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Notebook: September

06.09.2013

PCR

Investigator: Franzi

Aim: Construction of iBB14 → Adding of pre- and suffix via PCR.

Dissolved BioBrick BBa_E1010 (RFP) from spring distribution Kit 2013 with 10 µl H2O (P3, 12N). Used as template for PCR.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion-Polymerase	95	3 min
1 µl	Primer 50 fw (10pmol/µl)	95	30 sec
1 µl	Primer 51 rv (10pmol/µl)	60	1 min	x35
1 µl	Template	72	1 min
1 µl	dNTPs	72	5 min
10 µl	5xGC buffer	4	Hold
34 µl	ddH20

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl elution buffer.

09.09.2013

Transformation

Investigator: Franzi

Aim: Construction of iBB14 → Retransformation of BBa_E1010.

Retransformation of 1 µl pSB1C3 BBa_E1010 into E. coli DH5α (30 min ice, 60 sec 42°C, 90 min 37°C + 900 µl LB), plated on LB_CM, oN 37°C.

PCR

Investigator: Franzi

Aim: Construction of iBB15 (MTS) → Adding pre- and suffix via PCR.

Volume	Reagent	Temp (°C)	Time
1 µl	Phusion-Polymerase	95	3 min
1 µl	Primer 52 fw (10pmol/µl)	95	30 sec
1 µl	Primer 53 rv (10pmol/µl)	65	30 sec	x32
1 µl	Template (provided by AG Bange)	72	45 sec
1 µl	dNTPs	72	7 min
10 µl	5xGC buffer	4	Hold
35 µl	ddH20

Gel electrophoresis

Gel substances

2% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane	Content	Expectations
2-6	none
7	MTS-PCR (1)	1 band at 107 bp
8	MTS-PCR (2)	1 band at 107 bp

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl elution buffer.

10.09.2013

Digest

Investigator: Franzi

Aim: Construction of iBB14-15 → Digestion of single parts.

10 µl DNA template
1 µl enzyme 1
1 µl enzyme 2
2 µl Cut Smart buffer
6 µl H2O

Samples:

Digestion of iBB15 with XbaI and PstI-HF

Digestion of iBB14 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1,5 h at 37° C.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of iBB14-15 → Ligation of two single parts into pSB1C3.

23 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
44 ng iBB14 (EcoRI/PstI-cut)
41 ng iBB15 (EcoRI/PstI-cut)
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

The samples were incubated for 2 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of iBB14-15 → Transformation of combined BioBrick in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB_CM, oN 37°C.

Digest

Investigator: Franzi

Aim: Construction of iBB15 → Digestion with EcoRI and PstI.

10 µl DNA template (iBB15 from PCR)
1 µl EcoRI-HF
1 µl PstI-HF
2 µl Cut Smart buffer
6 µl H2O

The samples were incubated for 30 min at 37° C.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of iBB15 → Ligation of iBB15 into pSB1C3.

23 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
40 ng iBB15 (EcoRI/PstI-cut)
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

The samples were incubated for 30 min at RT.

Transformation

Investigator: Franzi

Aim: Construction of iBB15 → Transformation of iBB15 in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB_CM, oN 37°C.

11.09.2013

Ligation

Investigator: Franzi

Aim: Construction of iBB15 → Repeat of ligation of iBB15 into pSB1C3.

Reason: No colonies of pSB1C3 iBB15 on transformation plates.

46 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
40 ng iBB15 (EcoRI/PstI-cut)
3 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 30 µl H2O

The samples were incubated for 30 min at RT.

Transformation

Investigator: Franzi

Aim: Construction of iBB15 → Repeat of transformation of iBB15 in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 1 ml LB), plated on LB_amp, oN 37°C.

Inoculation

Investigator: Franzi

Aim: Construction of iBB14-15 and iBB14, inoculation of pSB1C3 iBB14-15 and pSB1C3 BBa_E1010 for miniprep.

5 colonies of pSB1C3 iBB14-15 and 2 colonies of pSB1C3 BBa_E1010 inoculated in 5 ml LB_CM, oN 37°C.

12.09.2013

Miniprep

Investigator: Franzi

Aim: Construction of iBB14-15 and iBB14 → Miniprep of pSB1C3 iBB14-15 and pSB1C3 BBa_E1010.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 50 µl elution buffer.

Digest

Investigator: Franzi

Aim: Construction of iBB14-15 → Control digestion of pSB1C3 iBB14-15.

2 µl DNA template
0,5 µl EcoRI-HF
5 µl PstI-HF
1 µl Cut Smart buffer
6 µl H2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane	Content	Expectations
2	iBB14	pSB1C3: 2 kb iBB14-15: 850 bp iBB14 E/S: 750 bp
3	iBB14-15 clone 1
4	iBB14-15 clone 2
5	iBB14-15 clone 3
6	iBB14-15 clone 4
7	iBB14-15 clone 5
8	iBB14-15 clone 6

All clones except clone 3 are positive.

Sequencing

Investigator: Franzi

Aim: Construction of iBB14-15 → K4 and K5 sent for sequencing.

IBB14-15 K4 and K5 sequenced with primers VF2_fw and VR_rv (AGBOOOK565-568).

Digest

Investigator: Franzi

Aim: Construction of iBB15 → Repeat of digestion with EcoRI and PstI.

Reason: Transformations of 11.09.13 unsuccessful.

9 µl DNA template (iBB15 from PCR)
1 µl EcoRI-HF
1 µl PstI-HF
2 µl Cut Smart buffer
7 µl H2O

The samples were incubated for 2 h at 37° C.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of iBB15 → Repeat of ligation of iBB15 into pSB1C3.

20 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
40 ng iBB15 (EcoRI/PstI-cut)
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

The samples were incubated for 2,5 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of iBB15 → Repeat of transformation of iBB15 in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB_CM, oN 37°C.

Transformation

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Retransformation of BBa_R0010 and BBa_B0034.

Retransformation of BBa_R0010 (PlacI, pSB1C3), BBa_B0034 (RBS, pSB1A2) into E.coli DH5α (30 min ice, 60 sec 42°C, 60 min (PlacI)/30 min (RBS) 37°C + 900 µl LB), plated on LB_CM (PlacI)/LB_amp, oN 37°C.

13.09.2013

Sequencing

Investigator: Franzi

Aim: Construction of iBB14-15 → Sequencing results.

Sequencing of pSB1C3 iBB14-15 negative (wrong restriction enzymes used, constructed with BamHI, not standard 23)

PCR

Investigator: Franzi

Aim: Construction of iBB14315 → Adding of pre- and suffix via PCR.

PCR of iBB15 repeated like 09.09.13.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane		Content
2		none
3		iBB14315
4		iBB14315

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl elution buffer.

Transformation

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Retransformation of BBa_R0010 and BBa_B0034.

Reason: All transformations (12.09.13) unsuccessful.

Retransformation of PlacI and RBS like 12.9.13, in addition into competent cells provided by AG Bange; RT over weekend.

All transformations will be unsuccessful (16.09.13).

16.09.2013

Digest

Investigator: Franzi

Aim: Construction of iBB14-15 → Digestion with EcoRI/PstI and BamHI.

10 µl DNA template
1 µl enzyme 1
1 µl enzyme 2
2 µl Cut Smart buffer
6 µl H2O

Samples:

Digestion of PCR-products, iBB14 with EcoRI-HF and BamHI-HF, iBB15 with BamHI-HF and PstI-HF.

The samples were incubated for 1,5 h at 37° C.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of iBB14-15 → Ligation of iBB14 and iBB15 into pSB1C3.

20 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
40 ng iBB14 (EcoRI/PstI-cut)
40 ng iBB15 (EcoRI/PstI-cut)
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

The samples were incubated for 2 h at RT.

Transformation

Investigator: Franzi

Aim: Transformation-tests, Construction of iBB14-15 and RFP-MTS-test → Transformation into E. coli (AG Bölker).

IBB14-15: Complete ligation samples were transformed into E. coli (AG Bölker) (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB_CM, oN 37°C.

Retransformation of PlacI and RBS like above, RBS 30 min 37°C, plated on LB_amp.

Testtransformation of pSB1A3 J04450 into E. coli (own, AG Bange and AG Bölker), like above, plated on LB_amp.

Testtransformation will be all positive (17.09.13).

17.09.2013

Inoculation

Investigator: Franzi

Aim: Construction of iBB14-15 and RFP-MTS-test → Inoculation of transformants.

2 colonies of pSB1A2 RBS inoculated in 5 ml LB_amp, 2 colonies of pSB1C3 PlacI and 6 colonies of pSB1C3 iBB14-15 inoculated in 5 ml LB_CM, oN 37°C.

18.09.2013

Miniprep

Investigator: Franzi

Aim: Construction of iBB14-15 and RFP-MTS-test → Miniprep of transformants.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl elution buffer.

Digest

Investigator: Franzi

Aim: Construction of iBB14-15 → Control digestion of pSB1C3 iBB14-15.

2 µl DNA template (pSB1C3 iBB14-15)
0,5 µl EcoRI-HF
0,5 µl PstI-HF
1 µl Cut Smart buffer
6 µl H2O

The samples were incubated for 2 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe per 50 ml gel
6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane	Content	Expectations
2	iBB14-15 clone 1	850 bp
3	iBB14-15 clone 2	850 bp
4	iBB14-15 clone 3	850 bp
5	iBB14-15 clone 4	850 bp
6	iBB14-15 clone 5	850 bp
7	iBB14-15 clone 6	850 bp
8	iBB14	750 bp

All positive.

Sequencing

Investigator: Franzi

Aim: Construction of iBB14-15 → Sequencing.

Sequencing K1 + K4 with primers VF2_fw and VR_rv (AGBOOOK 569-572).

Digest

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Digestion of PlacI and RBS.

10 µl pSB1C3 PlacI
1 µl SpeI
1 µl PstI-HF
2 µl Cut Smart buffer
6 µl H2O

15 µl pSB1A3 RBS
1 µl XbaI
1 µl PstI
2 µl buffer 3.1
1 µl H2O

Both samples were incubated for 2 h at 37° C.

RBS was heat-inactivated (80°C, 30 min).

The relevant fragments of PlacI were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Ligation of PlacI and RBS into pSB1C3.

67 ng pSB1C3 PlacI
10 µl RBS
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

The samples were incubated for 2 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Transformation of pSB1C3 PlacI RBS into E. coli DH5α.

IBB14-15: Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB_CM, oN 37°C.

19.09.2013

Sequencing

Investigator: Franzi

Aim: Construction of iBB14-15 → Sequencing results.

Point mutation at position 743 (G→A, silent) in both clones.

Shipping of the BioBricks

Investigator: Franzi

Aim: Finally sending the BioBricks to the registry. "☺"

Prepared all completed BioBricks for shipping: 250 ng Brick in 10 µl H2O.

PCR

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Control colony-PCR.

Colony-PCR of 7 colonies of pSB1C3 PlacI RBS.

Volume	Reagent	Temp (°C)	Time
0,5 µl	Q5 HF polymerase	95	3 min
0,5 µl	Primer fw (54, RBS fw)	95	30 sec
0,5 µl	Primer rv (38, VR_rv)	65	30 sec	x33
0,5 µl	dNTPs	72	45 sec
1 µl	Q5 buffer	72	7 min
18 µl	H2O	4	Hold

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
6x loading buffer (for samples)

Lane	Content	Expectations
2	pSB1C3 PlacI RBS colony 1	positive: 180 bp negative: none
3	pSB1C3 PlacI RBS colony 2
4	pSB1C3 PlacI RBS colony 3
5	pSB1C3 PlacI RBS colony 4
6	pSB1C3 PlacI RBS colony 5
7	pSB1C3 PlacI RBS colony 6
8	pSB1C3 PlacI RBS colony 7

Inoculation

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Inoculation of pSB1C3 PlacI RBS for miniprep.

K3, 4 and 7 inoculated in 5 ml LB_CM, oN 37°C.

20.09.2013

Miniprep

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Miniprep of pSB1C3 PlacI RBS.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl elution buffer.

Digest

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Digestion of pSB1C3 PlacI RBS and iBB14-15.

10 µl DNA template
1 µl enzyme 1
1 µl enzyme 2
2 µl Cut Smart buffer
6 µl H2O

Samples:

Digestion of pSB1C3 iBB14-15 with XbaI and PstI-HF

Digestion of pSB1C3 PlacI RBS K3,4,7 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1,5 h at 37° C.

The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

Ligation

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Ligation of PlacI RBS and iBB14-15 into pSB1A3.

40 ng pSB1C3
150 ng iBB14-15
160 ng PlacI RBS K3,4 or 7
2 µl 10x T4 DNA ligase buffer
1 µl T4 DNA ligase
ad 20 µl H2O

The samples were incubated for 1,5 h at RT.

Transformation

Investigator: Franzi

Aim: Construction of RFP-MTS-test → Transformation of pSB1A3 PlacI RBS iBB14-15 into E. coli DH5α.

IBB14-15: Complete ligation samples were transformed into E. coli DH5α (30 min ice, 60 sec 42°C, 30 min 37°C + 900 µl LB), plated on LB_amp, oN 37°C.