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.
The concentration of all samples was very week. Probably our shaking incubation was week.
We lost B0015 by our mistake. The concentration of pSB4K5 is high, so this condition of shaking incubation is moderate.
The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
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.
After PCR Purification, evaporated them and diluted 3µL.
Marker: 1kb.
At the condition 4 (4.5µL MgSO4) and 6 (6µL MgSO4), SRRz is amplified very much. So we decided to use them.
Marker: 1kb, 100bp
DpnI works correctly.
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.
Stored at -20℃.
pSB4K5 [EP] is concentrated 10µL and E0240(1) [XP], E0240(2) [XP] are concentrated 1µL.
Transformation
Name | Sample | Competent Cell | Total | Plate | Incubation | Colony
|
ML (1) | 1 µL | 20 | 21 | LB (Kan+) | 08/03-08/04 | ○
|
ML (2) | 1 | 20 | 21 | ○
|
MS | 1 | 20 | 21 | ○
|
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.
Name | Color | Incubation
|
ML (1-1) | Green Colony | 8/5-8/6
|
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
Name | Concentration
|
SΔTMD1-E0840(1) A | 28.9 ng/µL
|
SΔTMD1-E0840(1) B | 25.3
|
SΔTMD1-E0840(3) A | 26.6
|
SΔTMD1-E0840(3) B | 24.0
|
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
Name | Sample | 2 buffer | BSA | Enzyme 1 | Enzyme 2 | MilliQ | Total
|
ML (1-1) [EP] | 50 µL | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
ML (1-2) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
ML (1-3) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
ML (1-4) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
ML (2-1) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
ML (2-2) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
ML (2-3) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
ML (2-4) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
MS (1) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
MS (2) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
MS (3) [EP] | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60
|
Electrophoresis
No. | Name | Length | Results
|
1 | MS (1) [EP] | 960, 4339 |
|
2 | MS (2) [EP] | 960, 4339 |
|
3 | MS (3) [EP] | 960, 4339 |
|
4 | ML (1-1) [EP] | 980 3378 | ○
|
5 | ML (1-2) [EP] | 980 3378 | ○
|
6 | ML (1-3) [EP] | 980 3378 | ×
|
7 | ML (1-4) [EP] | 980 3378 | ×
|
8 | ML (2-1) [EP] | 980 3378 | ○
|
9 | ML (2-2) [EP] | 980 3378 | ×
|
10 | ML (2-3) [EP] | 980 3378 | ×
|
11 | ML (2-4) [EP] | 980 3378 | ×
|
12 | MS (1) [EP] | 960, 4339 | ○
|
13 | MS (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)
Name | Water | MgSO4 | dNTPs | 10xBuffer | Primer VF2 | Primer VR | Template SSam7,ΔTMD1-E0840 failed (50ng/µL) | KOD plus ver.2 | Total
|
SSam7,ΔTMD1-E0840 (1) | 32 | 3 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50
|
SSam7,ΔTMD1-E0840 (2) | 32 | 3 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50
|
94℃ | 2min |
|
98℃ | 10s | 25 cycles
|
68℃ | 4min
|
Add DpnI 2µL
|
Incubate | 1h |
|
4℃ | forever |
|
Transformation
Name | Well | Sample | Competent Cell | Total | Plate | Incubation | Colony
|
SSam7,ΔTMD1-E0840 (1) | - | 4 µL | 50 | 54 | LB (Kan+) | 08/06-08/09 | ○
|
SSam7,ΔTMD1-E0840 (2) | - | 4 | 50 | 54 | ○
|
I20260 | 2-17-F | 2 | 50 | 52 | ○
|
| 2-I-5 | 2 | 50 | 52 | LB (Amp+) | ○
|
Monday, August 9 By: Wataru, Tomonori, Ken, Takuya
Miniprep
Name | concentration
|
MS | 116.2 ng/µL
|
ML | 146.6
|
Transfotrmation
Sample | Sample | Competent Cell | Total | Plate | Incuvation | Results
|
MS | 2 µL | KRX | 50 | 52 | LB (Kan+) | 08/09 18:00-08/10 12:00 | ○
|
ML | 2 | KRX | 50 | 52 | ○
|
Restriction Eigestion and Ethanol Precipitation
To use R0011 for next ligation, we digested it by EcoRI and PstI
Name | Sample | 10x Buffer | BSA | Enzyme 1 | Enzyme 2 | MilliQ | Total | Incubation
|
R0011 [EP] | 50 | 6 | 0.6 | EcoRI | 0.5 | PstI | 0.5 | 2.4 | 60 | At 37℃ 08/09 16:20-18:20
|
After restriction enzyme digestion, we did ethanol precipitation.
Ligation and Transformation
Name | Sample | Competent cell | Total | Plate | Incuvation | Colony
|
R0011 [pSB4K5, KRX] | 2 µL | KRX | 50 | 52 | LB (Kan+) | 08/09 20:00-08/10 09:00 | ○
|
R0011 [pSB4K5</partrinfo>, C2] | 2 | C2 | 50 | 52 | ○
|
Tuesday, August 10 By: Wataru, Tomonori, Ken, Fumitaka
Culture
Cultured I20260 [pSB4K5, ML, R0011 [pSB4K5, KRX], and R0011 [pSB4K5</partrinfo>, C2].
Minprep
Name | Concentration
|
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. | Medium | Cloud | Incubation
|
1 | Kanamycin | ○ | At 37℃, 08/10 20:00-08/11 9:00
|
Ampicillin | ×
|
2 | Kanamycin | ○
|
Ampicillin | ○
|
3 | Kanamycin | ○
|
Ampicillin | ×
|
4 | Kanamycin | ○
|
Ampicillin | ×
|
5 | Kanamycin | ○
|
Ampicillin | ×
|
6 | Kanamycin | ○
|
Ampicillin | ○
|
7 | Kanamycin | ○
|
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'
Name | Concentration
|
R0011 [pSB4K5, C2] (1) | 31.2 ng/µL
|
R0011 [pSB4K5, C2] (3) | 29.9
|
Restriction Digestion and electrophoresis of R0011 [pSB4K5, C2]
Name | EcoRI | PstI
|
1 | 0.2 | -
|
2 | - | 0.2
|
3 | 0.2 | 0.2
|
N | - | -
|
No. | Name | Length | Results
|
1 | R0011 [pSB4K5, C2] (1-1) | |
|
2 | R0011 [pSB4K5, C2] (1-2) | |
|
3 | R0011 [pSB4K5, C2] (1-3) | |
|
4 | R0011 [pSB4K5, C2] (1-N) | |
|
5 | R0011 [pSB4K5, C2] (2-1) | |
|
6 | R0011 [pSB4K5, C2] (2-2) | |
|
7 | R0011 [pSB4K5, C2] (2-3) | |
|
8 | R0011 [pSB4K5, C2] (2-N) | |
|
Each enzyme correctly cut samples.
Screening PCR of SRRz
No. | Name | Results
|
1 | None |
|
2 | Control B0015 |
|
3 | Control J06702 |
|
4 | Control B0015 |
|
5-24 | SRRz-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
Name | Template | 10xbuffer | 100xbuffer | EcoRI | XbaI 1 | XbaI 2 | SpeI | PstI 1 | PstI 2 | Water | Total
|
1 | 3 | 1 | 0.1 | 0.2 | - | - | - | - | - | 5.7 | 10
|
2 | 3 | 1 | 0.1 | - | 0.2 | - | - | - | - | 5.7 | 10
|
3 | 3 | 1 | 0.1 | - | - | 0.2 | - | - | - | 5.7 | 10
|
4 | 3 | 1 | 0.1 | - | - | - | 0.2 | - | - | 5.7 | 10
|
5 | 3 | 1 | 0.1 | - | - | - | - | 0.2 | - | 5.7 | 10
|
6 | 3 | 1 | 0.1 | - | - | - | - | - | 0.2 | 5.7 | 10
|
N | 3 | 1 | 0.1 | - | - | - | - | - | - | 5.9 | 10
|
Maker: Lambda, 100bp
Discussion: Each enzyme correctly cut each sample and was active.
Thursday, August 19 By: Wataru, Tomo, Ken
Miniprep of SSam7,ΔTMD1-E0840
Name | Concentration
|
SSam7,ΔTMD1-E0840 | 29.6 ng/µL
|
Point mutation PCR of SSam7,ΔTMD1-E0840
Name | Template | 10xbuffer | dNTPs | MgSO4 | Primer Fwd. | Primer Rev. | MilliQ | KOD plus ver.2 | Total
|
SΔTMD1-E0840 (1) | 1.5 | 5 | 5 | 3 | 1.5 | 1.5 | 31.5 | 1 | 50
|
SΔTMD1-E0840 (2) | 1.5 | 5 | 5 | 3 | 1.5 | 1.5 | 31.5 | 1 | 50
|
Control | 1.5 | 5 | 5 | 3 | 1.5 | 1.5 | 32.5 | - | 50
|
94℃ | 2min |
|
98℃ | 10s | 30cycles
|
55℃ | 30s
|
68℃ | 3.5min
|
4℃ | forever |
|
Restriction Digestion by DpnI from 17:50 to 18:50
Electrophoresis
Name
|
SΔTMD1-E0840 (1)
|
SΔTMD1-E0840 (2)
|
Control
|
Marker: Lambda, 100bp
Ligation and Transformation
Name | Colony
|
SΔTMD1-E0840 (1) | ○
|
SΔTMD1-E0840 (2) | ○
|
Control | ×
|
Friday, August 20 By: Wataru, Ken
Making Culture and Master Plate of SΔTMD1-E0840
Miniprep
Name | Concentration
|
B0015 | 41.1 ng/µL
|
PCR of SRRz
Name | 10xBuffer | MgS04 | dNTP | Template | Primer Fwd. | Primer Rev. | MilliQ | KOD plus ver.2 | Total
|
SRRzSam7 (1) | 5 µL | 3 | 5 | 5 | F1 | 1.5 | 1.5 | 28 | 1 | 50
|
SRRzSam7 (2) | 5 | 3 | 5 | 5 | F2 | 1.5 | 1.5 | 28 | 1 | 50
|
SRRzSam7 (3) | 5 | 3 | 5 | 5 | F1 | 1.5 | 1.5 | 28 | 1 | 50
|
SRRzSam7 (4) | 5 | 3 | 5 | 5 | F2 | 1.5 | 1.5 | 28 | 1 | 50
|
SRRzSam7 (5) | 5 | 3 | 5 | 5 | F1 | 1.5 | 1.5 | 28 | 1 | 50
|
SRRzSam7 (6) | 5 | 3 | 5 | 5 | F2 | 1.5 | 1.5 | 28 | 1 | 50
|
94℃ | 2min |
|
98℃ | 10s | 30cycles
|
55℃ | 30s
|
68℃ | 2min
|
4℃ | forever |
|
Electrophoresis
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
Name | Concentration
|
SRRzSam7 (1) | 134.0 ng/µL
|
SRRzSam7 (3) | 69.0
|
Restriction Digestion
Name | Sample | 10xBuffer | 100xBuffer | EcoRI | XbaI | SpeI | MilliQ | Total | Incubation
|
B0015 [EX] | 50 µL | 6 | 0.6 | 0.4 | 0.4 | - | 2.6 | 60 | 17:45-18:45
|
SRRzSam7 (1) [EP] | 50 | 6 | 0.6 | 0.4 | - | 0.4 | 2.6 | 60
|
SRRzSam7 (3) [EP] | 50 | 6 | 0.6 | 0.4 | - | 0.4 | 2.6 | 60
|
Purification
Name | Concentration
|
SRRzSam7 (1) [EP] | 109.0 ng/µL
|
SRRzSam7 (2) [EP] | 110.0
|
B0015 | 25.5
|
Ligation and Transformation
Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku
Miniprep
Sample number | Concentration
|
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℃ | 30s | 35cycles
|
50℃ | 30s
|
72℃ | 1.5min
|
72℃ | 4min |
|
4℃ | hold |
|
No. | Name
|
1-13 | SRRzSam7-B0015
|
C | Control: B0015
|
N | None
|
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)
Name | Sample | 10xBuffer | dNTPs | Primer Fwd. | Primer Rev. | Template | Water | KOD-plus- | Total
|
rrSΔTMD1-E0840 (1) | 2 µL | 5 | 5 | 1.5 | 1.5 | 1 | 35 | 1 | 50
|
rrSΔTMD1-E0840 (2) | 2 | 5 | 5 | 1.5 | 1.5 | 1 | 35 | 1 | 50
|
rrSΔTMD1-E0840 (Control) | 2 | 5 | 5 | 1.5 | 1.5 | 1 | 35 | - | 50
|
94℃ | 2min |
|
94℃ | 10s | 35cycles
|
56℃ | 30s
|
68℃ | 3.5min
|
4℃ | forever |
|
Restriction Digestion (DpnI)
Sample | DpnI | Total | Incubation
|
25 µL | 1 | 26 | 19:00-20:10
|
Ligation
Name | Sample | Water | Ligation high | T4 Kinase | Total | Incubation
|
rrSΔTMD1-E0840 (1) | 3 µL | 6 | 5 | 1 | 15 | 20:15-21:15
|
rrSΔTMD1-E0840 (2) | 3 | 6 | 5 | 1 | 15
|
rrSΔTMD1-E0840 (Control) | 3 | 6 | 5 | 1 | 15
|
Transformation
Tuesday, August 24 By: Ken, Tomo, Tasuku, Takuya
Retry of deletion PCR of SΔTMD1-E0840
Name | Sample | 10xBuffer | dNTPs | MgSO4 | Primer1 | Primer2 | Template | Water | KOD-plus- | Total
|
rrSΔTMD1-E0840 (1) | 2 µL | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50
|
rrSΔTMD1-E0840 (2) | 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50
|
Control | 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50
|
94℃ | 2min |
|
94℃ | 10s | 35cycles
|
58℃ | 30s
|
68℃ | 3.5min
|
4℃ | hold |
|
Restriction Digestion (DpnI)
14:15-15:15
Electrophoreis
Lane | Name
|
1 | rrSΔTMD1-E0840 (1)
|
2 | rrSΔTMD1-E0840 (3)
|
C | rrSΔTMD1-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
Name | 10x | dNTPs | MgSO4 | Primer1 | Primer2 | Template | Water | KOD-plus- | total
|
SRRzSam7-B0015 (1) | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50
|
SRRzSam7-B0015 (2) | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50
|
SRRzSam7-B0015 (Control) | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50
|
94℃ | 2min |
|
98℃ | 10s | 30cycles
|
55℃ | 30s
|
68℃ | 4min
|
4℃ | hold |
|
Restriction Digestion (DpnI), Electrophoresis and Ligation
We could find point mutation PCR and restriction enzyme of DpnI was done.
PCR of E0240
Sample | 10xBuffer | dNTPs | MgSO4 | VF2 | VR | Template | Water | KOD-plus- | Total
|
E0240 (1) | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 31.5 | 1 | 50
|
E0240 (2) | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 31.5 | 1 | 50
|
PCR Purification
Name | Concentration
|
E0240 (1) | 5.5 x 50 ng/µL
|
E0240 (2) | 5.2 x 50
|
Restriction Digestion (EcoRI, PstI) and Gel Extraction
Name | Concentration
|
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
Name | Colony
|
rrSΔTMD1-E0840 (1) | ○
|
rrSΔTMD1-E0840 (2) | ○
|
rrSΔTMD1-E0840 (Control) | ×
|
SRRzSam7-B0015 (1) | ○
|
SRRzSam7-B0015 (2) | ○
|
SRRzSam7-B0015 (Control) | ×
|
Miniprep
Name | Concentration
|
pSB4K5 | 29.0 ng/µL
|
Restriction Digestion
Sample name | Template | 10xbuffer | 100xbuffer | EcoRI | SpeI | PstI | Water | Total
|
pSB4K5 | 50 | 6 | 0.6 | 0.4 | 0.4 | - | 2.6 | 60
|
R0011 [pSB4K5] | 10 | 4 | 0.4 | - | 0.3 | 0.3 | 25 | 40
|
Purification
Sample Name | Concentration
|
pSB4K5 | 18.4 ng/µL
|
R0011 [pSB4K5] | 8.6
|
Ligation of E0240 and pSB4K5, Transformation
Thursday, August 26 By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka
Miniprep
Sample name | Concentration
|
J23116 (RPU0.7) | 44.5 ng/µL
|
Restriction Digestion
Name | Template | 10xbuffer | 100xbuffer | SpeI | PstI | Water | Total
|
J23116 (RPU0.7) | 25 | 4 | 0.4 | 0.3 | 0.3 | 10 | 40
|
Purification of
Name | Concentration
|
J23116 (RPU0.7) | 49.8 ng/µL
|
Friday, August 27 By: Ken, Tomo, Kazuya, Fumitaka
Making master plate of E0240 [pSB4K5]
Sample Name | Concentration
|
rrSΔTMD1-E0840 (1-2) | 20.9 ng/µL
|
SRRz-B0015 (1-1) | 16.4
|
Restriction Digestion
Name | Template | 10xbuffer | 100xbuffer | XbaI | PstI | Water | Total | Incubation
|
rrSΔTMD1-E0840 (1-2) | 45 µL | 6 | 0.6 | 0.3 | 0.3 | 7.8 | 60 | 13:20-14:20
|
SRRz-B0015 (1-1) | 45 | 6 | 0.6 | 0.3 | 0.3 | 7.8 | 60
|
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
Name | Colony
|
R0011-rrSΔTMD1-E0840 | Many colonies
|
R0011-rrSΔTMD1-E0840 (Control) | Some colonies
|
J23116 (RPU0.7)- rrSΔTMD1-E0840 | Many 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
Name | Concentration
|
J23105 (RPU0.3) | 48.5 ng/µL
|
R0011-rrSΔTMD1-E0840 | 107.3
|
Restriction Digestion
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
Name | Concentration
|
J23105 (RPU0.3) | 5.8 ng/µL
|
R0011-rrSΔTMD1-E0840 | 7.8
|
Ligation and Transformation
Insert | Vector
|
R0011-rrSΔTMD1-E0840 | J23105 (RPU0.3)
|
Wednesday, September 1 By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken
Making culture and Master plate
Name | Colony
|
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
|
pSB4K5 | 56.0
|
Restriction Digestion of SRRz and pSB4K5
Name | Template | 10xbuffer | 100xbuffer | EcoRI | PstI | Water | Total | Incubation
|
SRRz-B0015 (1-1) | 20 µL | 4 | 0.4 | 0.3 | 0.3 | 15 | 40 | 13:25-14:30
|
pSB4K5 | 20 | 4 | 0.4 | 0.3 | 0.3 | 15 | 40
|
Purification
SRRz-B0015 (1-1) | 6.5 ng/µL
|
pSB4K5 | 16.8
|
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
Name | Concentration
|
SRRz | 29.6 ng/µL
|
R0011-rrSΔTMD1-E0840 | 70.2
|
J23105 (RPU0.3) | 75.3
|
rrSΔTMD1-E0840 (1-1) | 30.3
|
Restriction Digestion
- J23105 (RPU0.3): SpeI, PstI
- rrSΔTMD1-E0840: XbaI, PstI
Name | Concentration
|
J23105 (RPU0.3) [SP] | 20ng / 10µL
|
rrSΔTMD1-E0840 | 100ng / 1µL
|
Ligation
Vector | Insert | Ligation High | Total | Incubation
|
J23105 (RPU0.3) [SP] | 1 µL | rrSΔTMD1-E0840 [XP] | 1 | 2 | 4 | 30min
|
Transformation
Name | Competent Cell | Product of Ligation
|
J23105 (RPU0.3)-rrSΔTMD1-E0840 | 50 µL | 4 |
|
Tuesday, September 7 By: Wataru, Ken=
Insert Check
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
Name | Concentration
|
pSB4K5 | 48.8
|
SRRzSam7 | 33.4
|
Mutagenesis
We lost SRRz-B0015, so we decided to retry point mutation.
Name | MgS04 | dNTP | 10xBuffer | Template | KOD | MilliQ | Total
|
SRRz (1) | 3 | 5 | 5 | 1.5 | 1 | 34 | 50
|
SRRz (2) | 3 | 5 | 5 | 1.5 | 1 | 34 | 50
|
Control | 3 | 5 | 5 | 1.5 | 1 | 34 | 50
|
94℃ | 1min |
|
94℃ | 5s | 25 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
Name | Concentration
|
SRRz (1) | 61.3 ng/µL
|
SRRz (2) | 59.3
|
SRRz (3) | 69.3
|
Sequence Analysis
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
Name | Concentration
|
LTC (1) | 84.6 ng/µL
|
LTC (2) | 97.6
|
LTC (3) | 127.4
|
LTC (4) | 85.4
|
LTC (5) | 70.0
|
Thursday, 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.
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.
Ligation and Transformation
Inserted LTC, CTL, T, SRRz, LT or CT to pSB1C3.
Name | Colony
|
LTC | Some colonies
|
CTL | No colony
|
T | Some colonies
|
SRRz | Some colonies
|
LT | Some colonies
|
CT | Some colonies
|
Saturday, October 9 By: Ken
Making culture
Cultured LTC, T, SRRz, LT, and CT with LB (Cp/Amp+).
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
Name | Concentration
|
LTC1 | 210.6 ng/µL
|
T2 | 193.2
|
SRRz3 | 208.9
|
LT2 | 195.7
|
CT1 | 219.9
|
CTLS4-2 | 231.0
|
LTCS1 | 50.6
|
Restriction Digestion of LTC, T, SRRz, LT, CT, and CTLS
Template | 10xBuffer | 100xBuffer | EcoRI | PstI | MilliQ | Incubation
|
2 µL | 1 | 0.1 | 0.2 | 0.2 | 6.5 | 60min
|
Electrophoreis
- Lane 1, 8: Lambda, 100bp Marker
- Lane 2-7: LTC, T, SRRz, LT, CT, CTLS
We could observed that the each parts was correctly inserted to the pSB1C3.
Retry - Transformation of CTL (pSB1C3) and LTCS
Used KRX.
Deletion PCR of T2
Name | Template | 2xBuffer | dNTPs | MgSO4 | Primer1 | Primer2 | Primer3 | KOD-FX | MilliQ | Total
|
GFP-Deletion1, 2 | 0.25 µL | 25 | 10 | 3 | 1.5 | - | 1.5 | 1 | 8.75 | 50
|
GFP-DT-Deletion1, 2 | 0.25 µL | 25 | 10 | 3 | - | 1.5 | 1.5 | 1 | 8.75 | 50
|
94℃ | 2min |
|
98℃ | 10s | 30 cycles
|
59℃ | 30s
|
68℃ | 3.5min
|
4℃ | Hold |
|
Primer:
- ccaggcatcaaataaaacgaaaggctc
- tactagtagcggccgctgc
- ctctagtattattgatttctaccatcttctactcc
Isolation of R0011-SRRz (4)
Monday, October 11 Ken
Restriction Digestion, Electrophoresis, Ligation and Transformation of PCR products
Template | DpnI | Incubation
|
25 µL | 1 | 60min
|
Lane | Name
|
1,6 | Lambda, 100bp Marker
|
2 | GFP-Deletion1
|
3 | GFP-Deletion2
|
4 | GFP-DT-Deletion1
|
5 | GFP-DT-Deletion2
|
Deletion PCR was well performed.
Template | Ligation High | T4 Kinase | Incubation
|
9 µL | 5 | 1 | 90min
|
Sequence
Sequenced LTC1, T2, SRRz3, LT2, CT1, CTLS4-2 and LTCS1.
Template | 5xBuffer | Primer | Big Dye | MilliQ | Total
|
(200ng) | 2 µL | 1 | 0.5 | 6.5 | 10
|
Primer:
- VF2
- VR
- aggtgatgcaacatacggaaaacttacc
- tgctgggattacacatggcatggatg
- ttcctcgatatgctggcgtggtc
Used Primer 1 or 2 to each sample, and Primer 3, 4, or 5 only to CTLS4-2 and LTCS1.
96℃ | 1min |
|
96℃ | 10s | 40 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
Sunday, October 17 By: Ken
Retry of deletion PCR of T2
Deletion PCR of T2
Name | Template | 2xBuffer | dNTPs | Primer(fwd) | Primer(rev) | KOD-FX | MilliQ | Total
|
GFP-DT-Deletion1, 2 | 0.25 µL | 25 | 10 | 1.5 | 1.5 | 1 | 10.75 | 50
|
94℃ | 2min |
|
98℃ | 10s | 30 cycles
|
68℃ | 3.5min
|
4℃ | Hold |
|