540.40.40.428.4
Concentration after purification
NameConcentration
pSB1C3(E-P)16.1 ng/µL
Ligation and Transformation

Inserted LTC, CTL, T, SRRz, LT or CT to pSB1C3.

NameColony
LTCSome colonies
CTLNo colony
TSome colonies
SRRzSome colonies
LTSome colonies
CTSome colonies

Saturday, October 9 By: Ken

Making culture

Cultured LTC, T, SRRz, LT, and CT with LB (Cp/Amp+).

Restriction digestion of LTC and SRRz-low
NameTemplate10xbuffer100xbufferEcoRISpeIWaterTotal
LTC3.540.40.60.630.940
SRRz-low440.40.60.630.440
Ligation and Transformation of LTCS
NameColony
LTCSMany colonies
controlfew colonies

Used Top 10.

Sunday, October 10 By: Ken

Screening

Screened LTC, T, SRRz, LT, and CT. Except for LT1, the propagation could not be observed on each sample with LB (Amp+).

Miniprep
NameConcentration
LTC1210.6 ng/µL
T2193.2
SRRz3208.9
LT2195.7
CT1219.9
CTLS4-2231.0
LTCS150.6
Restriction Digestion of LTC, T, SRRz, LT, CT, and CTLS
Template10xBuffer100xBufferEcoRIPstIMilliQIncubation
2 µL10.10.20.26.560min
Electrophoreis
KyotoExp101010-1.png
  • Lane 1, 8: Lambda, 100bp Marker
  • Lane 2-7: LTC, T, SRRz, LT, CT, CTLS

We could observe that the each part was correctly inserted to the pSB1C3.

Retry - Transformation of CTL (pSB1C3) and LTCS

Used KRX.

Deletion PCR of T2
NameTemplate2xBufferdNTPsMgSO4Primer1Primer2Primer3KOD-FXMilliQTotal
GFP-Deletion1, 20.25 µL251031.5-1.518.7550
GFP-DT-Deletion1, 20.25 µL25103-1.51.518.7550
94℃2min
98℃10s30 cycles
59℃30s
68℃3.5min
4℃Hold

Primer:

  1. ccaggcatcaaataaaacgaaaggctc
  2. tactagtagcggccgctgc
  3. ctctagtattattgatttctaccatcttctactcc


Monday, October 11 By: Ken

Restriction Digestion, Electrophoresis, Ligation and Transformation of PCR products
TemplateDpnIIncubation
25 µL160min
KyotoExp101011-1.png
LaneName
1,6Lambda, 100bp Marker
2GFP-Deletion1
3GFP-Deletion2
4GFP-DT-Deletion1
5GFP-DT-Deletion2

Deletion PCR was well performed.

TemplateLigation HighT4 KinaseIncubation
9 µL5190min
NameColony
SΔTMD1-dTMany colonies
SΔTMD1Many colonies
  • Competent Cell: TOP10
Sequence

Sequenced LTC1, T2, SRRz3, LT2, CT1, CTLS4-2 and LTCS1.

Template5xBufferPrimerBig DyeMilliQTotal
(200ng)2 µL10.56.510

Primer:

  1. VF2
  2. VR
  3. aggtgatgcaacatacggaaaacttacc
  4. tgctgggattacacatggcatggatg
  5. ttcctcgatatgctggcgtggtc

Used Primer 1 or 2 to each sample, and Primer 3, 4, or 5 only to CTLS4-2 and LTCS1.

96℃1min
96℃10s40 cycles
50℃5s
60℃2min 5s
4℃Hold

Except for CTLS4-2 and LTCS1 with Primer2, the sequencing of each sample were well performed. And LCT1, T2, LT2, and CT1 were confirmed that the sequence were correct. There was, however, deletion of some base pairs on lactose promoter [R0011]. of CTLS402.

  • Result: AATTGTGAGCGGATAACAAGATACTGAGCACA
  • BBa_R0011: AATTGTGAGCGGATAACAATTGACATTGTGAGCGGATAACAAGATACTGAGCACA

There is repeated sequence on lactose promoter and the one was deleted. So, we supposed that the homologous recombination was occureed. From this result, we could say that the function check of CTLS was failed because SRRz gene wasn't induced by IPTG.

Making culture of LTCS and CTL

Tuesday, October 12 By: Ken

Screening of CTL

The propagation could not be observed with LB-Amp and could be observed with LB-Cp. It meant that the part, CTL, was correctly inserted to the plasmid, pSB1C3.

Making culture

Cultured two samples on each part: SΔTMD1-dT and SΔTMD1. Named T1,3,4 and 6.

Wednesday, October 13 By: Ken

Thursday, October 14 By: Ken

Retry of the construction of CTLS

Friday, October 15 By: Ken

Screening of CTLS

Cultured CTLS with M9+IPTG. And the propagation couldn't be observed on one sample. We decided to use this sample.

Sequence Analysis

We did the sequencing on ML, MS, CTLS, T4 and CTL. The sequencing of T4 and CTL were in success and the others were failed. The results of CTL was desirable, but that of T4 was bad sequence and there was a mutation on PstI site. Including the last time sequencing, we could say that the deletion of GFP-terminator from SΔTMD1-GFP was failed. So, we decided to retry the deletion PCR.


Sunday, October 17 By: Ken

Retry of deletion PCR of T2

We used PCR to delete GFP and terminator[E0840] from SΔTMD1-E0840.

NameTemplate2xBufferdNTPsPrimer(fwd)Primer(rev)KOD-FXMilliQTotal
GFP-DT-Deletion1, 20.25 µL25101.51.5110.7550
94℃2min
98℃10s30 cycles
68℃3.5min
4℃Hold

After the PCR, digestion by DpnI(37C/60min).

Electrophoresis
KyotoExp101017-1.png
  • Lane 1,2,5,6: Lambda, 100bp Maker
  • Lane 3,4 PCR products[T'1,T'2]

PCR was in success.

Ligation

Ligation PCR products.

Monday, October 18 By: Ken

Transformation

Transformed T'1 and T'2.

NameColony
T'1Some colonies
T'2Some colonies


Thursday, October 21 By: Ken

Making Culture

Cultured two samples on each plate, T'1 and T'2. Named dele1-4. And also cultured lac-SRRz.

Friday, Ocotber 22 By: Ken

Miniprep
NameConcentration
dele1117.9 ng/µL
dele2130.8
dele3108.7
dele4176.0
lac-SRRz67.2

Saturday, October 23 By: Ken

Sequence Analysis

We used VF2 on dele1-4 and lac-SRRz and VR on lac-SRRz. The sequencing of lac-SRRz with VR was failed, the others were in success. The results were desirable on dele1,3,4 and lac-SRRz.

Deletion PCR of lac-SRRz

We used deletion PCR to get lac-SΔTMD1RRz.

NameTemplate2xBufferdNTPsPrimer1Primer2KODMilliQTotal
lac-SΔTMD1RRz (1)0.825101.51.5110.250
lac-SΔTMD1RRz (2)0.825101.51.5110.250
control0.825101.51.5011.250
94℃2min
98℃10s30 cycles
68℃5min
4℃Hold

After the PCR, done restriction digestion by DpnI(37℃/60min).

Electrophoresis
KyotoExp101023-1.png
  • Lane 1,8,2,7: Lambda, 100bp Maker
  • Lane3,4: lac-SΔTMD1RRz (1,2)
  • Lane5: control
Ligation and Transformation
NameColony
lac-SΔTMD1RRz(1)some colonies
lac-SΔTMD1RRz(2)some colonies
controlno colony

Used KRX.

Sunday, October 24 By: Ken

Making Culture

Cultured CTLS, Lysisbox and three samples of lac-SΔTMD1RRz with LB-Tc(IPTG 0 or 1mM).

Monday, October 25 By: Ken

Miniprep
NameConcentration
CTLS62.9 ng/µL
Lysisbox31.9
lac-SΔTMD1RRz 1140.9
lac-SΔTMD1RRz 271.6

The propagation was not observed on lac-SΔTMD1RRz 3 with LB (IPTG 1mM). It meant this sample might be lac-SRRz. We decided not to use this.

PCR

We used PCR on CTLS and Lysisbox to change its own constitutive promoter to another.

NameTemplate2xBufferdNTPsPrimer1Primer2Primer3Primer4KOD-FXMilliQTotal
C'TLS(1)0.8 µL25101.51.5--110.250
C'TLS(2)0.8 µL25101.51.5--110.250
Lysisbox'(1)1.5 µL2510--1.51.519.550
Lysisbox'(2)1.5 µL2510--1.51.519.550
94℃2min
98℃10s30 cycles
68℃6min 20s
4℃Hold

Primer

  1. aggtacagtgctagctactagaggagc
  2. aggactgagctagccgtcaactc
  3. aggtattatgctagctactagaggagc
  4. aggactgagctagctgtaaactctag
Electrophoresis
KyotoExp101025-1.png
  • Lane 1,8,2,7: Lambda, 100bp Maker
  • Lane 3,4: C'TLS(1,2)
  • Lane 5,6: Lysisbox'(1,2)

Two bands were observed on lane 5 and 6. We decided to do gel extraction.

Ligation

We did ligation of C'TLS(1,2).

Tuesday, October 26 By: Ken

Gel Extraction of Lysisbox'
NameConcentration
Lysisbox'(1)33.6 ng/µL
Lysisbox'(2)43.4 ng/µL
Ligation

We did ligation of Lysisbox'(1,2).

Transformaiton

Transformed C'TLS(1,2) and Lysisbox'(1,2).

Retrieved from "http://2010.igem.org/Team:Kyoto/Notebook1"