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Latest revision as of 11:22, 17 October 2014

Notebook: July

01.07.2014

20.10 pMAD-Transformation of competent Bacillus subtilis WT 3610

Aim: Transformation of piGEM-016

100 µL of overnight culture were added to 10 mL of MNGE-Medium and incubated till an OD of 0,7 at 37°C which took 7 hours.

After reaching OD of 0,7 400 µL of the culture were transformed with 1,5 µg piGEM-021. After 1 hour incubation at 37°C 100 µL Expression mix was added and incubated for 1h as well.

In the end the 500 µL attempt were plated out on MLS-X-Gal plates and incubated at 30°C overnight until colonies could be seen.

Sequencing results of constructs from 30.06.2014

Premix	Label-Nr.	Construct	Result
1	AGB0023411	pET24d-Hag-SpeI construct cl. 2	Correct insertion of Hag-SpeI
2	AGB0023412	pET24d-Hag-DARPin construct cl. 7	Correct insertion of Hag-DARPin

14.48 PCR of PheA-single

Aim: Amplify PheA-single fragment for Gibson Assembly in pET-28a

The PCR with TG_PheA-Arc1p-C-8x FP und TG-PheA-single-GA RP should generate the 1716 bp fragment for Gibson Assembly from the PheA-Arc1p-C-2x-pET28a construct (14.23).

The used Primer concentration was 1 µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.

Content	Volume [µL]
Water	29.5
5x GC Buffer	10
DMSO	2.5
TG_PheA-Arc1p-C-8x FP	2.5
TG_PheA-Arc1p-C-8x RP	2.5
PheA-Arc1p-C-2x-pET28a	1
dNTPs	1
Phusion-Polymerase	1
Total Volume	50

Step	Temperature [°C]	Time [min:sec]
1	98	2:00
2	add Polymerase
3	98	0:10
4	67.5	0:30
5	72	1:15
6	go to 3	32x
7	72	10:00
8	4	hold

The amplified fragment was purified on an agarose gel on which it showed the correct size. It was extracted from the gel for further use in a Gibson Assembly yielding a concentration of 198 ng/µL.

02.07.2014

14.49 Gibson Assembly

Aim: Integrate PheA-single into linearized pET-28a(XhoI,NdeI) via Gibson Assembly

Content	Volume [µL]
Water	5.34
pET-28a(XhoI,NdeI) (24 ng/µL)	4.2
PheA-single (198 ng/µL)	0.46
2x Gibson Mastermix	10
Total Volume	20

The reaction mixture was incubated for 1 h at 50 °C and 350 rpm. E. coli Top10 electrocompetent cells were transformed with a 1:3 and a 1:6 dilution of the mixture, incubated for 1 h at 37 °C and plated out on LB-Kan⁵⁰ plates that were incubated overnight at 37 °C.

19.3 Co-Transformation of piGEM-019 & -020 into E. coli BL21(DE3) with FliS

Aim: Transformation of plasmids with flagellin modifications and chaperone for Flagellin into protein producing strain

piGEM-019 and -020 were used to transform E. coli BL21 (DE3) together with a plasmid containing the flagellin chaperone FlaS in order to express both proteins at the same time after induction so that the flagellin monomer accumulates inside the cells.

The cells were plated out on Can/Amp-LB plates and incubated overnight at 37°C.

03.07.2014

14.50 Overnight culture

Aim: Amplify Plasmid in an overnight culture

Six clones of the transformed Top10 cells from 14.49 were picked and used to inoculate overnight cultures (6 x 5 mL).

18.49 new PCR of isolated Gibson assembly clones ( pET24d-Hag (piGEM-019) and cup1-1)

Aim: screening clones for right insert

The isolated plasmid 1:10 diluted from the picked clones 1-4 were used as PCR template.

The PCR with Flo54 and iGEM-025 should generate a 785 bp fragment and the one with iGEM-024 and -025 the 164bp domain fragment.

Content	Volume (µL)	MM 1 for 5 attempts (µL)	MM 2 for 5 attempts (µL)
Plasmid from clones 1-4 (1:10)	1	-
Primer Flo 54	-	2,5	-
Primer iGEM-024	-	-	2,5
Primer iGEM-025	-	2,5	2,5
Q5 mastermix 2X	-	50	50
Water	-	45	45
Mastermix	19	-	-
Total Volume	20	100	100

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	30x
6	72	4min
7	8	infinite

The agarose gel showed bands at ca. 750 bp which fits to the expected 780 bp fragment with primers piGEM-025 (cup rv) and Flo54 ( Hag fw).

The gel's second row shows the amplified cup-fragment under the 200 bp ladder mark. The expected size is ca. 164 bp and fits to the gel result.

Plasmid from clone 4 was used to transform E.Coli DH5α and named piGEM-021.

19.4 Expression-Test with transformed E. coli BL21 (DE3)

Aim: Checking the overexpression of Hag-SpeI and Hag-SpeI-DARPin

In order to check the expression of the proteins a single clone containing piGEM-019/-20 was used to inoculate 20 mL of LB-Can/Amp. The culture was incubated at 37°C until an OD of 0,7.

A 1 mL preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL). After that the cultures were induced with 20 µL IPTG for 2 hours.

An induction sample (I) was taken (320 µL with an OD of 2,2; 350 µL with an OD of 2).

PI and I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and the pellet resuspended in 60 µL water and 40 µL SDS-Buffer.

The four samples were analysed on an SDS-PAGE gel with 5 and 10 µL volume per sample.

The gel showed that the induction with IPTG was successful and the cells overproduce a protein after 2h incubation.

19.5 Protein overexpression with transformed E. coli BL21 (DE3)

Aim: Overexpression of flagellin modifications for crystallization

After the positive expression test the protein expression was done on a higher level with an overnight lactose induction.

For that purpose 2 x 1 L LB were inoculated with a solution of LB and every clone on the cotransformation plate. All clones from the piGEM-019/ -020 + FlaS cotransformation plates were picked and resuspended in 1 mL LB. 500 µL were added t the 1 L culture flasks. Each flask was additionally mixed with 1 mL Ampicillin (100 mg/ mL) and 1 mL Canamycin (50 mg/ mL).

The expression was induced overnight with lactose (12,5 g/ L). 50 g lactose were solved in 200 mL millipore water. Each culture was mixed with 50 mL of the lactose/ water suspension.

The cultures were incubated overnight at 30°C.

	Lactose solution	BL21-piGEM-019 culture	BL21-piGEM-020 culture
LB	-	1 L	1 l
Ampicillin	-	1 ml	1 ml
Canamycin	-	1 ml	1 ml
Lactose	50g	-	-
Millipore water	200ml	-	-
Lactose solution	-	50 ml	50 ml
BL21-piGEM-019 clones	-	+	-
BL21-piGEM-020 clones	-	-	+

04.07.2014

Sequencing

Premix	Label-Nr.	Construct	Primers
1	AGB0023413	pET24d-Hag-SpeI-Cup constr. Cl. 4	T7 Term

14.51 Preparation of plasmids

Aim: Purify the amplified plasmids from overnight cultures

The plasmids were purified from the overnight cultures created in 14.50 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.

19.5 Protein overexpression with transformed E. coli BL21 (DE3)

1 mL of culture was spinned down, mixed with 60 µL water, 40 µL SDS-PAGE loading buffer and analysed on a SDS-PAGE gel.

The gel shows a high concentration of a specific protein.

The overnight induced cells were raised till an OD of 2-3 and spinned down at 4000 rpm. The pellets were frozen in liquid nitrogen and stored at -80µC.

19.6 Co-Transformation of piGEM-021 & with FlaiS E. coli BL21(DE3)

Aim: Transformation of plasmids with flagellin-cup modification and chaperone for Flagellin into protein producing strain

piGEM-021 and pFlaiS were used to transform into E.Coli BL21(DE3) and plated out on LB-Canamycin plates. The plate was incubated at 37µC overnight.

05.07.2014

19.7 Expression-Test with transformed E. coli BL21 (DE3) with piGEM-021

Aim: Checking the overexpression of Hag-SpeI-cup1-1

In order to check the expression of the proteins a single clone containing piGEM-021 was used to inoculate 20 mL of LB-Can/Amp. The culture was incubated at 37°C until an OD of 0,7.

A 1 mL preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL). After that the cultures were induced with 20 µL IPTG for 2h.

An induction sample (I) was taken (520 µL with an OD of 1,34).

PI and I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and the pellet resuspended in 60 µL water and 40 µL SDS-Buffer.

The samples were analysed on an SDS-PAGE gel with 5 and 10 µL volume per sample. Additionally PI and I from the expression test with transformed piGEM-021 were loaded on the gel as well as a comparison.

The gel shows a high concentration of a specific protein so that the test could be seen as successful.

19.6 Protein overexpression with transformed E. coli BL21 (DE3) (piGEM-021)

Aim: Overexpression of flagellin modifications for crystallization

After the positive expression test the protein expression was done on a higher level with an overnight lactose induction.

For that purpose 2 x 1 L LB were inoculated with a solution of LB and every clone on the cotransformation plate. All clones from the piGEM-021 + FlaiS cotransformation plates were picked and resuspended in 1 mL LB. 500 µL were added t the 1 L culture flasks. Each flask was additionally mixed with 1 mL Ampicillin (100 mg/ mL) and 1 mL Canamycin (50 mg/ mL).

The expression was induced overnight with lactose (12,5 g/ L). 25 g lactose were solved in 100 mL millipore water which was heated 1 min in the microwave for solvation. Each culture was mixed with 50 mL of the lactose/ water suspension.

The cultures were incubated overnight at 30°C.

06.07.2014

20.11 Overnight culture of blue clones

Aim: transformation of Bacillus subtilis WT3610 with plasmid

Colonies were grown on the plates with transformed piGEM-021. The blue/ white screening showed positive transformed blue clones. 3 clones of different morphology per plate were picked and used for inoculation of LB-MLS (4 mL LB, 4 µL lincomycin, 4 µL erythromycin). Incubation was carried out overnight at 30°C with the 3 cultures.

19.6 Protein overexpression with transformed E. coli BL21 (DE3) (piGEM-021)

The gel shows a high concentration of a specific protein after overnight induction with lactose referring to the Hag-Cup construct.

The overnight induced cells were raised till an OD of 2-3 and spinned down at 4000 rpm. The pellets were frozen in liquid nitrogen and stored at -80°C.

07.07.2014

20.12 First temperature shift

Aim: integration of pMAD-Insert into Bacillus chromosome via flanks

The 3 overnight cultures were used to inoculate 10 mL LB MLS until the culture obtained an OD of 0,1. The cultures were incubated at 30°C for 2 hours.

Then the temperature was shifted to 42°C for 6 hours. Unfortunately the temperature decreased to 33°C because of a wrong incubator setting. New overnight cultures were inoculated with 50 µL from those used in the morning. The new cultures were incubated overnight at 30°C.

14.52 Transformation of BL21 cells

Aim: Transform E. coli BL21(DE3) with PheA-single-pET28a

40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-single-pET28a from two different clones, incubated for 1 h at 37 °C and plated out on LB-Kan⁵⁰ plates that were incubated overnight at 37 °C.

08.07.2014

Sequencing results of constructs from 04.07.2014

Premix	Label-Nr.	Construct	Result
1	AGB0023413	pET24d-Hag-SpeI-Cup constr. Cl. 4	Correct insertion of Cup1-1 into Hag

14.53 Overnight culture

Aim: Inoculate Overnight culture of PheA-single-pET28a

5 mL of LB-Kan⁵⁰ medium were inoculated with a clone from 14.52 bearing the PheA-single-pET28a plasmid.

20.13 First temperature shift

Aim: integration of pMAD-Insert into Bacillus chromosome via flanks

The 3 overnight cultures were used to inoculate 10 mL LB MLS until the culture obtained an OD of 0,1. The cultures were incubated at 30°C for 2h.

Then the temperature was shifted to 42°C for 6h.

After the heat shock 100 µL dilutions from 10^-4 to 10^-6 of each culture were plated out on MLS-X-Gal so that nine plates could be incubated overnight at 42°C.

19.7 Purification of Flagellin-Hag-SpeI from frozen pellet

The pellets which were stored at -80°C were resuspended in 20 mL buffer A and cracked with the microfluidizer. The cell lysate was centrifuged at 20000 rpm for 20 min at 4&degC. The supernatant was transferred to a new 50 mL falcon and stored on ice.

The following steps were performed:

equilibration with Buffer A 10 min
taking 40 µL supernatant (load)+ 10 µL SDS-Buffer - L-Sample
50 mL load on column
taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample
first washing with 25ml Buffer A (half the load)
taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample
equilibrate with Buffer B (output pipe off the column into glas, input into B)
hanging pipe on column, 6 x 2 ml Evolutions - E 1-6
taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-sample 1-6

regeneration of column:

10min water
10min EDTA
10min water
10min NiSO4
10min water

Meanwhile the elutions 1-6 were pooled (combined 12 mL) in a 50 mL falcon and transferred in a concentrator column which was centrifuged until the volume in the filter was 1,5 mL for injection into the gel filtration station.

Parallel the L-, FL-, W- and E 1-6-samples were analysed via SDS-PAGE gel in order to proof the purification in the elution fractions.

The gel shows a huge protein concentration in E1 and E2 so that the purification could be continued with gel filtration. For that purpose the filtrate out of the concentrator was transferred into a new 2 mL tube resuspending very well without air bubbles.

17 mL of GeFi-buffer were injected into the loop. After that the protein filtrate was injected as well.

The GeFi was started according to the following settings:

Flow rate: 2500
max. pressure: 0,65 mPa
First endtimer at 90 mL volume before fractioning

After observing a peak on the screen 40 µL of different fractions (C7 , C9, C11, D1, D3, D5, D7) covering the peak were taken for analysis via SDS-PAGE gel.

The gel shows that the elutions contain the protein concentrated. The Elutions C7-D3 were pooled and transferred to the concentrator until an end volume of 200 µL.

The protein concentrate was used for crystallisation pure and in a 1:2 dilution with GeFi buffer.

Core I and II were used for setting drops.

Sequencing of purified cPCR samples from pMAD transformation

Premix	Label-Nr.	Construct	Primers
1	AGB0023414	Hag-Spe1 in B.s. genome clone 1	Hag-Nco-fw
2	AGB0023415	Hag-Spe1 in B.s. genome clone 3	Hag-Nco-fw
3	AGB0023416	Hag-Spe1-DARPin in B.s. genome clone 1	Hag-Nco-fw
4	AGB0023417	Hag-Spe1-DARPin in B.s. genome clone 3	Hag-Nco-fw

09.07.2014

20.14 Second temperature shift

Aim: flip out of the pMAD backbone

One blue colony per diluted clone was used to inoculate 4 mL LB. The 6 cultures were incubated at 30°C for 6 hours and afterwards for 3 hours at 42°C.

Dilutions from 10^-4 - to 10^-6 were plated out on 18 X-Gal plates without MLS selection. The positive clones should not contain the resistance inside the backbone as well as the galactosidase. The plates were incubated at 42°C overnight.

Results of Sequencing of cPCR samples from pMAD transformation from 08.07.14

Premix	Label-Nr.	Construct	Result
1	AGB0023414	Hag-SpeI in B. subtilis genome clone 1	No SpeI insertion
2	AGB0023415	Hag-SpeI in B. subtilis genome clone 3	No SpeI insertion
3	AGB0023416	Hag-SpeI-DARPin in B. subtilis genome clone 1	Not sequenced through
4	AGB0023417	Hag-SpeI-DARPin in B. subtilis genome clone 3	Not sequenced through

The sequencing will be repeated with the reverse Primer instead of the forward primer. In case of the Hag-SpeI clones, different clones have to be taken for sequencing.

19.7 Purification of Flagellin-Hag-SpeI from frozen pellet

After one day in well B1 Core I fine crystals could be seen.

14.54 Test Expression

Aim: Test the expression of PheA-single

Using the preculture from 14.53 60 mL of LB-Kan⁵⁰ medium were inoculated with 600 µL of the preculture and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The culture was cooled down to 18 °C and the protein production was induced with 12 µL of 500 mM IPTG. Over night the culture was incubated at 18 °C and 220 rpm. Afterwards the cell culture was centrifuged (17000 rpm, 20 min, 4 °C), the pellet resuspended in 3 mL buffer A and stored at -20 °C. A glycerolstock of the strain containing the PheA-single-pET28a plasmid was stored at -80 °C.

10.07.2014

Sequencing of cPCR samples from pMAD transformation

Premix	Label-Nr.	Construct	Primer
1	AGB0023439	Hag-SpeI in B. subtilis genome clone 7	Hag-BamHI-rv
2	AGB0023440	Hag-SpeI-DARPin in B. subtilis genome clone 1	Hag-BamHI-rv
3	AGB0023441	Hag-SpeI-DARPin in B. subtilis genome clone 3	Hag-BamHI-rv

19.8 Purification of Flagellin-Hag-Cup1-1 from frozen pellet

The pellets which were stored at -80°C were resuspended in 20 mL buffer A and cracked with the microfluidizer. The cell parts were spinned down at 20000 rpm for 20 min at 4°C. The supernatant was transferred to a new 50 mL falcon and stored on ice.

The following steps were performed:

equilibration with Buffer A 10 min
taking 40 µL supernatant (load)+ 10 µL SDS-Buffer - L-Sample
50 mL load on column
taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample
first washing with 25ml Buffer A (half the load)
taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample
equilibrate with Buffer B (output pipe off the column into glas, input into B)
hanging pipe on column, 6 x 2 ml Evolutions - E 1-6
taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-sample 1-6

regeneration of column:

10min water
10min EDTA
10min water
10min NiSO4
10min water

Meanwhile the Elutions 1-6 were pooled (combined 12 mL) in a 50 mL falcon and transferred in a concentrator column which was centrifuged until the volume in the filter was 1,5 mL for injection into the gel filtration station.

Parallel the L-, FL-, W- and E 1-6-samples were analysed on a SDS-PAGE gel in order to proof the purification in the elution fractions.

The gel shows a huge protein concentration in E1 and E2 so that the purification could be continued with the gel filtration. For that purpose the filtrate out of the concentrator was transferred into a new 2 mL tube resuspending very well without air bubbles.

17 mL of GeFi-buffer werd injected into the loop. After that the protein filtrate was injected as well.

The GeFi was started according to the following settings:

Flow rate: 2500-3000 µL / min
max. pressure: 0,60 - 0,65 mPa
First end timer at 80-90 mL volume before fractioning
Injectvalve

After observing a peak on the screen 40 µL of different fractions (C7 , C9, C11, D1, D3, D5, D7) covering the peak were taken for analysis via SDS-PAGE gel.

The gel shows that the elutions contain the protein concentrated. The Elutions C7-D7were pooled and transferred to the concentrator until an end volume of 200 µL.

The protein concentrate (27,4 mg/mL A280) was used for crystallization pure and in a 1:2 dilution with GeFi buffer.

Core I - IV were used for setting drops.

20.15 selection of positive clones

Aim: checking the correct flip out of the pMAD backbone

From the dilution plates was one WHITE clone picked and transferred on a Master X-Gal Plate as well as on a MLS plate so that nine clones were proven for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C overnight.

13.66 Digest of piGEM-002 NcoI/SpeI

Aim: digest for isolation of piGEM-002 without GFP, replacing it with mCherry out of pMA17

piGEM-002 was digested with NcoI/SpeI in order to isolate the backbone without the GFP so that an mCherry could be integrated via Gibson assembly.

Component	Volume (µl)
piGEM-002 (73 ng/µL)	14
CutSmart 10x	2
SpeI	0,2
NcoI	0,2
Water	3,6
Total Volume	20

The digested vector was isolated via gel extraction.

14.55 Purification of test expression

Aim: Purify PheA-single on a small scale

Lysozyme was added to the cell suspension from 14.54 to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 °C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.

60 mL LB-Kan⁵⁰ medium was inoculated from the glycerolstock prepared in 14.54 and incubated at 37 °C and 220 rpm over night.

13.67 mCherry PCR amplification

Aim: PCR for the amplification of mCherry

Mix	Master-Mix I	Master-Mix II
Template: pMA17 1:10 (25 ng/ µL)	5	5
Primer iGEM-027	5	-
Primer iGEM-028	5	-
Primer iGEM-029	-	5
Primer iGEM-030	-	5
Phusion DNA-polymerase	10	10
Phusion Buffer 5x	40	40
dNTP-mix	5	5
MgCl2	5	5
DMSO	5	5
Water	120	120
Total Volume	200	200

The PCR was ran as a gradient from 55-65°C annealing temperature overnight.

The gel shows the successful amplification of the two parts of mCherry.

11.07.2014

Sequencing results of cPCR samples from pMAD transformation

Premix	Lebel-Nr.	Construct	Primer
1	AGB0023439	Hag-SpeI in B. subtilis genome clone 7	Positive, but HagII deletion/ frame shift
2	AGB0023440	Hag-SpeI-DARPin in B. subtilis genome clone 1	1 point mutations in domain, deletion in HagII/ Frameshift
3	AGB0023441	Hag-SpeI-DARPin in B. subtilis genome clone 3	2 point mutations in domain, deletion in HagII/ Frameshift

Sequencing of pMAD-constructs

Aim: finding out if the mutations have been in the constructs or made by Bacillus

Premix	Label-Nr.	Construct	Primer
1	AGB0023442	piGEM-005 - Hag-SpeI	pMAD-seq-rv
2	AGB0023443	piGEM-016 - Hag-Cup	pMAD-seq-rv
3	AGB0023444	piGEM-018 - Hag-DARPin	pMAD-seq-rv

13.67 mCherry PCR amplification

Aim: digest for isolation of piGEM-002 without GFP, replacing it with mCherry

Gel analysis shows that the PCR was successful at every annealing temperature. MgCl₂ and DMSO were important for the function of the polymerase.

14.56 Expression of PheA-single

Aim: Express PheA-single for further use

10 x 500 mL LB-Kan⁵⁰ medium were inoculated with 5 mL of the overnight culture prepared in 14.55 and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The flasks were cooled to 18 °C and protein production was induced by adding 100 µL 500 mM IPTG. The cultures were incubated over night at 18 °C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 °C), resuspended in buffer A and stored at -20 °C until further use.

14.07.2014

13.68 Gibson Assembly with mCherry I and II & linearized piGEM-002

Aim: recombination of piGEM-002 and mCherry

Component	Volume (µl)
Gibson-mix	15
Insert I (mCherry I 460 ng/µL)	1,25
Insert II (mCherry II 460 ng/µL)	1,25
piGEM-002 NcoI/SpeI (5,5 ng/µL)	2,5
Total Volume	20

The mix was incubated 1 hour at 50°C, and used to transform E. coli XL1-Blue that were plated out on LB-Amp plates. Incubation overnight at 37°C.

14.57 Purification of PheA-single

Aim: Purify PheA-single in three steps

The resuspended cells from 14.56 were lysed using a french press. The lysate was incubated on ice with 5 µg/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C) to pellet cell debris. The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-0x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-0x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.

Sequencing results of pMAD-constructs

Aim: finding out if the mutations have been in the constructs or made by Bacillus

Premix	Label-Nr.	Construct	Primer
1	AGB0023442	piGEM-005 - Hag-SpeI	Deletion in FlankII
2	AGB0023443	piGEM-016 - Hag-Cup	Deletion in FlankII
3	AGB0023444	piGEM-018 - Hag-DARPin	Deletion in FlankII

The Flank was checked by sequencing and showed no deletion in the flank.

15.07.2014

13.68 Gibson Assembly with mCherry I and II & linearized piGEM-002

Aim: recombination of piGEM-002 and mCherry

No colonies were on the plates. The Gibson assembly was repeated with higher concentrated vector.

14.58 PCR of Arc1p-C-single

Aim: Amplify Arc1p-C-single fragment for Gibson Assembly in pET-28a

The PCR with TG_Arc1p-C FP and TG_PheA-Arc1p-C-8x RP should generate the 579 bp fragment for Gibson Assembly from the PheA-Arc1p-C-2x-pET28a construct (14.23).

The used primer concentration was 1 µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.

Content	Volume [µL]
Water	29.5
5x GC Buffer	10
DMSO	2.5
TG_Arc1p-C FP	2.5
TG_PheA-Arc1p-C-8x RP	2.5
PheA-Arc1p-C-2x-pET28a	1
dNTPs	1
Phusion-Polymerase	1
Total Volume	50

Step	Temperature [°C]	Time [min:sec]
1	98	2:00
2	add Polymerase
3	98	0:10
4	67.5	0:30
5	72	1:15
6	go to 3	32x
7	72	10:00
8	4	hold

The amplified fragment was purified on an agarose gel on which it showed the correct size. It was extracted from the gel for further use in a Gibson Assembly yielding a concentration of 122 ng/µL.

14.59 Gibson Assembly

Aim: Integrate Arc1p-C-single into linearized pET-28a(XhoI,NdeI) via Gibson Assembly

Content	Volume [µL]
Water	5.38
pET-28a(XhoI,NdeI) (24 ng/µL)	4.2
Arc1p-C-single (122 ng/µL)	0.42
2x Gibson Mastermix	10
Total Volume	20

The reaction mixture was incubated for 1 h at 50 °C and 350 rpm. E. coli Top10 electrocompetent cells were transformed with a 1:3 and a 1:6 dilution of the mixture, incubated for 1 h at 37 °C and plated out on LB-Kan⁵⁰ plates that were incubated overnight at 37 °C.

16.07.2014

14.60 Overnight culture

Aim: Amplify Plasmid in an overnight culture

Six clones of the transformed Top10 cells from 14.59 were picked and used to inoculate overnight cultures (6 x 5 mL).

17.07.2014

13.69 new Gibson Assembly with mCherry I and II & linearized piGEM-002

Aim: recombination of piGEM-002 and mCherry

In order to receive the construct more piGEM-002 a different ration of the used parts was used for a new Gibson assembly.

From Insert I was a higher amount taken becaue of the bigger fragment size. New attempts with 1:2 (vector:insert) and 1:3 ration:

Component	1:2 (µl)	1:3 (µl)
Gibson-mix	15	15
Insert I (mCherry I 460 ng/µL)	2,2	1,1
Insert II (mCherry II 460 ng/µL)	2,5	1,25
piGEM-002 NcoI/SpeI (42 ng/µL)	2,7	1,25
Total Volume	20	20

Gibson reactions were used to transform E. coli XL1-Blue after 1h incubation at 50°C and plated out on LB-Amp. Incubation was proceeded at 37°C overnight.

21.1 Swarming Assays

Aim: Check motility of Bacillus transformants

For testing the motility swarming agar (0,7% LB-Agar) and swimming agar (0,3% LB-Agar) were made and given away for autoclaving.

Overnight cultures of Bacillus transformants (DARPin clone 3 & 8, Cup1-1 clone 2 & 3, Hag-SpeI clone 7), PY79 and WT3610 were incubated at 37°C.

14.61 Isolation of plasmids

Aim: Purify the amplified plasmids from overnight cultures

The plasmids were purified from the overnight cultures created in 14.60 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.

18.07.2014

21.1 Swarming Assay

Aim: Check motility of Bacillus transformands

The overnight cultures were set to an OD of 10. 10 µL of each culture were spotted on a swarming and a swimming plate. The distance of the front line was measured after periods of time.

	DARPin cl. 3		DARPin cl. 8		Cup1-1 cl. 2		Cup1-1 cl. 3		Hag-SpeI cl. 7		WT3610		PY79
Time	Swim	Swarm	Swim	Swarm	Swim	Swarm	Swim	Swarm	Swim	Swarm	Swim	Swarm	Swim	Swarm
1h	-	-	0,5	2	-	1	-	1,5	3	6	2	3	-	-
2,5h	-	-	0,5	3,5	-	3	-	2,5	12	40<	7	20	-	-

19.07.2014

21.2 Swarming Assay

Aim: Check motility of Bacillus transformands

In order to repeat the swarming assay new overnight cultures of Bacillus subtilis WT3610, the mutants DARPin clone 3 & 8, Cup1-1 clone 2 , Hag-SpeI clone 7 in 10 mL LB and incubated at 37°C.

20.07.2014

21.2 Swarming Assay

Aim: Check motility of Bacillus transformants

The overnight cultures were set to an OD of 10. 10 µL of each culture were spotted on a swarming and a swimming plate. The plates with the drops were dried for half an hour on the bench. The distance of the front line was measured after periods of time. The measurement at t=0 is the radius of the start spot. The following measurements refer to the difference between the start spot and the new front line.

	Swarming	Swimming
WT 3610	+	+
Hag-SpeI cl. 7	+	+
DARPin cl. 3	-	-
DARPin cl. 8	-	-
Cup1-1 cl. 3	-	-
Cup1-1 cl. 8	-	-

21.07.2014

13.70 Screening clones of Gibson plates

Aim: checking success of Gibson assembly

On the plate with 1:2 ratio have been 2 clones, on the 1:3 ratio plate have been 3 clones. All clones were picked for inoculation of minipreps (5 mL LB-Amp, incubation overnight).

Sequencing cPCR Products from B. subtilis mutants with Flank fw/rv

Premix	Label-Nr.	Construct	Primer
1	AGB0023452	B. subtilis - Hag-SpeI Cl.7	Flo89 Flank-fw
2	AGB0023453	B. subtilis - Hag-SpeI Cl.7	Flo90 Flank-rv
3	AGB0023454	B. subtilis - Hag-Cup1-1 Cl.2	Flo89 Flank-fw
4	AGB0023455	B. subtilis - Hag-Cup1-1 Cl.2	Flo90 Flank-rv

14.62 Transformation of BL21 cells

Aim: Transform E. coli BL21(DE3) with Arc1p-C-single-pET28a

40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid Arc1p-C-single-pET28a from two different clones, incubated for 1 h at 37 °C and plated out on LB-Kan⁵⁰ plates that were incubated overnight at 37 °C.

19.9 Protein overexpression with transformed E. coli BL21 (DE3) (piGEM-021)

Aim: Overexpression of flagellin modifications for a second crystallization attempt

After freshly packed GeFi Columns we decided to purify the Fla-Cup again for crystallization.

For that purpose 2 x 1 L LB were inoculated with a solution of LB and every clone on the cotransformation plate. All clones from the piGEM-021 + FlaiS cotransformation plates were picked and resuspended in 1 mL LB. 500 µL were added to the 1 L culture flasks. Each flask was additionally mixed with 1 mL ampicillin (100 mg/ mL) and 1 mL canamycin (50 mg/ mL).

The expression was induced overnight with lactose (12,5 g/ L). 25 g lactose were solved in 100 mL millipore water which was heated 1 min in the microwave for solvation. Each culture was mixed with 50 mL of the lactose/ water suspension.

The cultures were incubated overnight at 30°C.

21.3 Expression of Flagellin

Aim: Check the expression of the modified flagella of Bacillus mutants

In order to check the expression of the flagellum in the Bacillus mutants cultures in 100 mL LB medium were grown until an OD of 0,8 at 37°C. 10 mL culture were centrifuged at 4000 rpm for 10 min to get the pellet. The pellet was resuspended in 60 µL water and 40 µl SDS-loading buffer. After that the sample was analysed via SDS-PAGE. Overexpressed E. coli flagellin was used as a positive control. The analysed samples should run on the same level as the ones produced in E. coli samples in case of a positive expression.

The gel showed that the Hag-SpeI mutant generates a band on the same level as the WT3610 which is a positive result because the modified flagellin with SpeI site has no remarkable change of molecule mass. The Bacillus transformation was successful though.

The mutants with the inserted Cup and DARPin showed no significant expression of the flagellin. Either the export of the flagellin is not possible for Bacillus because of the size/ folding of our constructs and ends in degradation of our modifications or the expression is too low to be analysed via SDS-PAGE. In that case it would not be efficient enough for further usage. The results of the SDS-PAGE fits to the previously made Swarming assays.

A new strategy had to be planned for further work.

22.07.2014

Sequencing cPCR Products from B. subtilis mutants with Flank fw/rv

Premix	Label-Nr.	Construct
1	AGB0023452	B. subtilis - Hag-SpeI Cl.7
2	AGB0023453	B. subtilis - Hag-SpeI Cl.7
3	AGB0023454	B. subtilis - Hag-Cup1-1 Cl.2
4	AGB0023455	B. subtilis - Hag-Cup1-1 Cl.2

19.9 Protein overexpression with transformed E. coli BL21 (DE3) (piGEM-021)

Aim: Overexpression of flagellin modifications for a second crystallization try

The overnight cultures were grown till an OD of 2-3 and centrifuged. The pellets were frozen away at -80°C.

13.70 screening clones of Gibson plates

Aim: checking success of Gibson assembly

The isolated plasmids were used for PCR analysis. The primers iGEM-27 and -30 were used for a PCR to amplify the whole mCherry with a size of ca. 711 bp.

Mix	Mastermix	PCR attempt
Template: plasmid from clones 1:10	-	0,5
Primer iGEM-027	3	-
Primer iGEM-030	3	-
dNTP-mix	3	-
Phusion DNA-polymerase	3	-
Phusion Buffer 5x	36	-
MgCl2	-	-
DMSO	-	-
Water	132	-
Mastermix	-	19,5
Total Volume	180	20

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	1 min
5	Go To 2	30x
6	72	4min
7	8	infinite

The gel shows that clone 1:3.2 might be positive for containing the mCherry fragment. The clone has to be sequenced.

21.2 Swarming Assay

Aim: Check motility of Bacillus transformands with D2-3-domain

The mutants showed expression on the SDS-gel. Swarming and swimming were tested to check the motility of his mutants for further planning of our domain constructions. The hag-construct contains the D2-3 domain of Salmonella.

WT3610 and the D2-3-mutant were used to inoculate 50 mL LB-medium for overnight culture incubated at 37&degC.

Sequencing cPCR Products from B. subtilis mutants with flank fw/rv

Premix	Label-Nr.	Construct	Results
1	AGB0023452	B. subtilis - Hag-SpeI Cl.7	Correct
2	AGB0023453	B. subtilis - Hag->SpeI Cl.7	Correct
3	AGB0023454	B. subtilis - Hag-Cup1-1 Cl.2	Correct sequencing till Hag
4	AGB0023455	B. subtilis - Hag-Cup1-1 Cl.2	Correct sequencing till Hag

14.63 Overnight culture

Aim: Inoculate Overnight culture of Arc1p-C-single-pET28a

5 mL of LB-Kan⁵⁰ medium were inoculated with a clone from 14.62 bearing the Arc1p-C-single-pET28a plasmid.

23.07.2014

14.64 Test Expression

Aim: Test the expression of Arc1p-C-single

Using the preculture from 14.63 60 mL of LB-Kan⁵⁰ medium were inoculated with 600 µL of the preculture and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The culture was cooled down to 18 °C and the protein production was induced with 12 µL of 500 mM IPTG. Over night the culture was incubated at 18 °C and 220 rpm. Afterwards the cell culture was centrifuged (17000 rpm, 20 min, 4 °C), the pellet resuspended in 3 mL buffer A and stored at -20 °C. A glycerolstock of the strain containing the Arc1p-C-single-pET28a plasmid was stored at -80 °C.

19.10 Purification of flagellin-Hag-Cup1-1 from frozen pellet

The pellets which were stored at -80°C were resuspended in 20 mL buffer A and cracked with the micro fluidizer. The cell parts were centrifuged at 20000 rpm for 20 min at 4°C. The supernatant was transferred to a new 50 mL falcon and stored on ice.

The following steps were performed:

equilibration with Buffer A 10 min
taking 40 µL supernatant (load)+ 10 µL SDS-Buffer - L-Sample
50 mL load on column
taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample
first washing with 25ml Buffer A (half the load)
taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample
equilibrate with Buffer B (output pipe off the column into glas, input into B)
hanging pipe on column, 6 x 2 ml elutions - E 1-6
taking 40 µL of elution 1-6 + 10 µL SDS-Buffer E-sample 1-6

regeneration of column:

10min water
10min EDTA
10min water
10min NiSO4
10min water

Meanwhile the Elutions 1 - 6 were pooled (combined 12 mL) in a 50 mL falcon and transferred into a concentrator column which was centrifuged until the volume in the filter was 1,5 mL for injection into the gel filtration station.

Parallel the L-, FL-, W- and E 1 - 6 -samples were analyzed on a SDS-PAGE in order to proof the purification in the elution fractions.

The gel shows a huge protein concentration in E1 and E2 so that the purification could be continued with gel filtration. For that purpose the filtrate out of the concentrator was transferred into a new 2 mL tube resuspending very well without air bubbles.

20 mL of GeFi-buffer were injected into the 2 mL loop. After that the protein filtrate was injected as well.

The GeFi was started according to the following settings:

Flow rate: 2500-3000 µL / min
max. pressure: 0,60 - 0,65 mPa
First endtimer at 80-90 mL volume before fractioning
Injectvalve

After observing a peak 40 µL of different fractions (C7 , C9, C11, D1, D3, D5, D7, D9) covering the peak were taken for analysis via SDS-PAGE.

The gel shows that the elutions contain the protein concentrated. The Elutions C7-D9 were pooled and transferred to a new concentrator until an end volume of 400 µL.

The concentrated protein had a pink color. The concentrated protein (38 mg/mL A280) was used for crystallization pure and in a 1:2 dilution with GeFi buffer.

Core I - IV were used for setting drops.

24.07.2014

Sequencing

Premix	Label-Nr.	Construct	Primer
1	AGB0023456	piGEM-002 Nose + mCherry 1:3 cl.2	TW260
2	AGB0023457	Hag-Cup1-1 cl. 2	Flo90 Flank-rv

13.71 Insertion of degradation tags into Nose- mCherry (piGEM-022 in case of pos. seq.)

Aim: insertion of degradation Tags

For insertion of the DTs via gibson assembly the newly generated piGEM-022 had to be linearized by digestion with SpeI:

Component	Volume (µl)
piGEM-022 (500 ng/µL)	4
CutSmart 10x	2
SpeI	0,2
Water	13,8
Total Volume	20

Incubation at 37°C for 2 hours.

Meanwhile the DTs were generated by a PCR:

Mix	Master Mix	PCR attempt DT 21	PCR attempt DT 22	PCR attempt DT 23
Template	-	-	-	-
Primer iGEM-021	-	0,5	-	-
Primer iGEM-022	-	-	0,5	-
Primer iGEM-023	-	-	-	0,5
Primer iGEM-031	2	-	-	-
dNTP-mix	2	-	-	-
Phusion DNA-polymerase	2	-	-	-
Phusion Buffer 5x	16	-	-	-
MgCl2	-	-	-	-
DMSO	-	-	-	-
Water	48	-	-	-
Mastermix	-	19,5	19,5	19,5
Total Volume	80	20	20	20

Step	Temperature °C	Time
1	98	5 min
2	98	30 sec
3	55	30 sec
4	72	20 sec
5	Go To 2	30x
6	72	4min
7	8	infinite

The DTs were integrated into the vector by Gibson assembly:

Component	Control	DT.21	DT.22	DT.23
Gibson-mix	15	15	15	15
Insert I (DT.x 600 ng/µL)	-	2,5	2,5	2,5
piGEM-002 Nco/SpeI (130 ng/µL)	2,5	2,5	2,5	2,5
Water	2,5	-	-	-
Total Volume	17,5	20	20	20

The reactions were incubated at 50°C for an hour and used to transform E. coli XLI-blue that were plated out on LB-Amp. Incubation was carried out over night.

14.65 Purification of test expression

Aim: Purify Arc1p-C-single on a small scale

Lysozyme was added to the cell suspension from 14.64 to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 °C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.

60 mL LB-Kan⁵⁰ medium was inoculated from the glycerolstock prepared in 14.64 and incubated at 37 °C and 220 rpm over night.

25.07.2014

14.66 Expression of Arc1p-C-single

Aim: Express Arc1p-C-single for further use

10 x 500 mL LB-Kan⁵⁰ medium were inoculated with 5 mL of the overnight culture prepared in 14.65 and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The flasks were cooled to 18 °C and protein production was induced by adding 100 µL 500 mM IPTG. The cultures were incubated over night at 18 °C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 °C), resuspended in buffer A and stored at -20 °C until further use.

13.72 Checking plates of Nose-mCherry-Dt containing clones from Gibson assembly

Aim: checking success of DT 21- 23 integration in Nose-mCherry (piGEM-022)

Sequencing results

Premix	Laber-Nr.	Construct	Result
1	AGB0023456	piGEM-002 Nose + mCherry 1:3 cl.2	2 point mutations, 1 silent, Phe -> Ile

28.07.2014

14.67 Purification of Arc1p-C-single

Aim: Purify Arc1p-C-single in three steps

The resuspended cells from 14.66 were lysed using a french press. The lysate was incubated on ice with 5 µg/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C) to pellet cell debris. The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-0x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-0x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.

31.07.2014

19.11 crystallization of Hag-Cup

Protein crystals can be seen in the wells of the first Hag-Cup crystallization attempt from 19.9

@@ Line 5: / Line 5: @@
 {{Team:Marburg/Template:StartContinue}}
 {{Team:Marburg/Template:Submenu:Notebook:July}}
+<html>
-...
+<!-- 01.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="01.07.2014">01.07.2014</a>
+</h2>
+<fieldset class="exp20">
+    <legend><a name="exp20.10">20.10 pMAD-Transformation of competent <i>Bacillus subtilis</i>  WT 3610</a></legend>
+    <div class="aim">
+    <p>Aim: Transformation of piGEM-016</p>
+    </div>
+    <div class="exp-content">
+    <p>100 &micro;L  of overnight culture were added to 10 mL of MNGE-Medium and incubated till an  OD of 0,7 at 37&deg;C which took  7 hours.</p>
+    <p>After  reaching OD of  0,7  400 &micro;L of the culture were transformed with  1,5 &micro;g piGEM-021. After 1 hour incubation at 37&deg;C 100 &micro;L Expression mix was added  and incubated for 1h as well.</p>
+    <p>In the  end the 500 &micro;L attempt were plated out on MLS-X-Gal plates and incubated at 30&deg;C overnight until colonies could be seen. </p>
+</div>
+</fieldset>
+<fieldset class="exp">
+    <legend><a name="exp_6">Sequencing  results of constructs from 30.06.2014</a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Result</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>411</strong></td>
+    <td>pET24d-Hag-<i>Spe</i>I  construct cl. 2</td>
+    <td>Correct insertion of  Hag-<i>Spe</i>I</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023<strong>412</strong></td>
+    <td>pET24d-Hag-DARPin  construct cl. 7</td>
+    <td>Correct insertion of  Hag-DARPin</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+    <legend><a name="exp14.48">14.48 PCR of PheA-single</a></legend>
+    <div class="aim">
+    <p>Aim: Amplify PheA-single fragment for Gibson Assembly in pET-28a</p>
+    </div>
+    <div class="exp-content">
+    <p>The PCR with TG_PheA-Arc1p-C-8x FP und TG-PheA-single-GA RP should generate the 1716 bp fragment for Gibson Assembly from the PheA-Arc1p-C-2x-pET28a construct (14.23).</p>
+    <p>The used Primer concentration was 1
+    &micro;M, the concentration of the template was 1 ng/&micro;L and we used a hot start to avoid unspecific annealing of primers.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Volume [&micro;L]</th>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>29.5</td>
+  </tr>
+  <tr>
+    <th scope="row">5x GC Buffer</th>
+    <td>10</td>
+  </tr>
+  <tr>
+    <th scope="row">DMSO</th>
+    <td>2.5</td>
+  </tr>
+  <tr>
+    <th scope="row">TG_PheA-Arc1p-C-8x FP</th>
+    <td>2.5</td>
+  </tr>
+  <tr>
+    <th scope="row">TG_PheA-Arc1p-C-8x RP</th>
+    <td>2.5</td>
+  </tr>
+  <tr>
+    <th scope="row">PheA-Arc1p-C-2x-pET28a</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion-Polymerase</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>50</td>
+  </tr>
+</table><br />
+<table width="100%" border="1">
+  <tr>
+    <th scope="col">Step</th>
+    <th scope="col">Temperature [&deg;C]</th>
+    <th scope="col">Time [min:sec]</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>98</td>
+    <td>2:00</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>add Polymerase</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>98</td>
+    <td>0:10</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>67.5</td>
+    <td>0:30</td>
+  </tr>
+  <tr>
+    <th scope="row">5</th>
+    <td>72</td>
+    <td>1:15</td>
+  </tr>
+  <tr>
+    <th scope="row">6</th>
+    <td>go to 3</td>
+    <td>32x</td>
+  </tr>
+  <tr>
+    <th scope="row">7</th>
+    <td>72</td>
+    <td>10:00</td>
+  </tr>
+  <tr>
+    <th scope="row">8</th>
+    <td>4</td>
+    <td>hold</td>
+  </tr>
+</table>
+<p>The amplified fragment was purified on an agarose gel on which it showed the correct size. It was extracted from the gel for further use in a Gibson Assembly yielding a concentration of 198 ng/&micro;L.</p>
+</div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 02.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="02.07.2014">02.07.2014</a>
+</h2>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.49">14.49 Gibson Assembly</a></legend>
+    <div class="aim">
+    <p>Aim: Integrate PheA-single into linearized pET-28a(<i>Xho</i>I,<i>Nde</i>I) via Gibson Assembly</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Volume [&micro;L]</th>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>5.34</td>
+  </tr>
+  <tr>
+    <th scope="row">pET-28a(XhoI,NdeI) (24 ng/&micro;L)</th>
+    <td>4.2</td>
+  </tr>
+  <tr>
+    <th scope="row">PheA-single (198 ng/&micro;L)</th>
+    <td>0.46</td>
+  </tr>
+  <tr>
+    <th scope="row">2x Gibson Mastermix</th>
+    <td>10</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+    <p>The reaction mixture was incubated for 1 h at 50 &deg;C and 350 rpm. E. coli Top10 electrocompetent cells were transformed with a 1:3 and a 1:6 dilution of the mixture, incubated for 1 h at 37 &deg;C and plated out on LB-Kan<sup>50</sup> plates that were incubated overnight at 37 &deg;C.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp19">
+    <legend><a name="exp19.3">19.3 Co-Transformation of piGEM-019 & -020 into <i>E. coli</i> BL21(DE3) with FliS</a></legend>
+    <div class="aim">
+    <p>Aim: Transformation of plasmids with flagellin modifications and chaperone for Flagellin into protein producing strain</p>
+    </div>
+    <div class="exp-content">
+    <p>piGEM-019  and -020 were used to transform <i>E. coli</i> BL21 (DE3) together with a plasmid  containing the flagellin chaperone FlaS in order to express both proteins at  the same time after induction so that the flagellin monomer accumulates inside  the cells.</p>
+    <p>The  cells were plated out on Can/Amp-LB plates and incubated overnight at 37&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 03.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="03.07.2014">03.07.2014</a>
+</h2>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.50">14.50 Overnight culture</a></legend>
+    <div class="aim">
+    <p>Aim: Amplify Plasmid in an overnight culture</p>
+    </div>
+    <div class="exp-content">
+    <p>Six clones of the transformed Top10 cells from 14.49 were picked and used to inoculate overnight cultures (6 x 5 mL).</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp18">
+    <legend><a name="exp18.49">18.49 new PCR of isolated Gibson assembly clones ( pET24d-Hag (piGEM-019) and cup1-1)</a></legend>
+    <div class="aim">
+    <p>Aim: screening clones for right insert</p>
+    </div>
+    <div class="exp-content">
+    <p>The isolated plasmid 1:10 diluted from the picked clones 1-4 were used as PCR template.</p>
+    <p>The PCR with Flo54 and iGEM-025  should generate a 785 bp fragment and the one with iGEM-024 and -025 the 164bp  domain fragment.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Volume (&micro;L)</th>
+    <th scope="col">MM 1 for 5 attempts (&micro;L)</th>
+    <th scope="col">MM 2 for 5 attempts (&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row"><p>Plasmid from clones 1-4 (1:10)</p></th>
+    <td>1</td>
+    <td>-</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer Flo 54 </th>
+    <td>-</td>
+    <td>2,5</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-024</th>
+    <td>-</td>
+    <td>-</td>
+    <td>2,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-025</th>
+    <td>-</td>
+    <td>2,5</td>
+    <td>2,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5 mastermix 2X</th>
+    <td>-</td>
+    <td>50</td>
+    <td>50</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>-</td>
+    <td>45</td>
+    <td>45</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>19</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>100</td>
+    <td>100</td>
+  </tr>
+</table>
+<br />
+<table width="100%" border="1">
+          <tr>
+            <th scope="col">Step</th>
+            <th scope="col">Temperature &deg;C</th>
+            <th scope="col">Time</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>3 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>1 min 45 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>30x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>4min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/6/64/MR_2014-07-03_18.49.jpg" width="30%" />
+        <p>The agarose gel showed bands at  ca. 750 bp which fits to the expected 780 bp fragment with primers piGEM-025  (cup rv) and Flo54 ( Hag fw).</p>
+        <p>The gel's second row shows the  amplified cup-fragment under the 200 bp ladder mark. The expected size is ca.  164 bp and fits to the gel result.</p>
+        <p>Plasmid from clone 4 was used to transform <em>E.Coli</em> DH5&alpha; and named piGEM-021.</p>
+</div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.4">19.4 Expression-Test with transformed <i>E. coli</i> BL21 (DE3)</a></legend>
+    <div class="aim">
+    <p>Aim: Checking the overexpression of Hag-<i>Spe</i>I and Hag-<i>Spe</i>I-DARPin</p>
+    </div>
+    <div class="exp-content">
+    <p>In  order to check the expression of the proteins a single clone containing  piGEM-019/-20 was used to inoculate 20 mL of LB-Can/Amp. The culture was  incubated at 37&deg;C until an OD of 0,7.</p>
+    <p>A 1 mL  preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL).  After that the cultures were induced with 20 &micro;L IPTG for 2 hours.</p>
+    <p>An  induction sample (I) was taken (320 &micro;L with an OD of 2,2; 350 &micro;L with an OD of  2).</p>
+    <p>PI and  I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and  the pellet resuspended in 60 &micro;L water and 40 &micro;L SDS-Buffer.</p>
+    <p>The  four samples were analysed on an SDS-PAGE gel with 5 and 10 &micro;L volume per sample.</p>
+    <img src="https://static.igem.org/mediawiki/2014/0/0f/MR_2014-07-03_19.4.jpg" width="40%" />
+    <p>The gel showed that the induction with IPTG was successful and the cells overproduce a protein after 2h incubation.</p>
+</div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.a5">19.5 Protein overexpression with transformed <i> E. coli</i> BL21 (DE3)</a></legend>
+    <div class="aim">
+    <p>Aim: Overexpression of flagellin modifications for crystallization</p>
+    </div>
+    <div class="exp-content">
+    <p>After the positive expression test the protein expression was done on a higher level with an overnight lactose induction. </p>
+    <p>For  that purpose 2 x 1 L LB were inoculated with a solution of LB and every clone  on the cotransformation plate. All clones from the piGEM-019/ -020 + FlaS  cotransformation plates were picked and resuspended in 1 mL LB. 500 &micro;L were  added t the 1 L culture flasks. Each flask was additionally mixed with 1 mL  Ampicillin (100 mg/ mL) and 1 mL Canamycin (50 mg/ mL).</p>
+    <p>The  expression was induced overnight with lactose (12,5 g/ L). 50 g lactose were  solved in 200 mL millipore water. Each culture was mixed with 50 mL of the  lactose/ water suspension.</p>
+    <p>The  cultures were incubated overnight at 30&deg;C.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">&nbsp;</th>
+    <th scope="col">Lactose solution</th>
+    <th scope="col">BL21-piGEM-019 culture</th>
+    <th scope="col">BL21-piGEM-020 culture</th>
+  </tr>
+  <tr>
+    <th scope="row">LB</th>
+    <td>-</td>
+    <td>1 L</td>
+    <td>1 l</td>
+  </tr>
+  <tr>
+    <th scope="row">Ampicillin</th>
+    <td>-</td>
+    <td>1 ml</td>
+    <td>1 ml</td>
+  </tr>
+  <tr>
+    <th scope="row">Canamycin</th>
+    <td>-</td>
+    <td>1 ml</td>
+    <td>1 ml</td>
+  </tr>
+  <tr>
+    <th scope="row">Lactose</th>
+    <td>50g</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Millipore water</th>
+    <td>200ml</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Lactose solution</th>
+    <td>-</td>
+    <td>50 ml</td>
+    <td>50 ml</td>
+  </tr>
+  <tr>
+    <th scope="row">BL21-piGEM-019 clones</th>
+    <td>-</td>
+    <td>+</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">BL21-piGEM-020 clones</th>
+    <td>-</td>
+    <td>-</td>
+    <td>+</td>
+  </tr>
+</table>
+</div>
+</fieldset>
+</div>
+<!-- 04.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="04.07.2014">04.07.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_7">Sequencing</a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primers</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>413</strong></td>
+    <td>pET24d-Hag-<i>Spe</i>I-Cup  constr. Cl. 4</td>
+    <td>T7 Term</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.51">14.51 Preparation of plasmids</a></legend>
+    <div class="aim">
+    <p>Aim: Purify the amplified plasmids from overnight cultures</p>
+    </div>
+    <div class="exp-content">
+    <p>The plasmids were purified from the overnight cultures created in 14.50 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 &micro;L water.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp19">
+    <legend><a name="exp19.5b">19.5 Protein overexpression with transformed <i>E. coli</i> BL21 (DE3)</a></legend>
+    <div class="exp-content">
+    <p>1 mL of  culture was spinned down, mixed with 60 &micro;L water, 40 &micro;L SDS-PAGE loading buffer  and analysed on a SDS-PAGE gel.</p>
+    <img src="https://static.igem.org/mediawiki/2014/b/b0/MR_2014-07-04_19.5.jpg" width="30%" />
+    <p>The gel shows a high  concentration of a specific protein.</p>
+    <p> The overnight induced cells were raised till  an OD of 2-3  and spinned down at 4000  rpm.  The pellets were frozen in liquid  nitrogen and stored at -80&micro;C.</p>
+</div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.6a"> 19.6 Co-Transformation of piGEM-021 & with FlaiS  <i>E. coli</i> BL21(DE3) </a></legend>
+    <div class="aim">
+    <p>Aim: Transformation of plasmids with flagellin-cup modification and chaperone for Flagellin into protein producing strain</p>
+    </div>
+    <div class="exp-content">
+    <p>piGEM-021  and pFlaiS were used to transform into <em>E.Coli</em> BL21(DE3) and plated out  on LB-Canamycin plates. The plate was incubated at 37&micro;C overnight. </p>
+</div>
+</fieldset>
+</div>
+<!-- 05.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="05.07.2014">05.07.2014</a>
+</h2>
+<fieldset class="exp19">
+    <legend><a name="exp19.7a">19.7 Expression-Test with transformed <i>E. coli</i> BL21 (DE3) with piGEM-021</a></legend>
+    <div class="aim">
+    <p>Aim: Checking the overexpression of Hag-<i>Spe</i>I-cup1-1</p>
+    </div>
+    <div class="exp-content">
+    <p>In  order to check the expression of the proteins a single clone containing  piGEM-021 was used to inoculate 20 mL of LB-Can/Amp. The culture was incubated  at 37&deg;C until an OD of 0,7.</p>
+    <p>A 1 mL  preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL).  After that the cultures were induced with 20 &micro;L IPTG for 2h.</p>
+    <p>An  induction sample (I) was taken (520 &micro;L with an OD of 1,34).</p>
+    <p>PI and  I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and  the pellet resuspended in 60 &micro;L water and 40 &micro;L SDS-Buffer.</p>
+    <p>The  samples were analysed on an SDS-PAGE gel with 5 and 10 &micro;L volume per sample.  Additionally PI and I from the expression test with transformed piGEM-021 were  loaded on the gel as well as a comparison.</p>
+    <img src="https://static.igem.org/mediawiki/2014/b/be/MR_2014-07-05_19.7.jpg" width="30%" />
+    <p>The gel shows a high concentration of a specific protein so that the test could be seen as successful.</p>
+</div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.6b">19.6 Protein overexpression with transformed <i>E. coli</i> BL21 (DE3) (piGEM-021)</a></legend>
+    <div class="aim">
+    <p>Aim: Overexpression of flagellin modifications for crystallization</p>
+    </div>
+    <div class="exp-content">
+    <p>After  the positive expression test the protein expression was done on a higher level  with an overnight lactose induction. </p>
+    <p>For  that purpose 2 x 1 L LB were inoculated with a solution of LB and every clone  on the cotransformation plate. All clones from the piGEM-021 + FlaiS  cotransformation plates were picked and resuspended in 1 mL LB. 500 &micro;L were  added t the 1 L culture flasks. Each flask was additionally mixed with 1 mL Ampicillin  (100 mg/ mL) and 1 mL Canamycin (50 mg/ mL).</p>
+    <p>The  expression was induced overnight with lactose (12,5 g/ L). 25 g lactose were  solved in 100 mL millipore water which was heated 1 min in the microwave for  solvation. Each culture was mixed with 50 mL of the lactose/ water suspension.</p>
+    <p>The  cultures were incubated overnight at 30&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 06.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="06.07.2014">06.07.2014</a>
+</h2>
+<fieldset class="exp20">
+    <legend><a name="exp20.11">20.11 Overnight culture of blue clones</a></legend>
+    <div class="aim">
+    <p>Aim: transformation of <i>Bacillus subtilis</i> WT3610 with plasmid</p>
+    </div>
+    <div class="exp-content">
+    <p>Colonies  were grown on the plates with transformed piGEM-021. The blue/ white screening  showed positive transformed blue clones.   3 clones of different morphology per plate were picked and used for  inoculation of LB-MLS (4 mL LB, 4 &micro;L lincomycin, 4 &micro;L erythromycin). Incubation  was carried out overnight at 30&deg;C with the 3 cultures.<strong> </strong></p>
+</div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.6">19.6 Protein overexpression with transformed <i>E. coli</i> BL21 (DE3) (piGEM-021)</a></legend>
+    <div class="exp-content">
+    <br />
+    <img src="https://static.igem.org/mediawiki/2014/9/94/MR_2014-07-06_19.6.jpg" width="30%" />
+    <p>The gel  shows a high concentration of a specific protein after overnight induction with  lactose referring to the Hag-Cup construct.</p>
+    <p>The  overnight induced cells were raised till an OD of 2-3  and spinned down at 4000 rpm.  The pellets were frozen in liquid nitrogen  and stored at -80&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 07.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="07.07.2014">07.07.2014</a>
+</h2>
+<fieldset class="exp20">
+    <legend><a name="exp20.12">20.12 First temperature shift</a></legend>
+    <div class="aim">
+    <p>Aim: integration of  pMAD-Insert into Bacillus chromosome via flanks</p>
+    </div>
+    <div class="exp-content">
+    <p>The 3  overnight cultures were used to inoculate 10 mL LB MLS until  the culture obtained an OD of 0,1. The  cultures were incubated at 30&deg;C for 2 hours.</p>
+    <p>Then  the temperature was shifted to 42&deg;C for 6 hours. Unfortunately the temperature  decreased to 33&deg;C because of a wrong incubator setting. New overnight cultures  were inoculated with 50 &micro;L from those used in the morning. The new cultures  were incubated overnight at 30&deg;C.</p>
+</div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.52">14.52 Transformation of BL21 cells</a></legend>
+    <div class="aim">
+    <p>Aim: Transform <i>E. coli</i> BL21(DE3) with PheA-single-pET28a</p>
+    </div>
+    <div class="exp-content">
+    <p>40 &micro;L <i>E. coli</i> BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-single-pET28a from two different clones, incubated for 1 h at 37 &deg;C and plated out on LB-Kan<sup>50</sup> plates that were incubated overnight at 37 &deg;C.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 08.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="08.07.2014">08.07.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_8">Sequencing  results of constructs from 04.07.2014</a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Result</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>413</strong></td>
+    <td>pET24d-Hag-<i>Spe</i>I-Cup  constr. Cl. 4</td>
+    <td>Correct insertion of  Cup1-1 into Hag</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.53">14.53 Overnight culture</a></legend>
+    <div class="aim">
+    <p>Aim: Inoculate Overnight culture of PheA-single-pET28a</p>
+    </div>
+    <div class="exp-content">
+    <p>5 mL of LB-Kan<sup>50</sup> medium were inoculated with a clone from 14.52 bearing the PheA-single-pET28a plasmid.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp20">
+    <legend><a name="exp20.13">20.13 First temperature shift</a></legend>
+    <div class="aim">
+    <p>Aim: integration of  pMAD-Insert into Bacillus chromosome via flanks</p>
+    </div>
+    <div class="exp-content">
+    <p>The 3  overnight cultures were used to inoculate 10 mL LB MLS until  the culture obtained an OD of 0,1. The  cultures were incubated at 30&deg;C for 2h.</p>
+    <p>Then  the temperature was shifted to 42&deg;C for 6h.</p>
+    <p>After  the heat shock 100 &micro;L dilutions from 10<sup>-4</sup> to 10<sup>-6</sup> of each  culture were plated out on MLS-X-Gal so that nine plates could be incubated  overnight at 42&deg;C.</p>
+</div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.7b">19.7 Purification of Flagellin-Hag-<i>Spe</i>I from frozen pellet</a></legend>
+    <div class="exp-content">
+    <p>The  pellets which were stored at -80&deg;C were resuspended in 20 mL buffer A and  cracked with the microfluidizer. The cell lysate was centrifuged at 20000 rpm  for 20 min at 4&degC. The supernatant was transferred to a new 50 mL falcon and  stored on ice.</p>
+    <p>The following steps were performed:</p>
+    <ul class="list">
+    <li>equilibration with Buffer A 10 min</li>
+    <li>taking 40 &micro;L supernatant  (load)+ 10 &micro;L SDS-Buffer - L-Sample</li>
+    <li>50 mL load on column</li>
+    <li>taking 40 &micro;L of flow through + 10 &micro;L SDS-buffer - FT-Sample</li>
+    <li>first washing with 25ml Buffer A (half the load)</li>
+    <li>taking 40 &micro;L of washing flow through + 10 &micro;L SDS-Buffer - W-Sample</li>
+    <li>equilibrate with Buffer B (output pipe off the column into glas, input into B)</li>
+    <li>hanging pipe on column, 6 x 2 ml Evolutions - E 1-6</li>
+    <li>taking 40 &micro;L of Elution 1-6 + 10 &micro;L SDS-Buffer E-sample 1-6</li>
+    </ul>
+    <p>regeneration of column:</p>
+    <ul class="list">
+    <li>10min water</li>
+    <li>10min EDTA</li>
+    <li>10min water</li>
+    <li>10min NiSO4</li>
+    <li>10min water</li>
+    </ul>
+    <p>Meanwhile  the elutions 1-6 were pooled (combined 12 mL) in a 50 mL falcon and transferred  in a concentrator column which was centrifuged until the volume in the filter  was 1,5 mL for injection into the gel filtration station. </p>
+    <p>Parallel  the L-, FL-, W- and E 1-6-samples were analysed via SDS-PAGE gel in order to  proof the purification in the elution fractions.</p>
+    <img src="https://static.igem.org/mediawiki/2014/8/8a/MR_2014-07-08_19.7_1.jpg" width="30%"/>
+    <p>The gel  shows a huge protein concentration in E1 and E2 so that the purification could  be continued with gel filtration. For that purpose the filtrate  out of the concentrator was transferred into a new 2 mL tube resuspending very  well without air bubbles.</p>
+    <p>17 mL  of GeFi-buffer were injected into the loop. After that the protein filtrate was  injected as well. </p>
+    <p>The  GeFi was started according to the following settings:</p>
+    <ul class="list">
+    <li>Flow rate: 2500</li>
+    <li>max. pressure: 0,65 mPa</li>
+    <li>First endtimer at 90 mL volume before fractioning</li>
+    </ul>
+    <p>After  observing a peak on the screen 40 &micro;L of different fractions (C7 , C9, C11, D1,  D3, D5, D7) covering the peak were taken for analysis via SDS-PAGE gel.</p>
+        <img src="https://static.igem.org/mediawiki/2014/8/87/MR_2014-07-08_19.7_2.jpg" width="30%"/>
+    <p>The gel  shows that the elutions contain the protein concentrated. The Elutions C7-D3  were pooled and transferred to the concentrator until an end volume of 200 &micro;L.</p>
+    <p>The  protein concentrate was used for crystallisation pure and in a 1:2 dilution  with GeFi buffer.</p>
+    <p>Core I  and II were used for setting drops.</p>
+</div>
+</fieldset>
+<fieldset class="exp">
+    <legend><a name="exp_9">Sequencing  of purified cPCR samples from pMAD transformation</a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primers</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>414</strong></td>
+    <td>Hag-Spe1 in B.s.  genome clone 1</td>
+    <td>Hag-Nco-fw</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023<strong>415</strong></td>
+    <td>Hag-Spe1 in B.s.  genome clone 3</td>
+    <td>Hag-Nco-fw</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023<strong>416</strong></td>
+    <td>Hag-Spe1-DARPin  in B.s. genome clone 1</td>
+    <td>Hag-Nco-fw</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB0023<strong>417</strong></td>
+    <td>Hag-Spe1-DARPin  in B.s. genome clone 3</td>
+    <td>Hag-Nco-fw</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+</div>
+<!-- 09.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="09.07.2014">09.07.2014</a>
+</h2>
+<fieldset class="exp20">
+    <legend><a name="exp20.14">20.14 Second temperature shift</a></legend>
+    <div class="aim">
+    <p>Aim: flip out of the pMAD backbone</p>
+    </div>
+    <div class="exp-content">
+    <p>One  blue colony per diluted clone was used to inoculate 4 mL LB. The 6 cultures  were incubated at 30&deg;C for 6 hours and afterwards for 3 hours at 42&deg;C.</p>
+    <p>Dilutions  from 10<sup>-4</sup> - to 10<sup>-6</sup> were plated out on 18 X-Gal plates  without MLS selection. The positive clones should not contain the resistance  inside the backbone as well as the galactosidase. The plates were incubated at  42&deg;C overnight.</p>
+</div>
+</fieldset>
+<fieldset class="exp">
+    <legend><a name="exp_10">Results of Sequencing  of cPCR samples from pMAD transformation from 08.07.14</a></legend>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Result</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>414</strong></td>
+    <td>Hag-<i>Spe</i>I in <i>B. subtilis</i> genome clone 1</td>
+    <td>No <i>Spe</i>I insertion</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023<strong>415</strong></td>
+    <td>Hag-<i>Spe</i>I in <i>B. subtilis</i> genome clone 3</td>
+    <td>No <i>Spe</i>I insertion</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023<strong>416</strong></td>
+    <td>Hag-<i>Spe</i>I-DARPin  in <i>B. subtilis</i> genome clone 1</td>
+    <td>Not sequenced through</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB0023<strong>417</strong></td>
+    <td>Hag-<i>Spe</i>I-DARPin  in <i>B. subtilis</i> genome clone 3</td>
+    <td>Not sequenced through</td>
+  </tr>
+</table>
+	<p>The sequencing will be repeated with the reverse Primer instead of the  forward primer. In case of the Hag-<i>Spe</i>I clones, different clones have to be  taken for sequencing.</p>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.7">19.7 Purification of Flagellin-Hag-SpeI from frozen pellet</a></legend>
+    <div class="exp-content">
+    <p>After one day in well B1 Core I fine crystals could be seen.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.54">14.54 Test Expression</a></legend>
+    <div class="aim">
+    <p>Aim: Test the expression of PheA-single</p>
+    </div>
+    <div class="exp-content">
+    <p>Using the preculture from 14.53 60 mL of LB-Kan<sup>50</sup> medium were inoculated with 600 &micro;L of the preculture and incubated at 37 &deg;C and 220 rpm until OD<sub>600</sub> reached 0.5. The culture was cooled down to 18 &deg;C and the protein production was induced with 12 &micro;L of 500 mM IPTG. Over night the culture was incubated at 18 &deg;C and 220 rpm. Afterwards the cell culture was centrifuged (17000 rpm, 20 min, 4 &deg;C), the pellet resuspended in 3 mL buffer A and stored at -20 &deg;C. A glycerolstock of the strain containing the PheA-single-pET28a plasmid was stored at -80 &deg;C.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 10.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="10.07.2014">10.07.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_11">Sequencing  of cPCR samples from pMAD transformation </a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primer</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>439</strong></td>
+    <td>Hag-<i>Spe</i>I in <i>B. subtilis</i>  genome clone 7</td>
+    <td>Hag-<i>Bam</i>HI-rv</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023<strong>440</strong></td>
+    <td>Hag-<i>Spe</i>I-DARPin  in <i>B. subtilis</i> genome clone 1</td>
+    <td>Hag-<i>Bam</i>HI-rv</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023<strong>441</strong></td>
+    <td>Hag-<i>Spe</i>I-DARPin  in <i>B. subtilis</i> genome clone 3</td>
+    <td>Hag-<i>Bam</i>HI-rv</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.8">19.8 Purification of Flagellin-Hag-Cup1-1 from frozen pellet</a></legend>
+    <div class="exp-content">
+    <p>The  pellets which were stored at -80&deg;C were resuspended in 20 mL buffer A and  cracked with the microfluidizer. The cell parts were spinned down at 20000 rpm  for 20 min at 4&deg;C. The supernatant was transferred to a new 50 mL falcon and  stored on ice.</p>
+<p>The following steps were performed:</p>
+    <ul class="list">
+    <li>equilibration with Buffer A 10 min</li>
+    <li>taking 40 &micro;L supernatant  (load)+ 10 &micro;L SDS-Buffer - L-Sample</li>
+    <li>50 mL load on column</li>
+    <li>taking 40 &micro;L of flow through + 10 &micro;L SDS-buffer - FT-Sample</li>
+    <li>first washing with 25ml Buffer A (half the load)</li>
+    <li>taking 40 &micro;L of washing flow through + 10 &micro;L SDS-Buffer - W-Sample</li>
+    <li>equilibrate with Buffer B (output pipe off the column into glas, input into B)</li>
+    <li>hanging pipe on column, 6 x 2 ml Evolutions - E 1-6</li>
+    <li>taking 40 &micro;L of Elution 1-6 + 10 &micro;L SDS-Buffer E-sample 1-6</li>
+    </ul>
+    <p>regeneration of column:</p>
+    <ul class="list">
+    <li>10min water</li>
+    <li>10min EDTA</li>
+    <li>10min water</li>
+    <li>10min NiSO4</li>
+    <li>10min water</li>
+    </ul>
+    <p>Meanwhile  the Elutions 1-6 were pooled (combined 12 mL) in a 50 mL falcon and transferred  in a concentrator column which was centrifuged until the volume in the filter  was 1,5 mL for injection into the gel filtration station. </p>
+    <p>Parallel  the L-, FL-, W- and E 1-6-samples were analysed on a SDS-PAGE gel in order to  proof the purification in the elution fractions.</p>
+        <img src="https://static.igem.org/mediawiki/2014/c/ce/MR_2014-07-10_19.8_1.jpg" width="30%"/>
+<p>The gel  shows a huge protein concentration in E1 and E2 so that the purification could  be continued with the gel filtration. For that purpose the filtrate  out of the concentrator was transferred into a new 2 mL tube resuspending very  well without air bubbles. </p>
+<p>17 mL  of GeFi-buffer werd injected into the loop. After that the protein filtrate was  injected as well. </p>
+<p>The  GeFi was started according to the following settings:</p>
+<ul class="list">
+  <li>Flow rate: 2500-3000  &micro;L / min</li>
+  <li>max. pressure: 0,60 -  0,65 mPa</li>
+  <li>First  end timer at 80-90 mL volume before fractioning </li>
+  <li>Injectvalve</li>
+</ul>
+    <p>After  observing a peak on the screen 40 &micro;L of different fractions (C7 , C9, C11, D1,  D3, D5, D7) covering the peak were taken for analysis via SDS-PAGE gel.</p>
+        <img src="https://static.igem.org/mediawiki/2014/3/33/MR_2014-07-10_19.8_2.jpg" width="30%"/>
+<p>The gel  shows that the elutions contain the protein concentrated. The Elutions  C7-D7were pooled and transferred to the concentrator until an end volume of 200 &micro;L.</p>
+<p>The protein  concentrate (27,4 mg/mL A280) was used for crystallization pure and in a 1:2  dilution with GeFi buffer.</p>
+<p>Core I  - IV were used for setting drops.</p>
+</div>
+</fieldset>
+<fieldset class="exp20">
+    <legend><a name="exp20.15">20.15 selection of positive clones</a></legend>
+    <div class="aim">
+    <p>Aim: checking the correct flip out of the pMAD backbone</p>
+    </div>
+    <div class="exp-content">
+    <p>From  the dilution plates was one WHITE clone picked and transferred on a Master  X-Gal Plate as well as on a MLS plate so that nine clones were proven for the right  integration of the insert although flipping out the pMAD backbone.</p>
+    <p>The  plates were incubated at 42&deg;C overnight.</p>
+</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.66">13.66 Digest of piGEM-002 <i>Nco</i>I/<i>Spe</i>I</a></legend>
+    <div class="aim">
+    <p>Aim: digest for isolation of piGEM-002 without GFP, replacing it with mCherry out of pMA17</p>
+    </div>
+    <div class="exp-content">
+    <p>piGEM-002 was digested with <i>Nco</i>I/<i>Spe</i>I in order to isolate the backbone without the GFP so that an mCherry could be integrated via Gibson assembly.</p>
+	<table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Volume (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 (73 ng/&micro;L)</th>
+    <td>14</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart 10x</th>
+    <td>2</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Spe</i>I</th>
+    <td>0,2</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I</th>
+    <td>0,2</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>3,6</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+	<p>The digested vector was isolated via gel extraction.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.55">14.55 Purification of test expression</a></legend>
+    <div class="aim">
+    <p>Aim: Purify PheA-single on a small scale</p>
+    </div>
+    <div class="exp-content">
+    <p>Lysozyme was added to the cell suspension from 14.54 to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 &deg;C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.</p>
+    <p>60 mL LB-Kan<sup>50</sup> medium was inoculated from the glycerolstock prepared in 14.54 and incubated at 37 &deg;C and 220 rpm over night.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp13">
+    <legend><a name="exp13.67">13.67 mCherry PCR amplification</a></legend>
+    <div class="aim">
+    <p>Aim: PCR for the amplification of mCherry</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">Master-Mix I</th>
+    <th scope="col">Master-Mix II</th>
+  </tr>
+  <tr>
+    <th scope="row">Template: pMA17 1:10 (25 ng/ &micro;L)</th>
+    <td>5</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-027</th>
+    <td>5</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-028</th>
+    <td>5</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-029</th>
+    <td>-</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-030</th>
+    <td>-</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>10</td>
+    <td>10</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>40</td>
+    <td>40</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTP-mix</th>
+    <td>5</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">MgCl2</th>
+    <td>5</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">DMSO</th>
+    <td>5</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>120</td>
+    <td>120</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>200</td>
+    <td>200</td>
+  </tr>
+</table>
+	<p>The PCR was ran as a gradient from 55-65&deg;C annealing temperature overnight.</p>
+    <br />
+	<img src="https://static.igem.org/mediawiki/2014/b/bd/MR_20140710_amplification_mCherry_parts.jpg" width="50%" />
+	<br />
+	<p>The gel shows the successful amplification of the two parts of mCherry.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 11.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="11.07.2014">11.07.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_12">Sequencing results  of cPCR samples from pMAD transformation </a></legend>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Lebel-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primer</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>439</strong></td>
+    <td>Hag-<i>Spe</i>I in <i>B. subtilis</i>  genome clone 7</td>
+    <td>Positive, but HagII deletion/ frame shift</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023<strong>440</strong></td>
+    <td>Hag-<i>Spe</i>I-DARPin  in <i>B. subtilis</i> genome clone 1</td>
+    <td>1 point mutations in domain, deletion in HagII/ Frameshift</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023<strong>441</strong></td>
+    <td>Hag-<i>Spe</i>I-DARPin  in <i>B. subtilis</i> genome clone 3</td>
+    <td>2 point mutations in domain, deletion in HagII/ Frameshift</td>
+  </tr>
+</table>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp_13">Sequencing  of pMAD-constructs </a></legend>
+    <div class="aim">
+    <p>Aim: finding out if the mutations have been in the constructs or made by Bacillus</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primer</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>442</strong></td>
+    <td>piGEM-005 - Hag-<i>Spe</i>I</td>
+    <td>pMAD-seq-rv</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023<strong>443</strong></td>
+    <td>piGEM-016 - Hag-Cup</td>
+    <td>pMAD-seq-rv</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023<strong>444</strong></td>
+    <td>piGEM-018 - Hag-DARPin</td>
+    <td>pMAD-seq-rv</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.67">13.67 mCherry PCR amplification</a></legend>
+    <div class="aim">
+    <p>Aim: digest for isolation of piGEM-002 without GFP, replacing it with mCherry</p>
+    </div>
+    <div class="exp-contetn">
+    <p>Gel analysis shows that the PCR was successful at every annealing temperature. MgCl<sub>2</sub> and DMSO were important for the function of the polymerase. </p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.56">14.56 Expression of PheA-single</a></legend>
+    <div class="aim">
+    <p>Aim: Express PheA-single for further use</p>
+    </div>
+    <div class="exp-content">
+    <p>10 x 500 mL LB-Kan<sup>50</sup> medium were inoculated with 5 mL of the overnight culture prepared in 14.55 and incubated at 37 &deg;C and 220 rpm until OD<sub>600</sub> reached 0.5. The flasks were cooled to 18 &deg;C and protein production was induced by adding 100 &micro;L 500 mM IPTG. The cultures were incubated over night at 18 &deg;C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 &deg;C), resuspended in buffer A and stored at -20 &deg;C until further use.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 14.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="14.07.2014">14.07.2014</a>
+</h2>
+<fieldset class="exp13">
+    <legend><a name="exp13.68">13.68 Gibson Assembly with mCherry I and II & linearized piGEM-002</a></legend>
+    <div class="aim">
+    <p>Aim: recombination of piGEM-002 and mCherry</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Volume (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">Gibson-mix</th>
+    <td>15</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert I (mCherry I 460 ng/&micro;L)</th>
+    <td>1,25</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert II (mCherry II 460 ng/&micro;L)</th>
+    <td>1,25</td>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 <i>Nco</i>I/<i>Spe</i>I (5,5 ng/&micro;L)</th>
+    <td>2,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+	<p>The mix was incubated 1 hour at 50&deg;C, and used to transform <i>E. coli</i> XL1-Blue that were plated out on LB-Amp plates. Incubation overnight at 37&deg;C. </p>
+</div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.57">14.57 Purification of PheA-single</a></legend>
+    <div class="aim">
+    <p>Aim: Purify PheA-single in three steps</p>
+    </div>
+    <div class="exp-content">
+    <p>The resuspended cells from 14.56 were lysed using a french press. The lysate was incubated on ice with 5 &micro;g/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 &deg;C) to pellet cell debris. The supernatant was filtered (0.45 &micro;m) and PheA-Arc1p-C-0x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-0x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 &deg;C in aliquods for further use.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp">
+    <legend><a name="exp_14">Sequencing results of pMAD-constructs </a></legend>
+    <div class="aim">
+    <p>Aim: finding out if the mutations have been in the constructs or made by Bacillus</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primer</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023<strong>442</strong></td>
+    <td>piGEM-005 - Hag-<i>Spe</i>I</td>
+    <td>Deletion in FlankII</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023<strong>443</strong></td>
+    <td>piGEM-016 - Hag-Cup</td>
+    <td>Deletion in FlankII</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023<strong>444</strong></td>
+    <td>piGEM-018 - Hag-DARPin</td>
+    <td>Deletion in FlankII</td>
+  </tr>
+</table>
+    <p>The Flank was checked by sequencing and showed no deletion in the flank.</p>
+    </div>
+</fieldset>
+</div>
+<!-- 15.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="15.07.2014">15.07.2014</a>
+</h2>
+<fieldset class="exp13">
+    <legend><a name="exp13.68">13.68 Gibson Assembly with mCherry I and II & linearized piGEM-002</a></legend>
+    <div class="aim">
+    <p>Aim: recombination of piGEM-002 and mCherry</p>
+    </div>
+    <div class="exp-content">
+    <p>No colonies were on the plates. The Gibson assembly was repeated with higher concentrated vector.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+    <legend><a name="exp14.58">14.58 PCR of Arc1p-C-single</a></legend>
+    <div class="aim">
+    <p>Aim: Amplify Arc1p-C-single fragment for Gibson Assembly in pET-28a</p>
+    </div>
+    <div class="exp-content">
+    <p>The PCR with TG_Arc1p-C FP and TG_PheA-Arc1p-C-8x RP should generate the 579 bp fragment for Gibson Assembly from the PheA-Arc1p-C-2x-pET28a construct (14.23).</p>
+    <p>The used primer concentration was 1
+    &micro;M, the concentration of the template was 1 ng/&micro;L and we used a hot start to avoid unspecific annealing of primers.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Volume [&micro;L]</th>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>29.5</td>
+  </tr>
+  <tr>
+    <th scope="row">5x GC Buffer</th>
+    <td>10</td>
+  </tr>
+  <tr>
+    <th scope="row">DMSO</th>
+    <td>2.5</td>
+  </tr>
+  <tr>
+    <th scope="row">TG_Arc1p-C FP</th>
+    <td>2.5</td>
+  </tr>
+  <tr>
+    <th scope="row">TG_PheA-Arc1p-C-8x RP</th>
+    <td>2.5</td>
+  </tr>
+  <tr>
+    <th scope="row">PheA-Arc1p-C-2x-pET28a</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion-Polymerase</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>50</td>
+  </tr>
+</table><br />
+<table width="100%" border="1">
+  <tr>
+    <th scope="col">Step</th>
+    <th scope="col">Temperature [&deg;C]</th>
+    <th scope="col">Time [min:sec]</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>98</td>
+    <td>2:00</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>add Polymerase</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>98</td>
+    <td>0:10</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>67.5</td>
+    <td>0:30</td>
+  </tr>
+  <tr>
+    <th scope="row">5</th>
+    <td>72</td>
+    <td>1:15</td>
+  </tr>
+  <tr>
+    <th scope="row">6</th>
+    <td>go to 3</td>
+    <td>32x</td>
+  </tr>
+  <tr>
+    <th scope="row">7</th>
+    <td>72</td>
+    <td>10:00</td>
+  </tr>
+  <tr>
+    <th scope="row">8</th>
+    <td>4</td>
+    <td>hold</td>
+  </tr>
+</table>
+<p>The amplified fragment was purified on an agarose gel on which it showed the correct size. It was extracted from the gel for further use in a Gibson Assembly yielding a concentration of 122 ng/&micro;L.</p>
+</div>
+</fieldset>
+<fieldset class="exp14">
+ <legend><a name="exp14.59">14.59 Gibson Assembly</a></legend>
+    <div class="aim">
+    <p>Aim: Integrate Arc1p-C-single into linearized pET-28a(XhoI,NdeI) via Gibson Assembly</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Volume [&micro;L]</th>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>5.38</td>
+  </tr>
+  <tr>
+    <th scope="row">pET-28a(XhoI,NdeI) (24 ng/&micro;L)</th>
+    <td>4.2</td>
+  </tr>
+  <tr>
+    <th scope="row">Arc1p-C-single (122 ng/&micro;L)</th>
+    <td>0.42</td>
+  </tr>
+  <tr>
+    <th scope="row">2x Gibson Mastermix</th>
+    <td>10</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+    <p>The reaction mixture was incubated for 1 h at 50 &deg;C and 350 rpm. E. coli Top10 electrocompetent cells were transformed with a 1:3 and a 1:6 dilution of the mixture, incubated for 1 h at 37 &deg;C and plated out on LB-Kan<sup>50</sup> plates that were incubated overnight at 37 &deg;C.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- ROMAN -->
+<!-- 16.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="16.07.2014">16.07.2014</a>
+</h2>
+<fieldset class="exp14">
+ <legend><a name="exp14.60">14.60 Overnight culture</a></legend>
+    <div class="aim">
+    <p>Aim: Amplify Plasmid in an overnight culture</p>
+    </div>
+    <div class="exp-content">
+    <p>Six clones of the transformed Top10 cells from 14.59 were picked and used to inoculate overnight cultures (6 x 5 mL).</p>
+    </div>
+</fieldset>
+</div>
+<!-- ROMAN -->
+<!-- 17.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="17.07.2014">17.07.2014</a>
+</h2>
+<fieldset class="exp13">
+    <legend><a name="exp13.69">13.69 new Gibson Assembly with mCherry I and II & linearized piGEM-002</a></legend>
+    <div class="aim">
+    <p>Aim: recombination of piGEM-002 and mCherry</p>
+    </div>
+    <div class="exp-content">
+    <p>In order to receive the construct more piGEM-002  a different ration of the used parts was used for a new Gibson assembly.</p>
+    <p>From Insert I was a higher amount taken becaue of the bigger fragment size. New attempts with 1:2 (vector:insert) and 1:3 ration:</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">1:2 (&micro;l)</th>
+    <th scope="col">1:3 (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">Gibson-mix</th>
+    <td>15</td>
+    <td>15</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert I (mCherry  I 460 ng/&micro;L)</th>
+    <td>2,2</td>
+    <td>1,1</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert II (mCherry  II 460 ng/&micro;L)</th>
+    <td>2,5</td>
+    <td>1,25</td>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 <i>Nco</i>I/<i>Spe</i>I (42 ng/&micro;L)</th>
+    <td>2,7</td>
+    <td>1,25</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+  </tr>
+</table>
+	<p>Gibson reactions were used to transform <i>E. coli</i>  XL1-Blue after 1h incubation at 50&deg;C and plated out on LB-Amp. Incubation was  proceeded at 37&deg;C overnight.</p>
+</div>
+</fieldset>
+<fieldset class="exp21">
+    <legend><a name="exp21.1a">21.1 Swarming Assays</a></legend>
+    <div class="aim">
+    <p>Aim: Check motility of Bacillus transformants</p>
+    </div>
+    <div class="exp-content">
+    <p>For  testing the motility swarming agar (0,7% LB-Agar) and swimming agar (0,3%  LB-Agar) were made and given away for autoclaving.</p>
+    <p>Overnight  cultures of Bacillus transformants (DARPin clone 3 &amp; 8, Cup1-1 clone 2  &amp; 3, Hag-<i>Spe</i>I clone 7), PY79 and WT3610 were incubated at 37&deg;C.</p>
+</div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.61">14.61 Isolation of plasmids</a></legend>
+    <div class="aim">
+    <p>Aim: Purify the amplified plasmids from overnight cultures</p>
+    </div>
+    <div class="exp-content">
+    <p>The plasmids were purified from the overnight cultures created in 14.60 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 &micro;L water.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 18.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="18.07.2014">18.07.2014</a>
+</h2>
+<fieldset class="exp21">
+    <legend><a name="exp21.1b">21.1 Swarming Assay</a></legend>
+    <div class="aim">
+    <p>Aim: Check motility of Bacillus transformands</p>
+    </div>
+    <div class="exp-content">
+    <p>The overnight cultures were set to an OD of 10. 10 &micro;L of each culture were spotted on a swarming and a swimming plate. The distance of the front line was measured after periods of time.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">&nbsp;</th>
+    <th colspan="2" scope="col">DARPin cl. 3</th>
+    <th colspan="2" scope="col">DARPin cl. 8</th>
+    <th colspan="2" scope="col">Cup1-1 cl. 2</th>
+    <th colspan="2" scope="col">Cup1-1 cl. 3</th>
+    <th colspan="2" scope="col">Hag-<i>Spe</i>I cl. 7</th>
+    <th colspan="2" scope="col">WT3610</th>
+    <th colspan="2" scope="col">PY79</th>
+    </tr>
+  <tr>
+    <th scope="row">Time</th>
+    <td>Swim</td>
+    <td>Swarm</td>
+    <td>Swim</td>
+    <td>Swarm</td>
+    <td>Swim</td>
+    <td>Swarm</td>
+    <td>Swim</td>
+    <td>Swarm</td>
+    <td>Swim</td>
+    <td>Swarm</td>
+    <td>Swim</td>
+    <td>Swarm</td>
+    <td>Swim</td>
+    <td>Swarm</td>
+    </tr>
+  <tr>
+    <th scope="row">1h</th>
+    <td>-</td>
+    <td>-</td>
+    <td>0,5</td>
+    <td>2</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>1,5</td>
+    <td>3</td>
+    <td>6</td>
+    <td>2</td>
+    <td>3</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">2,5h</th>
+    <td>-</td>
+    <td>-</td>
+    <td>0,5</td>
+    <td>3,5</td>
+    <td>-</td>
+    <td>3</td>
+    <td>-</td>
+    <td>2,5</td>
+    <td>12</td>
+    <td>40&lt;</td>
+    <td>7</td>
+    <td>20</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+</div>
+<!-- 19.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="19.07.2014">19.07.2014</a>
+</h2>
+<fieldset class="exp21">
+    <legend><a name="exp21.2a">21.2 Swarming Assay</a></legend>
+    <div class="aim">
+    <p>Aim: Check motility of Bacillus transformands</p>
+    </div>
+    <div class="exp-content">
+    <p>In  order to repeat the swarming assay new overnight cultures of  <i>Bacillus subtilis</i> WT3610, the mutants DARPin  clone 3 &amp; 8, Cup1-1 clone 2 , Hag-SpeI clone 7 in 10 mL LB and incubated at  37&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 20.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="20.07.2014">20.07.2014</a>
+</h2>
+<fieldset class="exp21">
+    <legend><a name="exp21.2">21.2 Swarming Assay</a></legend>
+    <div class="aim">
+    <p>Aim: Check motility of Bacillus transformants</p>
+    </div>
+    <div class="exp-content">
+    <p>The  overnight cultures were set to an OD of 10. 10 &micro;L of each culture were spotted  on a swarming and a swimming plate. The plates with the drops were dried for  half an hour on the bench. The distance of the front line was measured after  periods of time. The measurement at t=0 is the radius of the start spot. The  following measurements refer to the difference between the start spot and the  new front line.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">&nbsp;</th>
+    <th scope="col">Swarming</th>
+    <th scope="col">Swimming</th>
+  </tr>
+  <tr>
+    <th scope="row">WT 3610</th>
+    <td>+</td>
+    <td>+</td>
+  </tr>
+  <tr>
+    <th scope="row">Hag-<i>Spe</i>I cl. 7</th>
+    <td>+</td>
+    <td>+</td>
+  </tr>
+  <tr>
+    <th scope="row">DARPin cl. 3</th>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">DARPin cl. 8</th>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Cup1-1 cl. 3</th>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Cup1-1 cl. 8</th>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+</table>
+</div>
+</fieldset>
+</div>
+<!-- 21.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="21.07.2014">21.07.2014</a>
+</h2>
+<fieldset class="exp13">
+    <legend><a name="exp13.70">13.70 Screening clones of Gibson plates</a></legend>
+    <div class="aim">
+    <p>Aim: checking success of Gibson assembly</p>
+    </div>
+    <div class="exp-content">
+    <p>On the plate with 1:2 ratio have been 2 clones, on the 1:3 ratio plate have been 3 clones. All clones were picked for inoculation of minipreps (5 mL LB-Amp, incubation overnight).</p>
+    </div>
+</fieldset>
+<fieldset class="exp">
+    <legend><a name="exp_15">Sequencing cPCR Products from <i>B. subtilis</i> mutants with Flank fw/rv</a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primer</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB00234<strong>52</strong></td>
+    <td><i>B. subtilis</i> - Hag-<i>Spe</i>I Cl.7</td>
+    <td>Flo89 Flank-fw</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB00234<strong>53</strong></td>
+    <td><i>B. subtilis</i>  - Hag-<i>Spe</i>I Cl.7</td>
+    <td>Flo90 Flank-rv</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB00234<strong>54</strong></td>
+    <td><i>B. subtilis</i> - Hag-Cup1-1 Cl.2</td>
+    <td>Flo89 Flank-fw</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB00234<strong>55</strong></td>
+    <td><i>B. subtilis</i> - Hag-Cup1-1 Cl.2</td>
+    <td>Flo90 Flank-rv</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.62">14.62 Transformation of BL21 cells</a></legend>
+    <div class="aim">
+    <p>Aim: Transform <i>E. coli</i> BL21(DE3) with Arc1p-C-single-pET28a</p>
+    </div>
+    <div class="exp-content">
+    <p>40 &micro;L <i>E. coli</i> BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid Arc1p-C-single-pET28a from two different clones, incubated for 1 h at 37 &deg;C and plated out on LB-Kan<sup>50</sup> plates that were incubated overnight at 37 &deg;C.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp19">
+    <legend><a name="exp19.9">19.9 Protein overexpression with transformed <i>E. coli</i> BL21 (DE3) (piGEM-021)</a></legend>
+    <div class="aim">
+    <p>Aim: Overexpression of flagellin modifications for a second crystallization attempt</p>
+    </div>
+    <div class="exp-content">
+    <p>After freshly packed GeFi Columns we decided to purify the Fla-Cup again for crystallization.</p>
+    <p>For that purpose 2 x 1 L LB were inoculated with a solution of LB and  every clone on the cotransformation plate. All clones from the piGEM-021 +  FlaiS cotransformation plates were picked and resuspended in 1 mL LB. 500 &micro;L  were added to the 1 L culture flasks. Each flask was additionally mixed with 1  mL ampicillin (100 mg/ mL) and 1 mL canamycin (50 mg/ mL).</p>
+    <p>The expression was induced overnight with lactose (12,5 g/ L). 25 g  lactose were solved in 100 mL millipore water which was heated 1 min in the  microwave for solvation. Each culture was mixed with 50 mL of the lactose/  water suspension.</p>
+    <p>The cultures were incubated overnight at 30&deg;C.</p>
+</div>
+</fieldset>
+<fieldset class="exp21">
+    <legend><a name="exp21.3">21.3 Expression of Flagellin</a></legend>
+    <div class="aim">
+    <p>Aim: Check the expression of the modified flagella of Bacillus mutants</p>
+    </div>
+    <div class="exp-content">
+    <p>In order to check the expression of  the flagellum in the Bacillus mutants cultures  in 100 mL LB medium were grown until an OD of 0,8 at 37&deg;C. 10 mL culture were centrifuged  at 4000 rpm for 10 min to get the pellet. The pellet was resuspended in 60 &micro;L  water and 40 &micro;l SDS-loading buffer. After that the sample was analysed via  SDS-PAGE. Overexpressed <i>E. coli</i> flagellin was used as a positive control. The analysed samples should run on the same level as the ones produced in <i>E. coli</i> samples in case of a positive expression.</p>
+    <img src="https://static.igem.org/mediawiki/2014/0/08/MR_2014-07-21_21.3.jpg" width="30%" />
+    <p>The gel showed that the Hag-<i>Spe</i>I mutant generates a band on the same level as the WT3610 which is a positive result because the modified flagellin with <i>Spe</i>I site has no remarkable change of molecule mass. The Bacillus transformation was successful though.</p>
+    <p>The mutants with the inserted Cup and DARPin  showed no significant expression of the flagellin. Either the export of the flagellin  is not possible for Bacillus because of the size/ folding of our constructs and  ends in degradation of our modifications or the expression is too low to be  analysed via SDS-PAGE. In that case it would not be efficient enough for further usage. The results of the SDS-PAGE fits to the  previously made Swarming assays. </p>
+    <p>A new strategy had to be planned for further  work.</p>
+</div>
+</fieldset>
+</div>
+<!-- 22.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="22.07.2014">22.07.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_16">Sequencing cPCR Products from <i>B. subtilis</i> mutants with Flank fw/rv</a></legend>
+    <div class="exp-content">
+        <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Results</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB00234<strong>52</strong></td>
+    <td><i>B. subtilis</i>  - Hag-<i>Spe</i>I Cl.7</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB00234<strong>53</strong></td>
+    <td><i>B. subtilis</i>  - Hag-<i>Spe</i>I Cl.7</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB00234<strong>54</strong></td>
+    <td><i>B. subtilis</i> - Hag-Cup1-1 Cl.2</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB00234<strong>55</strong></td>
+    <td><i>B. subtilis</i> - Hag-Cup1-1 Cl.2</td>
+    <td>&nbsp;</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<fieldset class="exp19">
+    <legend><a name="exp19.9">19.9 Protein overexpression with transformed <i>E. coli</i> BL21 (DE3) (piGEM-021)</a></legend>
+    <div class="aim">
+    <p>Aim: Overexpression of flagellin modifications for a second crystallization try</p>
+    </div>
+    <div class="exp-content">
+    <p>The  overnight cultures were grown till an OD of 2-3 and centrifuged. The pellets  were frozen away at -80&deg;C.</p>
+</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.70">13.70 screening clones of Gibson plates</a></legend>
+    <div class="aim">
+    <p>Aim: checking success of Gibson assembly</p>
+    </div>
+    <div class="exp-content">
+    <p>The isolated plasmids were used for PCR analysis. The primers iGEM-27 and -30 were used for a PCR to amplify the whole mCherry with a size of ca. 711 bp.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">Mastermix</th>
+    <th scope="col">PCR attempt</th>
+  </tr>
+  <tr>
+    <th scope="row">Template: plasmid from clones 1:10 </th>
+    <td>-</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-027</th>
+    <td>3</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-030</th>
+    <td>3</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTP-mix</th>
+    <td>3</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>3</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>36</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">MgCl2</th>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">DMSO</th>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>132</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>19,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>180</td>
+    <td>20</td>
+  </tr>
+</table>
+	<br />
+        <table width="100%" border="1">
+          <tr>
+            <th scope="col">Step</th>
+            <th scope="col">Temperature &deg;C</th>
+            <th scope="col">Time</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>3 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>1 min </td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>30x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>4min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/e/e1/MR_2014-07-22_13.70.jpg" width="30%" />
+        <p>The gel shows that clone 1:3.2 might be positive for containing the mCherry fragment. The clone has to be sequenced.</p>
+        </div>
+</fieldset>
+<fieldset class="exp21">
+    <legend><a name="exp21.2b">21.2 Swarming Assay</a></legend>
+    <div class="aim">
+    <p>Aim: Check motility of Bacillus transformands with D2-3-domain</p>
+    </div>
+    <div class="exp-content">
+    <p>The mutants showed expression on the SDS-gel. Swarming and swimming were tested to  check the motility of his mutants for further planning of our domain  constructions. The hag-construct contains the D2-3 domain of Salmonella.</p>
+    <p>WT3610  and the D2-3-mutant were used to inoculate 50 mL LB-medium for overnight culture  incubated at 37&degC.</p>
+</div>
+</fieldset>
+<fieldset class="exp">
+    <legend><a name="exp_17">Sequencing cPCR Products from <i>B. subtilis</i> mutants with flank fw/rv</a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Results</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB00234<strong>52</strong></td>
+    <td><i>B. subtilis</i>  - Hag-<i>Spe</i>I Cl.7</td>
+    <td>Correct</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB00234<strong>53</strong></td>
+    <td><i>B. subtilis</i>  - Hag->Spe</i>I Cl.7</td>
+    <td>Correct</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB00234<strong>54</strong></td>
+    <td><i>B. subtilis</i> - Hag-Cup1-1 Cl.2</td>
+    <td>Correct sequencing  till Hag</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB00234<strong>55</strong></td>
+    <td><i>B. subtilis</i> - Hag-Cup1-1 Cl.2</td>
+    <td>Correct sequencing  till Hag</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.63">14.63 Overnight culture</a></legend>
+    <div class="aim">
+    <p>Aim: Inoculate Overnight culture of Arc1p-C-single-pET28a</p>
+    </div>
+    <div class="exp-content">
+    <p>5 mL of LB-Kan<sup>50</sup> medium were inoculated with a clone from 14.62 bearing the Arc1p-C-single-pET28a plasmid.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 23.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="23.07.2014">23.07.2014</a>
+</h2>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.64">14.64 Test Expression</a></legend>
+    <div class="aim">
+    <p>Aim: Test the expression of Arc1p-C-single</p>
+    </div>
+    <div class="exp-content">
+    <p>Using the preculture from 14.63 60 mL of LB-Kan<sup>50</sup> medium were inoculated with 600 &micro;L of the preculture and incubated at 37 &deg;C and 220 rpm until OD<sub>600</sub> reached 0.5. The culture was cooled down to 18 &deg;C and the protein production was induced with 12 &micro;L of 500 mM IPTG. Over night the culture was incubated at 18 &deg;C and 220 rpm. Afterwards the cell culture was centrifuged (17000 rpm, 20 min, 4 &deg;C), the pellet resuspended in 3 mL buffer A and stored at -20 &deg;C. A glycerolstock of the strain containing the Arc1p-C-single-pET28a plasmid was stored at -80 &deg;C.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp19">
+    <legend><a name="exp19.10">19.10 Purification of flagellin-Hag-Cup1-1 from frozen pellet</a></legend>
+        <div class="exp-content">
+    <p>The  pellets which were stored at -80&deg;C were resuspended in 20 mL buffer A and  cracked with the micro fluidizer. The cell parts were centrifuged at 20000 rpm  for 20 min at 4&deg;C. The supernatant was transferred to a new 50 mL falcon and  stored on ice.</p>
+<p>The following steps were performed:</p>
+    <ul class="list">
+    <li>equilibration with Buffer A 10 min</li>
+    <li>taking 40 &micro;L supernatant  (load)+ 10 &micro;L SDS-Buffer - L-Sample</li>
+    <li>50 mL load on column</li>
+    <li>taking 40 &micro;L of flow through + 10 &micro;L SDS-buffer - FT-Sample</li>
+    <li>first washing with 25ml Buffer A (half the load)</li>
+    <li>taking 40 &micro;L of washing flow through + 10 &micro;L SDS-Buffer - W-Sample</li>
+    <li>equilibrate with Buffer B (output pipe off the column into glas, input into B)</li>
+    <li>hanging pipe on column, 6 x 2 ml elutions - E 1-6</li>
+    <li>taking 40 &micro;L of elution 1-6 + 10 &micro;L SDS-Buffer E-sample 1-6</li>
+    </ul>
+    <p>regeneration of column:</p>
+    <ul class="list">
+    <li>10min water</li>
+    <li>10min EDTA</li>
+    <li>10min water</li>
+    <li>10min NiSO4</li>
+    <li>10min water</li>
+    </ul>
+    <p>Meanwhile  the Elutions 1 - 6 were pooled (combined 12 mL) in a 50 mL falcon and transferred  into a concentrator column which was centrifuged until the volume in the filter  was 1,5 mL for injection into the gel filtration station. </p>
+<p>Parallel  the L-, FL-, W- and E 1 - 6 -samples were analyzed on a SDS-PAGE in order to  proof the purification in the elution fractions.</p>
+<img src="https://static.igem.org/mediawiki/2014/e/ee/MR_2014-07-23_19.10.jpg" width="30%" />
+<p>The gel  shows a huge protein concentration in E1 and E2 so that the purification could  be continued with gel filtration. For that purpose the filtrate  out of the concentrator was transferred into a new 2 mL tube resuspending very  well without air bubbles. </p>
+<p>20 mL  of GeFi-buffer were injected into the 2 mL loop. After that the protein filtrate  was injected as well. </p>
+<p>The  GeFi was started according to the following settings:</p>
+    <ul class="list">
+    <li>Flow rate: 2500-3000 &micro;L / min</li>
+    <li>max. pressure: 0,60 - 0,65 mPa</li>
+    <li>First endtimer at 80-90 mL volume before fractioning</li>
+    <li>Injectvalve</li>
+    </ul>
+    <p>After  observing a peak 40 &micro;L of different fractions (C7 , C9, C11, D1,  D3, D5, D7, D9) covering the peak were taken for analysis via SDS-PAGE.</p>
+    <img src="https://static.igem.org/mediawiki/2014/2/29/MR_2014-07-23_19.10_2.jpg" width="30%" />
+<p>The gel  shows that the elutions contain the protein concentrated. The Elutions C7-D9  were pooled and transferred to a new concentrator until an end volume of 400 &micro;L.</p>
+<p>The  concentrated protein had a pink color. The concentrated protein (38 mg/mL A280)  was used for crystallization pure and in a 1:2 dilution with GeFi buffer.</p>
+<p>Core I  - IV were used for setting drops.</p>
+</div>
+</fieldset>
+</div>
+<!-- 24.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="24.07.2014">24.07.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_18">Sequencing </a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Label-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Primer</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB00234<strong>56</strong></td>
+    <td>piGEM-002 Nose +  mCherry 1:3 cl.2</td>
+    <td>TW260</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB00234<strong>57</strong></td>
+    <td>Hag-Cup1-1 cl. 2</td>
+    <td>Flo90 Flank-rv</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.71">13.71 Insertion of degradation tags into Nose- mCherry (piGEM-022 in case of pos. seq.)</a></legend>
+    <div class="aim">
+    <p>Aim: insertion of degradation Tags</p>
+    </div>
+    <div class="exp-content">
+    <p>For insertion of the DTs via gibson assembly the newly generated piGEM-022 had to be linearized by digestion with <i>Spe</i>I:</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Volume (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-022 (500 ng/&micro;L)</th>
+    <td>4</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart 10x</th>
+    <td>2</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Spe</i>I</th>
+    <td>0,2</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>13,8</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+	<p>Incubation at 37&deg;C for 2 hours.</p>
+	<p>Meanwhile the DTs were generated by a PCR:</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">Master Mix</th>
+    <th scope="col">PCR attempt DT 21</th>
+    <th scope="col">PCR attempt DT 22</th>
+    <th scope="col">PCR attempt DT 23</th>
+  </tr>
+  <tr>
+    <th scope="row">Template </th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-021</th>
+    <td>-</td>
+    <td>0,5</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-022</th>
+    <td>-</td>
+    <td>-</td>
+    <td>0,5</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-023</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-031</th>
+    <td>2</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTP-mix</th>
+    <td>2</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>2</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>16</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">MgCl2</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">DMSO</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>48</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>19,5</td>
+    <td>19,5</td>
+    <td>19,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>80</td>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+  </tr>
+</table>
+<br />
+        <table width="100%" border="1">
+          <tr>
+            <th scope="col">Step</th>
+            <th scope="col">Temperature &deg;C</th>
+            <th scope="col">Time</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>30x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>4min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+      <p>The DTs were integrated into the vector by Gibson assembly:</p>
+      <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Control</th>
+    <th scope="col">DT.21</th>
+    <th scope="col">DT.22</th>
+    <th scope="col">DT.23</th>
+  </tr>
+  <tr>
+    <th scope="row">Gibson-mix</th>
+    <td>15</td>
+    <td>15</td>
+    <td>15</td>
+    <td>15</td>
+    </tr>
+  <tr>
+    <th scope="row">Insert I (DT.x 600 ng/&micro;L)</th>
+    <td>-</td>
+    <td>2,5</td>
+    <td>2,5</td>
+    <td>2,5</td>
+    </tr>
+  <tr>
+    <th scope="row">piGEM-002 <i>Nco</i>/<i>Spe</i>I (130 ng/&micro;L)</th>
+    <td>2,5</td>
+    <td>2,5</td>
+    <td>2,5</td>
+    <td>2,5</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>2,5</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>17,5</td>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    </tr>
+</table>
+<p>The reactions  were incubated at 50&deg;C for an hour and used to transform <i>E. coli</i> XLI-blue that were plated out on LB-Amp. Incubation was carried out over night.</p>
+</div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.65">14.65 Purification of test expression</a></legend>
+    <div class="aim">
+    <p>Aim: Purify Arc1p-C-single on a small scale</p>
+    </div>
+    <div class="exp-content">
+    <p>Lysozyme was added to the cell suspension from 14.64 to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 &deg;C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.</p>
+    <p>60 mL LB-Kan<sup>50</sup> medium was inoculated from the glycerolstock prepared in 14.64 and incubated at 37 &deg;C and 220 rpm over night.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 25.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="25.07.2014">25.07.2014</a>
+</h2>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.66">14.66 Expression of Arc1p-C-single</a></legend>
+    <div class="aim">
+    <p>Aim: Express Arc1p-C-single for further use</p>
+    </div>
+    <div class="exp-content">
+    <p>10 x 500 mL LB-Kan<sup>50</sup> medium were inoculated with 5 mL of the overnight culture prepared in 14.65 and incubated at 37 &deg;C and 220 rpm until OD<sub>600</sub> reached 0.5. The flasks were cooled to 18 &deg;C and protein production was induced by adding 100 &micro;L 500 mM IPTG. The cultures were incubated over night at 18 &deg;C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 &deg;C), resuspended in buffer A and stored at -20 &deg;C until further use.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp13">
+    <legend><a name="exp13.72">13.72 Checking plates of Nose-mCherry-Dt containing clones from Gibson assembly</a></legend>
+    <div class="aim">
+    <p>Aim: checking success of DT 21- 23 integration in Nose-mCherry (piGEM-022)</p>
+    </div>
+</fieldset>
+<fieldset class="exp">
+    <legend><a name="exp_19">Sequencing results </a></legend>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Premix</th>
+    <th scope="col">Laber-Nr.</th>
+    <th scope="col">Construct</th>
+    <th scope="col">Result</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB00234<strong>56</strong></td>
+    <td>piGEM-002 Nose +  mCherry 1:3 cl.2</td>
+    <td>2 point mutations, 1  silent, Phe -&gt; Ile</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+</div>
+<!-- ROMAN -->
+<!-- 28.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="28.07.2014">28.07.2014</a>
+</h2>
+<fieldset class="exp14">
+ <legend><a name="exp14.67">14.67 Purification of Arc1p-C-single</a></legend>
+    <div class="aim">
+    <p>Aim: Purify Arc1p-C-single in three steps</p>
+    </div>
+    <div class="exp-content">
+    <p>The resuspended cells from 14.66 were lysed using a french press. The lysate was incubated on ice with 5 &micro;g/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 &deg;C) to pellet cell debris. The supernatant was filtered (0.45 &micro;m) and PheA-Arc1p-C-0x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-0x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 &deg;C in aliquods for further use.</p>
+    </div>
+</fieldset>
+</div>
+<!-- ROMAN -->
+<!-- 31.07.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="31.07.2014">31.07.2014</a>
+</h2>
+<fieldset class="exp19">
+    <legend><a name="exp19.11">19.11 crystallization of Hag-Cup</a></legend>
+    <div class="exp-content">
+    <p>Protein crystals can be seen in the wells of the first Hag-Cup crystallization attempt from 19.9</p>
+    </div>
+</fieldset>
+</div>
+</html>
 {{Team:Marburg/Template:End}}

Team:Marburg:Project:Notebook:July

From 2014.igem.org

Latest revision as of 11:22, 17 October 2014

Notebook: July