Team:Kyoto/Notebook

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Notebook

Tuesday, July 20

By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

1. Solubilization of antibiotics.

For Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).

For Kanamycin(kan): add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml).

Dispense 1.1ml of the solution into 1.5ml tubes.

Store in the freezer (-20℃).

2. Making plates for LB (Amp+) and LB (Kan+).

3. Transformation of iGEM Parts.

NameWellSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>J23100</partinfo>1-18-C12021LB (Amp+)At 37℃ 7/20 20:50 - 7/21 17:00○
<partinfo>J23105</partinfo>1-18-M12021○
<partinfo>J23116</partinfo>1-20-M12021○
<partinfo>R0011</partinfo>1-6-G12021○
<partinfo>E0840</partinfo>1-12-O12021○
<partinfo>J06702</partinfo>2-8-E12021○
<partinfo>pSB4K5</partinfo>1-5-G12021×
<partinfo>B0015</partinfo>1-23-L12021LB (Kanamycin+)×

A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+). 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 Yamamoto

1. Culture plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.

2. Make a master plate of the above plates.

3. Retry Transformation of iGEM Parts.

NameWellSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>pSB4K5</partinfo>1-5-G12021LB (Kanamycin+)At 37℃ 7/21 20:50 - 7/22 16:30○
<partinfo>B0015</partinfo>1-23-L12021○

4. PCR for S-R-Rz/Rz1 and S

Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.

No.Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2TemplateDNA (5ng/µl)Primer S-R-Rz/Rz1 Forward (10µM)Primer S-R-Rz/Rz1 Reverse (10µM)Primer S Reverse (10µM)KOD Plus ver.2Total
128µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
228µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
328µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
428µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
528µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
628µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
728µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
828µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl

Forward Primer of S-R-Rz/Rz1 and S is common.

PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

Thursday, July 22

By: Wataru

1. Electrophoresis of the PCR products for 40min.

File:KyotoEXP100722-1.png

Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.

2. Miniprep of iGEM Parts.

NameConcentration(ng/µl)
<partinfo>J23100</partinfo>18.5
<partinfo>J23105</partinfo>12.5
<partinfo>J23116</partinfo>14.6
<partinfo>R0011</partinfo>8.6
<partinfo>E0840</partinfo>12.1
<partinfo>J06702</partinfo>14.7

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

3. Culture plates and make master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 7/22 17:00 to 7/23 10:00.

Friday, July 23

By: Wataru, Tomo, Makoto

1. Miniprep of iGEM Parts.

NameConcentration(ng/µl)
<partinfo>pSB4K5</partinfo>79.2
<partinfo>B0015</partinfo>-

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

2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.

SampleConcentration (ng/µl)New Name
118.6-
377.6S1
533.6-
765.4S2

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

3. Retry of PCR of S-R-Rz/Rz1.

SampleWater25mmol/l MgSO42mmol/l dNTPs10×Buffer for KOD plus ver.2Template DNA (5ng/µl)Primer S-R-Rz/Rz1 Forward (10µmol/l)Primer S-R-Rz/Rz1 Reverse (10µmol/l)KOD plus ver.2Total
128µl35551.51.5150
22835551.51.5150
326.54.55551.51.5150
426.54.55551.51.5150
52565551.51.5150
62565551.51.5150

PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.

Sample10xBufferBSAEnzymeMilliQTotalIncubation
15µl1EcoRI 0.13.610At 37℃ 7/23 18:00 - 7/23 18:30
251XbaI 0.13.610
351SpeI 0.13.610
451PstI 0.13.610
551-3.710

5. Electrophoresis of above sample for 35min.

KyotoExp100723-1.png

Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

6. To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.

Sample10×BufferEnzyme 1Enzyme 2MilliQTotalIncubation
S111µl5EcoRI 0.2SpeI 0.233.650At 37℃ for 2h
S2115EcoRI 0.2SpeI 0.233.650
<partinfo>E0840</partinfo>(GFP)455EcoRI 0.2XbaI 0.2050

After PCR purification, evaporated them and diluted 3ul.

7. Ligated over night.

SampleVectorInsertLigation HighTotal
S-GFP1<partinfo>E0840</partinfo> 0.5µlS1 0.512
S-GFP2<partinfo>E0840</partinfo> 0.5S2 0.512

Monday, July 26

By: Wataru, Tomonori, Makoto

1. Electrophoresis of PCR products

KyotoExp100726-1.png

At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.

2. PCR purification

SampleConcentration (ng/µl)New Name
451.6
559.3
659.6

3. Transformation of iGEM Parts

NameWellSample (µl)Competent Cell (µl)Total (µl)PlateIncubationResult
1-12-M12021LB (Amplicillin+)At 37℃ 7/26 - 7/27×
2-17-F12021LB (Kanamycin+)×
1-5-E12021×

4. Culture of 1-6-G, 1-12-O, and 1-23-L

Monday, July 26

By: Wataru, Tomonari, Makoto

Electrophoresis of PCR products

3①3②4.5①4.5②6①6②

KyotoExp100726-1.png

Discussion

At the condition 4.5②　and 6②、S-R-Rz is amplified very much. So we decided to use them inexperience.

PCR purification

Sample numberConcentration(ng/µL)
4.5②51.6
6①59.3
6②59.6

Transformation

SampleConc(/µL)Sample Volume(µL)Competent Cell(µL)TotalPlateIncubation
1-12-M-12021LB amp7/26~7/27
2-17-F-12021LB kan
1-5-E-12021

Culture

1-6-G, 1-12-O, 1-23-L

Tuesday, July 27

By: Wataru, Tomo, Kazuya, Ken, Naoi

Result of transformation

1-12-MNo colony
1-5-E
2-17-F

Colony of PCR of S-E840

To check that S GFP is correctly inserted, we did colony PCR.

Gel: Agarose Time: 35min Voltage: 100V Maker: 1K 100 1～6 S-GFP① 7～13 S-GFP② Posi E840（about 900bp） Nega None

1K 1 2 3 4 5 6 7 8 9 10 11 12 13 posi nega 100 File:KyotoExp100727.png

As a result, 1,3,5,6,11,12,13 are inserted S gene correctly. So, we decided to use 6 as S-E840①　and 11 as S-E840②.

Miniprep

Sample numberConcentration(ng/µL)
1-6-G26.9
1-23-L120.0
1-12-O120.1

RE

Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
1-23-L3050.5EcoRI0.4XbaI0.313.750
4.5②4050.50.40.43.850
6②4050.50.40.43.850

Incubate 37℃ 16:45~18:00

Ligation

Transformation

Wednesday, July 28

1. Result of Transformation

SRRz①-DTMany colonies
SRRz②-DT

2. Deletion PCR

To delete functional domain of S gene, we did deletion PCR.

Miniprep
Sample numberConcentration(ng/µ)
S-E840①95.5
S-E840②98.6

Diluted S-GFP①　and S-GFP②　20 times with water, and used as template DNA.

Deletion PCR

To delete functional region of S gene, we did deletion PCR.

Water25mM MgSO42mM dNTPs10x buffer for KOD Plus ver.2Primer Deletion F(10µM)Primer Deletion R(10µM)Template S-E840①Template S-E840②KOD Plus ver.2Total
Δ1-1283551.51.55-150
Δ1-2283551.51.55-150
Δ2-1283551.51.5-5150

PCR program

94℃2min
98℃10sec35 cycles
55℃30sec
68℃4min
4℃forever

3. RE

To check function of our Restriction enzymes, we digested S-E840① and S-E-840② by DpnI.

Samplefast digestion bufferDpnIMilliQTotal
S-E840①310.15.810
S-E840②310.15.810
Electrophoresis

Gel: Agarose Time: 35min Voltage: 100V Maker: 1K 100

1 1k marker 2 not digested S-E840① 3 not digested S-E840② 4 digested S-E840① 5 digested S-E840② 6 100bp marker

1k 1 2 3 4 100

File:KyotoExp100728.png

Discussion

DpnI works correctly

Thursday, July 29

RE

Sample volumeFastdigestion bufferEnzyme 1MilliQTotal
Δ1-1506DpnI 0.23.860
Δ2-1506DpnI 0.23.860

Incubate 7/29 9:40~7/29 11:00

Ligation and Pospholylation

SampleMilliQLigation HighT4 KinaseTotal
Δ1-1275115
Δ2-1275115

Incubate 7/29 11:30~7/29 13:00

Transformation

SampleConc(/µL)Sample Volume(µL)Competent Cell(µL)TotalPlateIncubation
Δ1-1-33033LB amp7/29~7/30
Δ1-1-33033

Friday, July 30

Result of transformation of Δ1 and Δ2 Many colonies are observed.

Monday, August 2

By: Wataru, Ken

1. Miniprep

Sample numberConcentration(ng/µL)
Δ152.7
Δ254.4
Δ389.5
pSB4K550.7
LacP18.6

2. PCR and RE of E240

E240 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.

Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template E240KOD Pllus ver.2Total
E240①283551.51.55150
E240②283551.51.55150

PCR program

94℃2min
98℃10sec35 cycles
55℃30sec
68℃4min
4℃forever

Electrophoresis

PCR purification

Sample numberConcentration(ng/µL)
E240①42.6
E240②55.3

RE

To insert E240 to pSB4K5 by 3A assembly, we digested the PCR products of E240 by XbaI and PstI

Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
E240①X-P3050.5XbaI0.2PstI0.214.150
E240②X-P3050.5XbaI0.2PstI0.214.150

PCR purification

Sample numberConcentration(ng/µL)Volume(µL)
E240①X-P21.840
E240②X-P32.445

-20℃ freezer

3. Error PCR

Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template Δ1TemplateTemplateKOD Pllus ver.2Total
ΔTMD①323551.51.51--150
ΔTMD②323551.51.5-1-150
ΔTMD③323551.51.5--1150
94℃2min
98℃10sec20 cycles
68℃4min
4℃forever

After the digestion by DpnI, we transfected 2µL of sample to 20µL of competent cell.

Tuesday, August 3

1. The result of transformation

ΔTMD①Many colonies
ΔTMD②No colony
ΔTMD③Many colonies

We picked two colonies from ΔTMD① and ΔTMD③, and cultured 37℃ 8/3~8/4.

2. The construction of ML and MS

Miniprep
Sample numberConcentration(ng/µL)
pSB4K560.7
LacP26.8
RE
Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
LacP5060.6EcoRI0.2SpeI0.2360
pSB4K5(E-P)5060.6EcoRI0.2PstI0.2360
E240①(X-P)5060.6XbaI0.2PstI0.2360
E240②(X-P)5060.6XbaI0.2PstI0.2360

Incubate

Purification

PCR purification
Sample numberConcentration(ng/µL)
pSB4K5 E-P39.5
E240①X-P21.8
E240②X-P32.4

pSB4K5 E-P is concentrated 10µL and E240①X-P, E240②X-P are concentrated 1µL.

Ethanol precipitation

1-5-G dilute milliQ 2µL

Ligation
VectorInsert 1Insert 2Ligation HighTotal
ML1pSB4K5 E-P 1LacI E-S 1E240①X-P 1315
ML2pSB4K5 E-P 1LacI E-S 1E240②X-P 1315

Incubation 17:30~20:20

PCR of J23101-E240

J23101-E240 is important in the measurement of RPU, so we amplified this parts by PCR.

Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template J23101-E240KOD plus ver.2 Total
MS①323551.51.51150
MS②323551.51.5-150
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever
PCR purification
Sample numberConcentration(ng/µL)
J23101-E24040.6

Discussion J23101-E240(MS) is amplified corrrecly.

RE
Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
J23101-E240(E-P)4560.6EcoRI0.2PstI0.2860
PCR purification
Sample numberConcentration(ng/µL)Volume(µL)
J23101-E240 E-P 74.130

J23101-E240 is concentrated 7µL

Ligation
VectorInsertLigation HighTotal
MSpSB4K5 E-P 1J23101-E240 E-P 124

Incubation 20:00~20:30

Transformation
SampleConc(/µL)Sample Volume(µL)Competent Cell(µL)TotalPlateIncubation
ML1-12021LB kan8/3~8/4
ML2-12021
MS-12021

Thursday, August 5

1. Result of transformation

ML1Many colonies
ML2
MS

ML1 and ML2

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 ML1 and ML2 plate. We cultured both white and green colonies.

MS Self-ligated colony is red Many of colonies are red, however, green colonies are observed.

Culture and Master

Green colony ML1-1 ML1-2 ML2-1 MS1-1 MS1-2 MS1-3 White colony ML1-3 ML1-4 ML2-2 ML2-3 ML2-4

J23100 and LacP 8/5~8/6

2. Sequence

Sample numberConcentration(ng/µL)
ΔTMD①A28.9
ΔTMD①B25.3
ΔTMD③A26.6
ΔTMD③B24.0

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

1. Miniprep

ML1-1 ML1-2 ML1-3 ML1-4 ML2-1 ML2-2 ML2-3 ML2-4 MS1 MS2 MS3

2. RE

Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
5060.6EcoRI0.3PstI0.32.860
Electrophoresis

1 100bp 2λ 3λ 4100bp 5MS1 6MS2 7MS3 8ML1-1 9ML1-2 10ML1-3 11ML1-4 12ML2-1 13ML2-2 14ML2-3 15ML2-4 16MS1 17MS2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

KyotoExp100806-1.png

Discussion MS1 and MS2 are inserted correctly. ML1-1 and ML1-2 are inserted correctly. ML2-1 are inserted correctly. White colonies are inserted not lacP but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of LacI.

Error PCR(Retry)
Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template ΔTMD failed(50ng/µL)KOD plus ver.2Total
ΔTMD①323551.51.51150
ΔTMD②323551.51.51150
94℃2min
98℃10sec25 cycles
68℃4min
4℃forever

Add DpnI 2µL and incubate 1h.

Transformation
SampleConc(/µL)Sample Volum(µL)Competent Cell(µL)TotalPlateIncubation
ΔTMD①-45054LB kan8/6~8/9
ΔTMD②-45054
2-17-F-25052
2-I-525052LB amp
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