Team:HokkaidoU Japan/Notebook/aggregation Week 11

Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 by EcoRI and SpeI, Ag43-dT-pSB1AK3 (previously digested by HindIII) by XbaI and NotI, and pSTV28 by EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 by XbaI and SpeI as a control for confirmation of the ability of restriction enzyme.
Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)

DNA solution (35 ng/ul)	17 ul
EcoRI	1 ul
SpeI	1 ul
10xH buffer	3 ul
DW	8 ul
Total	30 ul

Insert2 (Ag43-dT-pSB1AK3)

DNA solution (35 ng/ul)	25 ul
XbaI	1 ul
NotI	1 ul
10xK buffer	1.5 ul
100xBSA	0.3ul
DW	1.5 ul
Total	30 ul

Vector(pSTV28)

DNA solution (15 ng/ul)	9 ul
EcoRI	1 ul
NotI	1 ul
10xH buffer	2 ul
10xBSA	0.2 ul
DW	7 ul
Total	20 ul

control (pT7-RBS-pSB1C3)

DNA solution (30~40 ng/ul)	10 ul
XbaI	1 ul
SpeI	1 ul
10xM buffer	2 ul
DW	6 ul
Total	20 ul

Number	Degree	Minute
1	37	120
2	70	20
3	4	HOLD

Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 14000 rpm, 30 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 15 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

We confirmed that the concentration of Insert1 DNA solution is 50 ng/ul, Insert2 DNA solution is 10~15 ng/ul and Vector DNA solution is 10 ng/ul.

Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28

Vector DNA (10 ng/ul)	4 ul
Insert1 DNA (50 ng/ul)	2 ul
Insert2 DNA (10~15 ng/ul)	4 ul
Ligation Mighty Mix	10 ul
Total	20 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

Transformation ligation product into DH5α.

1. Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Incubated for 2 hrs to get the resistance to Chloramphenicol.
5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
6. Plated 300 ul of the culture onto first dish and spread.
7. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
8. Incubated the plates at 37C for 14 hrs.

Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2

DNA solution	4 ul
Kapa-Taq(Taq polymerase)	5 ul
Forward Primer(pbad-f2 primer)	0.5 ul
Reverse Primer(PS-R down primer)	0.5 ul
Total	10 ul

Number	Degree	Second
1	95	120
2	95	30
3	53.3	30
4	72	60
5	72	60
6	4	HOLD

Cycle:2~4 x 35

• We noticed that this step was actually 68.9 degree after reaction.

We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls. Desired product is about 542bp.

Because of mis-setting the PCR program, we failed to amplify desired DNA fragment so we retried the colony PCR in same reagent, correct PCR machine program.

We noticed that the ligated DNA contains at least Ag43-dT-BioBrick_Suffix complex. We selected No. 2,4 for incubation for plasmid extraction and No. 5,6 for Aggregation check.

Analysis nucleotide sequence

We analyzed the nucleotide sequence of pBAD-RBS-Ag43-dT on pSB1AK3 and pSB1C3. We used these 6 kinds of primers. 100b up EX-F primer, pBAD-f1 primer, pBAD-f2 primer, Ag43-f1 primer, Ag43-f2 primer, Ag43-f3 primer, Ag43-f4 primer, 200b down PS-R primer

Aggregation check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

Aggregation check for No. 5,6 colonies selected by colony PCR at August 11th.

1. Prepared 5 ml LBA into glass tubes.
2. Re-suspended 2 colony mixture (No. 2 and No. 5 respectively).
3. Incubated at 37C for 18 hrs.

Digestion of RBS-phaB-pSB1A2 as Vector and RBS-eYFP-dT as Insert

To make a construct of RBS-phaB-RBS-eYFP-dT-pSB1A2, we digested RBS-phaB-pSB1A2 with SpeI & PstI, RBS-eYFP-dT with XbaI & PstI.

Insert (RBS-eYFP-dT)

DNA solution (40 ng/ul)	25 ul
XbaI	1 ul
NotI	1 ul
10xK buffer	1.5 ul
100xBSA	0.3 ul
DW	1.5 ul
Total	30 ul

Vector(RBS-phaB-pSB1A2)

DNA solution (35 ng/ul)	6 ul
SpeI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	10 ul
Total	20 ul

Number	Degree	Minute
1	37	120
2	60	15
3	4	HOLD

Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 15 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

We confirmed that the concentration of Insert DNA solution is 40 ng/ul and Vector DNA solution is 30 ng/ul.

Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28

Vector DNA (40 ng/ul)	3 ul
Insert DNA (30 ng/ul)	3 ul
Ligation Mighty Mix	6 ul
Total	12 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of RBS-phaB-RBS-eYFP-dT-pSB1A2

Transformation of ligation product into JM109.

1. Mixed 2 ul ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).
5. Plated 300 ul of the culture onto first dish and spread.
6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
7. Incubated the plates at 37C for 14 hrs.

Aggregation Check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

1. Mixed 25 ul Arabinose solution (20 %) to 5 ml LBC in glass tube.
2. Incubated for 24 hrs at 37C.

Result: The construct has aggregated not forming large cluster but small dot cluster, and the supernatant has muddiness.

Colony PCR of RBS-phaB-RBS-eYFP-dT-pSB1A2

DNA solution	4 ul
Kapa-Taq(Taq polymerase)	10 ul
Forward Primer(pbad-f2 primer: 10 ng/ul)	0.8 ul
Reverse Primer(PS-R down primer: 10 ng/ul)	0.8 ul
DW	4.4 ul
Total	20 ul

Number	Degree	Second
1	95	120
2	95	30
3	64.4	30
4	72	180
5	72	120
6	4	HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2(RBS-phaB-pSB1A2)as controls. Desired product is about 1800~2000bp.

We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.

Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction

1. Prepared 2 ml LBC into culture tubes.
2. Re-suspended 2 colony mixture (No. 1 and No. 2 respectively).
3. Incubated at 37C for 14 hrs.

Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.

Insert (RBS-phaB-RBS-eYFP-dT)

DNA solution (30 ng/ul)	21 ul
XbaI	1 ul
PstI	1 ul
10xM buffer	3 ul
DW	4 ul
Total	30 ul

Vector(RBS-phaC-RBS-phaA-pSB1A2)

DNA solution (about 30~40 ng/ul)	6 ul
SpeI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	10 ul
Total	20 ul

Number	Degree	Minute
1	37	180
2	60	15
3	4	HOLD

Number	Degree	Minute
1	37	120
2	70	20
3	4	HOLD

We suppose the Insert DNA (RBS-phaB-RBS-eYFP-dT) has contained Vector-dimer DNA as contamination. But desired DNA fragment (about 1600 bp) bond also appeared in the result of migration. We picked up the fragment and extracted from gel.

Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged in 14000 rpm, 30 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged in 15000 rpm, 15 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.

Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

Insert DNA (20 ng/ul)	4 ul
Vector DNA (50~60 ng/ul)	1.5 ul
Ligation Mighty Mix	6 ul
DW	0.5 ul
Total	20 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2

Transformation ligation product into JM109.

1. Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).
5. Plated 300 ul of the culture onto first dish and spread.
6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
7. Incubated the plates at 37C for 13 hrs.

We succeeded to transform the cells but noticed that we have used wrong DNA solution. We'll try this DNA synthesis again tomorrow.

Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.

Insert (RBS-phaB-RBS-eYFP-dT)

DNA solution (30 ng/ul)	21 ul
XbaI	1 ul
PstI	1 ul
10xM buffer	3 ul
DW	4 ul
Total	30 ul

Vector(RBS-phaC-RBS-phaA-pSB1A2)

DNA solution (about 30~40 ng/ul)	6 ul
SpeI	1 ul
PstI	1 ul
10xH buffer	2 ul
DW	10 ul
Total	20 ul

Number	Degree	Minute
1	37	120
2	60	15
3	4	HOLD

Number	Degree	Minute
1	37	120
2	70	20
3	4	HOLD

Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged in 14000 rpm, 30 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged in 15000 rpm, 15 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.

Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

Insert DNA (20 ng/ul)	4 ul
Vector DNA (50~60 ng/ul)	1.5 ul
Ligation Mighty Mix	6 ul
DW	0.5 ul
Total	20 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2

Transformation of ligation product into JM109.

1. Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).
5. Plated 300 ul of the culture onto first dish and spread.
6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
7. Incubated the plates at 37C for 17 hrs.

Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pT7-RBS-pSB1C3

To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI, and pT7-RBS-pSB1C3 with EcoRI and SpeI. As a control, we digested pT7-RBS-pSB1C3 with only EcoRI to confirm the digestibility of This DNA (The digestibility with SpeI has confirmed in August 26th).

Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)

DNA solution (about 35ng/ul)	41 ul
EcoRI	1 ul
SpeI	1 ul
10xH buffer	5 ul
DW	2 ul
Total	50 ul

Vector(pT7-RBS-pSB1C3)

DNA solution (about 30~40 ng/ul)	2 ul
EcoRI	1 ul
SpeI	1 ul
10xH buffer	2 ul
DW	14 ul
Total	20 ul

control (pT7-RBS-pSB1C3)

DNA solution (about 30~40 ng/ul)	2 ul
EcoRI	1 ul
10xH buffer	1 ul
DW	6 ul
Total	10 ul

Number	Degree	Minute
1	37	120
2	60	15
3	4	HOLD

We confirmed 4 bonds in the result of Inset DNA digestion. A Bond which shows the highest concentration and places about 8k ~ 10k bp area would be pBAD-RBS-eCFP-RBS-Ag43-pSTV28 construct. We considered about it bond whether remained from digestion, or at first formed dimer and separated by digestion. In either case, our target product has about 5200 bp and there is appropriate bond. We extracted the target bond from TBE gel and went next step.

Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT on pSB1C3

1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
2. Centrifuged in 15000 rpm, 15 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged in 15000 rpm, 10 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3

Insert DNA	4 ul
Vector DNA	1 ul
Ligation Mighty Mix	5 ul
Total	10 ul

Ligation reaction time was in detail below.

Degree	Minute
16	30
65	10
4	Hold

Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3

Transformation ligation product into DH5α.

1. Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Incubated for 2 hrs to get the resistance to Chloramphenicol.
5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
6. Plated 300 ul of the culture onto first dish and spread.
7. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
8. Incubated the plates at 37C for 13 hrs.

Colony PCR of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2

DNA solution	4 ul
Kapa-Taq(Taq polymerase)	10 ul
Forward Primer(phaA-1083bp-F primer: 10 ng/ul)	0.8 ul
Reverse Primer(PS-R down primer: 10 ng/ul)	0.8 ul
DW	4.4 ul
Total	20 ul

Number	Degree	Second
1	95	120
2	95	30
3	68.9	30
4	72	180
5	72	120
6	4	HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2(RBS-phaC-RBS-phaA-RBS-phaB-dT-pSB1A2)as controls. Desired product is about 1800~2000bp.

We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.

Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction

1. Prepared 2 ml LBA into culture tubes.
2. Re-suspended 2 colony mixture (No. 1 and No. 2 respectively).
3. Incubated at 37C for 20 hrs.