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Latest revision as of 15:45, 17 October 2014

Notebook: August

01.08.2014

18.50 Design of a Strep-Tag/ StrepDARPidin system

Aim: Designing a new system for using the Flagellin-DARPin-Konstrukt

A new plan was made to obtain the flagellas multi-functionality, using an affinity tag. We want to integrate a Strep-Tag into the flagellin so that the DARPin domain can be fused to the flagellum via streptavidin.

The D2 domain of Salmonella typhimurium's flagellin was used as a linker between the hag of Bacillus subtilis and the Strep-Tag, furthermore GSGS linkers were used between the D2 and the StrepTag.

Additionally the DARPin was designed with an N-terminal His-Tag and C-Terminal streptavidin, so a chimeric protein would be generated.

The designed structures were synthesized by Integrated DNA Technologies.

15.61 Design of Hag-D2-Cup

Aim: Designing a new domain construct for expression of cup1-1-Hag

We decided to design the flagellin as well with the D2 domain and the cup1-1.

The designed structures were synthesized by Integrated DNA Technologies

04.08.2014

14.68 Crystallization of Arc1p-C

Aim: Crystallize the Arc1p-C-single domain

For the experiments the sitting drop method in SWISSCI MRC 2 well crystallization plates with a drop size of 1 µL was used (1:1 mixture of protein and crystallization solution) with an reservoir volume of 50 µL. Crystals of Arc1p-C-single were obtained by room temperature and at 6 mg/mL protein concentration after 1 week in 0.1 M MES, pH 6.0, 10% (v/v) glycerol, 30% (w/v) PEG 600 and 5% (w/v) PEG 1000. To collect data the crystals were flash frozen in liquid nitrogen and measured at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France at beamline ID23-1. The structure of Arc1p-C-single was determined by molecular replacement (MR) using the crystal structure of human Arc1p-C (PDB ID: 1FL0).

05.08.2014

18.51 Isolation of flagella from Bacillus subtilis WT3610

Aim: isolation of filaments of WT3610 as negative control for e.g. FACS analysis & microscopy

1 L LB was inoculated with WT 3610 and incubated over night at 37°C.

15.62 Restriction digest of piGEM-005 SpeI

Aim: linearizing vector for Gibson assembly with Hag-D2-Cup and -D2-Strep

Component	Volume (µl)
piGEM-005 (100 ng/µL	20
CutSmart 10x	2,5
SpeI	0,2
Water	2,3
Total Volume	25

Incubation at 37°C for 2 hours – Inactivation by heat shock at 85° for 10 min.

End concentration: 50 ng/µL

18.51 NcoI/BamHI & NcoI/XhoI digest of pET24d

Aim: purification of vector for ligation with StrepDARPidin gene

Component	Volume [µl]	Volume [µl]
pET24d (80 ng/µL)	30	30
CutSmart 10x	3,5	3,5
NcoI	0,25	0,25
BamHI	0,25	-
XhoI	-	0,25
Water	1	1
Total Volume	35	35

Incubation at 37°C for 2h .

The fragments were purified via gel extraction.

End concentration: 25 ng/µL pET24d-N/B & 15 ng/µL-N/X

06.08.2014

18.52 Isolation of flagella from Bacillus subtilis WT3610

Aim: isolation of filaments of WT3610 as negative control for e.g. FACS analysis & microscopy

The preculture was centrifuged at 4000 rpm for 15 minutes and the pellet was resuspended in 33 mL TRIS/HCl buffer (pH 8.0) with 0,5% Brij-58. A stock solution of lysozyme with 10 mg/mL was made. 330 µL were added to the TRIS/HCl buffer mix. The lysis was performed at 4°C while rotating the tubes until the pellet was completely solved which took approx. 2 - 3 hours. DNase (5 µg/ mL) in presence of 0,01 M magnesium chloride was added and incubated for half an hour at 4°C in the rotator as well.

After that the suspension was centrifuged at 10000x g for 10 min. 80 µL of the supernatant were transferred into a 1,5 mL tube. The supernatant was then centrifuged at 87000xg for 90min.

The pellet was resuspended in 10 mL standard saline citrate (0,01M trisodium citrate & 0,1M sodium chloride, pH 7,3) on ice and 80 µL were taken as well. For fractioning, 2 g of ammonium sulfate (20%) were added slowly until precipitation could be seen.

After some minutes of resting the suspension was centrifuged at 20000 rpm for 15 min. The pellet was then resuspended in 2 mL of standard saline citrate buffer. After taking a sample of 80 µL the 4 samples ware mixed with 20 µL SDS-loading buffer each and analysed on an SDS-PAGE.

The purified filaments were frozen in liquid nitrogen and then at -80°C.

The flagella purification was successful. Besides the flagellin the samples also contain the hook proteins, the smaller proteins were removed from the purified pellet.

14.69 tRNA^Phe assay

Aim: Measure aminoacylation levels

To measure aminoacylation of tRNA^Phe the components were mixed.

Content	Final concentration
Dialysis-buffer	ad 250 µL
DTT	1 mM
MgCl₂	20 mM
KCl	10 mM
tRNA^Phe	10 µM
L- or D-Phenylalanine	1 mM
catalytic fusion Protein	10 µM

After mixing, reaction was initiated by the addition of 2 mM ATP and then incubated for 1 to 60 min by 37 °C and 350 rpm. Assays were stopped by the addition of 25 µL 3 M sodium acetate solution. Afterwards tRNAs were precipitated with ethanol. Dissolved pellets were further purified using size-exclusion columns. The elution fraction was split into two aliquots. One samples was treated with 0.2 M NaOH (final concentration: 10 mM) and incubated at 50 °C for 10 min, while the other aliquot was kept on ice. The base treated sample was again acidified by the addition of 3 M sodium acetate (pH 5.2, final concentration: 300 mM). The volume of the untreated sample was adjusted using 2 mM sodium acetate (pH 5.2). Subsequently, both samples were subjected to LCMS analysis using 100 µL for injection.

11.08.2014

25. Cell culture assays

25.1 Splitting of Caco-2-cells

Aim: Splitting cell culture for growing

The Caco-2-cells were kindly provided by Dr. Hartmann Reifert (BMFZ). In order to let them grow, the cells had to be splitted according to a specific protocol. Caco-2-cells require DMEM (Dulbecco’s Modified Eagle Medium) with 20% FCS and L-Glutamine.

A centrifuge was precooled to 4°C.

The medium of the culture flask was discarded from the side opposite to the adherent cells and washed with 1X PBS from the opposite site as well.

The PBS was aspirated with a glass pipette until the supernatant was clear. After adding 1 - 2 mL 1x trypsin from the opposite of the adherent cells the flask was incubated for 5 - 10 min at 37°C depending on how fast the cells unfastened of from the ground. Under the microscope the free floating cells were checked.

The cells were transferred into a 15 mL falcon and rinsed with 10 mL DMEM to collect all remaining cells in the flask. The 15 mL falcon was filled with DMEM and the culture centrifuged at 1500 rpm for 5 min at 4°C.

The supernatant was discarded and resuspended in 2 mL DMEM. The cells were splitted 1:3 which means that 666 µL were taken from the suspension and transferred into a new culture flask. After adding 20-25 mL DMEM the flask was incubated at 37°C and checked on the 3rd / 4th day under the microscope.

On the medium flask were the following notes:
Date, Name, Cell Line, Medium, Ratio of splitting, passage number

15.63 PCR Amplification of Hag-D2-Cup

Aim: Amplification of insert for Gibson assembly into piGEM-005

The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged and resuspended in 20 µL Millipore water to get an end concentration of 10 ng/µL as a Stock solution.

0,5 µL were used as a PCR template.

Mix (µl)	Mastermix	D2-Cup	D2-Strep
Template	-	2	2
Primer Flo96 D23-fw	3	-	-
Primer Flo97 D23-rv	3	-	-
Q5-Master mix	75	-	-
Water	63	-	-
Mastermix	-	48	48
Total Volume	144	50	20

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	30 sec
5	Go To 2	31x
6	72	4min
7	8	infinite

The expected bands could be seen.

15.64 Gibson assembly with linearized piGEM-005 & PCR products from 15.63

Aim: Insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005

2

Component	Hag-D2-Cup(µL)	Hag-D2-Strep (µL)
Gibson-mix	15	15
Insert I: (Hag-D2-Cup 431 ng/µL)	2,5	-
Insert II: (Hag-D2-Strep 423 ng/µL)	-	2,5
piGEM-005 SpeI (50 ng/ µL)	2,5	2,5
Total	20	20

Incubation was done at 50°C for 1 hour with following transformation into E. coli XL1-Blue plated out on LB-Amp plates and incubated overnight at 37°C.

12.08.2014

15.64 Screening clones from 15.63

Aim: Checking insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005

In order to check the successful integration of D2-Cup and D2-Strep, clones were picked for cPCR and transferred onto a LB-Amp plate. The primers Flo96 (D23-fw) and Flo97 (D23-v) were used.

Mix (µl)	Mastermix	D2-Cup	D2-Strep
Template	-	Colony	Colony
Primer Flo96 D23-fw	4,5	-	-
Primer Flo97 D23-rv	4,5	-	-
dNTP	4,5	-	-
5x Phusion Buffer	36	-	-
Phusion DNA-polymerase	4,5	-	-
Water	126	-	-
Mastermix	-	20	20
Total Volume	180	20	20

Step	Temperature °C	Time
1	98	10 min
2	98	30 sec
3	55	30 sec
4	72	40 sec
5	Go To 2	33x
6	72	4min
7	8	infinite

The gel shows that clone 2 from the Gibson Hag-D2-Cup clones and clones 3 & 4 from the Gibson Hag-D2-Strep clones contain the integrated plasmid constructs. All 3 clones were picked for a miniprep and used for the inoculation of 10 mL LB-Amp. Incubation was performed over night at 37°C.

The new constructs were labelled with piGEM-026 (pMAD-Hag-D2-Cup) and piGEM-027 (pMAD-Hag-D2-Strep).

15.65 Overnight culture for WT3610

Aim:Preparation for mainculture the next day

5 mL of LB were inoculated with Bacillus subtilis WT3610 from an LB plate and incubated at 37°C over night.

13.08.2014

15.66 Transformation of competent Bacillus subtilis WT3610

Aim: transformation of plasmid into Bacillus subtilis WT3610

100 µL preculture were added to 10 mL of MNGE-Medium and incubated to an OD₆₀₀ of 1,1-1,3 at 37°C which could take 4-5h.

After reaching an OD₆₀₀ of 1,1-1,3 2 x 400 µL of the culture were transformed with 1,5 µg plasmid (piGEM-026 & -027). After 1 hour of incubation at 37°C 100 µL expression mix were added and incubated for 1 hour as well.

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

Sequencing

Premix	Label-Nr.	Construct	Primer
1	AGB0023458	piGEM-023	TW260
2	AGB0023459	piGEM-024	TW260
3	AGB0023460	piGEM-025	TW260
4	AGB0023461	piGEM-026 D2-Cup cl.2	Flo96-D2 3 for
5	AGB0023462	piGEM-026 D2-Cup cl.2	Flo56-Hag rv
6	AGB0023463	piGEM-027 D2-Strep cl.3	Flo96-D2 3 for
7	AGB0023464	piGEM-027 D2-Strep cl.3	Flo56-Hag rv
8	AGB0023465	piGEM-027 D2-Strep cl.4	Flo96-D2 3 for
9	AGB0023466	piGEM-027 D2-Strep cl.4	Flo56-Hag rv

13.72 Restriction of Nose plasmids with EcoRV

Aim: check if amyE and cat are still in plasmid, since B. subtilis could not successfully be transformed with the plasmids yet

All Nose-plasmids (iGEM002-004 and 007-015) were digested with EcoRV to check if amyE and cat are still present in these plasmids, since B. subtilis could not be transformed successfully.

Per sample 1 µl 2x CutSmart Buffer, 7.9 µl H2O, 0.1 µl EcoRV and 2 µl of plasmids were added. The reaction was incubated over night at RT.

14.08.2014

13.72 Restriction of Nose plasmids with EcoRV - continuation

Aim: Analysis of the restriction on an agarose gel

The samples were mixed with 2 µl of 5x Loading dye and 8 µl of it was loaded on a 1% agarose gel.

All plasmids but piGEM004 and piGEM011 show the expected bands of ca 2000 and 4000 bp.

The plasmids were used to transform Bacillus subtilis, which was incubated at 30°C.

18.08.2014

15.67 Transformation of competent WT3610

Aim: Bacillus subtilis pMad Trafo mit D2-Strep and D2-Cup

Next step: inoculate overnight culture from the X-gal plate with 3 ml LB-MLS.

As the cultures were recognizable blue (thin or old plate) wildtype cultures were inoculated for a new transformation. Therefore the plasmids piGEM-026 and -027 were isolated from frozen pellets.

19.08.2014

15.68 Transformation of competent WT3610

Bacillus subtilis pMad trafo mit D2-Strep and D2-Cup

For the experimental procedure see the Materials section. Anyway, two transformations were running currently that will be referred to as 'old' and 'new' transformation from now on.

For the old transformation the first temperature shift was performed in LB-MLS medium. The culture was plated on LB-MLS-X-Gal plates and incubated at 42°C over night.

The new transformation was started with a culture grown in MNGE medium, inoculated with a wildtype overnight culture. The culture was grown until an optical density of 1,2 that is normally reached after 4-5 h. Strangely the culture reached this OD after almost 7 h today. Plasmids (1.5µg) piGEM026 and -027 were transformed and the protocol was followed as described below.

20.08.2014

15.69 Transformation of competent WT3610

Bacillus subtilis pMad Trafo mit D2-Strep and D2-Cup

New trafo showed no colonies so far. It was further incubated.

For the old trafo 2nd temperature shift of positive colonies (only light blue).

21.08.2014

15.70 Transformation of competent WT3610

Bacillus subtilis pMad Trafo mit D2-Strep and D2-Cup

Old trafo: white colonies were re-streaked on x-Gal plates and on MLS plates to check for MLS sensitivity. Incubation at 42°C.

22.08.2014

15.70 Transformation of competent WT3610

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

The blue/ white screening showed positive transformed blue clones from the new transformation. Three piGEM026 and -027 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 over night at 30°C with the cultures.

22. The Killswitch

22.1 Amplification of flanks

Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the amyE and the lacA/ganA locus of Bacillus subtilis after cloning into pMAD

Primers arrived and have been dissolved in millipore water, dilution 1:10. Q5 Master Mix was used for the following PCRs to create the flanks of the killswitch parts I-III:

Fragment	Fw primer (iGEM-0xx)	Rv primer (iGEM-0xx)
AmyE flankI	iGEM-034	iGEM-035
AmyE flankII	iGEM-036	iGEM-037
lacA flankI	iGEM-040	iGEM-041
lacA flankII	iGEM-042	iGEM-043
KSIII AmyE-yvyd FlankII	iGEM-048	-

Mix	Master Mix	amyE FlankI	amyE FlankII	lacA FlankI	lacA FlankII	amyE-yvyD FlankII
Template (B. subtilis gDNA)	11	-	-	-	-	-
Primer iGEM-34	-	1	-	-	-	-
Primer iGEM-35	-	1	-	-	-	-
Primer iGEM-36	-	-	1	-	-	-
Primer iGEM-37	-	-	1	-	-	-
Primer iGEM-40	-	-	-	1	-	-
Primer iGEM-41	-	-	-	1	-	-
Primer iGEM-42	-	-	-	-	1	-
Primer iGEM-43	-	-	-	-	1	-
Primer iGEM-48	-	-	-	-	-	1
Primer iGEM-37	-	-	-	-	-	1
Q5-Master mix	165	-	-	-	-	-
Water	132	-	-	-	-	-
Mastermix	-	28	28	28	28	28
Total Volume [µl]	308	30	30	30	30	30

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	60 sec
5	Go To 2	30x
6	72	4min
7	8	infinite

All bands had the expected size (500 /1000 bp)

23.08.2014

15.71 Transformation of competent WT3610

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

The 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 dilutions from 10-5 to 10-6 of each culture were plated out on MLS-X-Gal so that plates could be incubated overnight at 42°C.

24.08.2014

22.2 Amplification of Killswitch modules I-III

Aim: The modules had to be amplified via PCR in order to proceed with a fusion PCR linking the modules with the flanks for integration into the Bacillus Loci.

The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged down and resuspended in 20 µL millipore water to get an end concentration of 10 ng/µL as a stock solution.

0,5 µL were used as a PCR template. The PCR fragments should be ca. 350, 650 & 2000 bp long.

Mix	KS module I	KS module II	KS module III
Template (gBlock DNA)	0,5	0,5	0,5
Primer iGEM-38	1	-	-
Primer iGEM-39	1	-	-
Primer iGEM-44	-	1	-
Primer iGEM-45	-	1	-
Primer iGEM-49	-	-	1
Primer iGEM-50	-	-	1
Q5-Mastermix	25	25	25
Water	22,5	22,5	22,5
Total Volume (µl)	50	50	50

q

Step	Temperature °C	Time KSI & III	Time KSII
1	98	3 min	3 min
2	98	20 sec	30 sec
3	55	20 sec	20 sec
4	72	60 sec	120 sec
5	Go To 2	31x	31x
6	72	4min	4min
7	8	infinite	infinite

The modules I and III could be amplified without problems, but KSII showed no distinct band and many unspecific fragments.

15.71 Transformation of competent WT3610

Aim: Second heat shock -flip out of the pMAD backbone

One blue colony per diluted piGEM026 and -027 clone was used to inoculate 4 mL LB. The cultures were incubated at 30°C for 6 hours and afterwards for 3 hours at 42°C.

Dilutions from 10^-5 to 10^-6 were plated out on 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. Some clones (piGEM027.1 and one of 27.2) did not grow and will be inoculated again. They will be inoculated in LB-MLS for the first temperature shift tomorrow.

22.3 Transformation of competent DH5α with pMAD

Aim: Gain E.coli on plate for mini preps of pMAD

Standard heat shock transformation protocol was followed, incubation over night at 37°C. pMAD from the plasmid box was used for transformation.

22.4 Restriction of pMAD with NcoI and BamHI over night

Aim: Linearizing Vector for Gibson Assembly with KS modules

Component	Volume (µl)
pMAD (108 ng/µL)	9
CutSmart 10x	2
NcoI	0,2
BamHI	0,2
Water	8,6
Total Volume	20

Incubation over night at room temperature.

25.08.2014

15.72 Transformation of competent WT3610

Aim: checking the correct flip out of the pMAD backbone and MLS sensitivity

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

The plates were incubated at 42°C overnight.

22.5a Fusion PCR

Aim: Assembly of Killswitch modules I & II with amyE and lacA flanks

Segment	Concentraction (ng/µL)
KS module I	401
KS module II	414
KS module III	369
amyE flank I	446
amyE flank II	403
lacA flank I	368
lacA flank II	369
amyE yvyD flank II	372

Mix	amyE KS I	lacA KS II
KS module I (40,1 ng/µL)	1	-
KS module II (41,4 ng/µL)	-	1
amyE flank I ( 44,6 ng/µL)	1	-
amyE flank II (40,3 ng/µL)	1	-
lacA flank I (36,8 ng/µL)	-	1
lacA flank II (39,6 ng/µL)	-	1
Primer iGEM-34	1	-
Primer iGEM-37	1	-
Primer iGEM-40	-	1
Primer iGEM-43	-	1
Phusion Buffer 5x	10	10
Phusion DNA-polymerase	1	1
dNTPs	1	1
Water	33	33
Total Volume [µl]	50	50

q

Step	Temperature °C	Time amyE KSI	Time amyE KSII
1	98	3 min	3 min
2	98	20 sec	30 sec
3	55	20 sec	20 sec
4	72	120 sec	190 sec
5	Go To 2	31x	31x
6	72	4min	4min
7	8	infinite	infinite

No PCR amplificates could be seen on the gel.

18.53 PCR amplification of StrepDARPidin

Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in E. coli BL21(DE3)

The synthesized fragments by Integrated DNA technologies arrived as a dried 200n g DNA pellet. In order to use it as a PCR template the pellet was centrifuged and resuspended in 20 µL millipore water to get an end concentration of 10 ng/µL as a stock solution.

0,5 µL were used as a PCR template. The PCR fragment should be ca. 900 bp long.

Mix	PCR attempt
Template	0,5
Primer iGEM-33	1
Primer iGEM-34	1
Q5-Mastermix	25
Water	22,5
Mastermix	-
Total Volume	50

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	60 sec
5	Go To 2	31x
6	72	4min
7	8	infinite

Both samples had a concentration of 380 ng/µL.

18.54 Digestion of amplified of StrepDARPidin for cloning into pET24d

Aim: digest with NcoI and XhoI of the StrepDARPidin PCR product for cloning into the expression vector pet24d

The PCR product was cut with NcoI and XhoI to get the restriction sites for cloning into pET24d NcoI/XhoI cut.

Component	StrepDARPidin (µl)
PCR product (380 ng/µL)	6
CutSmart 10x	2
NcoI	0,25
XhoI	0,25
Water	11,5
Total Volume	20

Incubation for 1h at 37°C with heat shock at 85°C for 10 min.

18.55 Ligation of digested StrepDARPidin for cloning into pET24d

Aim: ligation of StrepDARPidin PCR product with the expression vector pET24d

The PCR product was ligated into digested pET24d NcoI/XhoI. Two samples were made for a transformation of E. coli XL1-Blue and BL21.

Component	Ligation I (µl)	Ligation II (µl)
NcoI and XhoI StrepDARPidin (64 ng/µL)	2	2
pET24d NcoI and XhoI (15 ng/µL)	5	5
Ligation Buffer 10x	2	2
Ligase	1	1
Water	10	10
Total Volume	20	20

Incubation for 1,5h at room temperature with heat shock at 85°C for 10 min. The 20 µL from ligation I were used to transform E. coli XLI-Blue, 10 µL from Ligation II were used to transform E. coli BL21(DE3) and the rest of ligation II into Xl1-Blue as well and selected on LB-Canamycin plates.

26.08.2014

15.73 Transformation of competent WT3610

Aim: checking the correct flip out of the pMAD backbone and MLS sensitivity

All clones on the master plate grew on x-gal plates but were MLS sensitive and could be seen as positive. In order to check the correct insertion of D2-Cup an D2-Strep into the Bacillus subtilis genome the clones were cooked in 100 µL 1x PBS at 95°C for 10 min and used for colony PCR. Because of the repetition of the temperature shifts with clones 27.1.1, 27.1.2, 27.1.3 yesterday and today and 27.2.3, 6 clones from 26.1 -26.3, 27.2.1 & 27.2.2 were picked for the cPCR (30 reactions).

Mix (µl)	Master-Mix for 30 attempts (µL)	Mix for PCR attempt (µL)
Colony PBS suspension	-	2
Primer Flo54 Hag-fw	15	-
Primer Flo56 Hag-fw	15	-
Phusion DNA-polymerase	15	-
Phusion Buffer 5x	120	-
dNTP-mix	15	-
Water	410	-
DMSO	10	-
Mastermix	-	18
Total Volume	600	20

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

There were no bands visible on the gel.

18.56 cPCR with plates from ligation

Aim: checking the success of the ligation

The grown clones from the ligation the day before were inoculated for plasmid isolation and then checked via cPCR with primers iGEM-032 and -033.

Content (µl)	Master mix for 20 clones (µL)	Clones from Ligation I (µL)	Clones from Ligation II (µL)	Clones from Ligation III (µL)
Template (colony)	-	1	1	1
Primer iGEM-32	10	-	-	-
Primer iGEM-32	10	-	-	-
Phusion Buffer 5x	80	-	-	-
Phusion DNA-polymerase	10	-	-	-
dNTP-mix	10	-	-	-
Water	280	-	-	-
Mastermix	-	19	19	19
Total Volume	400	20	20	20

Step	Temperature °C	Time
1	98	10 min
2	98	20 sec
3	55	20 sec
4	72	60 sec
5	Go To 2	33x
6	72	4min
7	8	infinite

The gel shows that the clones I 2-6, II 1-6 and III 2, 3, 4 and 6 are positive due to the band at approx. 900 bp. The other bands might be plasmid which was used as template.

The clones I6, II3, III3 were used to transform E. coli BL21(DE3) and selected on LB-Can plates. III4 and III6 were already on a plate. The clones were inoculated for plasmid isolation and sent for sequencing.

22.5b Fusion PCR Attempt 3

Aim: Fusion of amyE and lacA flanks to the killswitch fragments

The Fusion PCR was repeated since it did not work the first two times. The fragments of the killswitch I and II were diluted 10fold. A gradient was carried out from 52°C to 62°C.

Mix (µl)	amyE KSI	lacA KS II
KS module I (40,1 ng/µL)	1	-
KS module II (41,4 ng/µL)	-	1
amyE flank I ( 44,6 ng/µL)	1	-
amyE flank II (40,3 ng/µL)	1	-
lacA flank I (36,8 ng/µL)	-	1
lacA flank II (39,6 ng/µL)	-	1
Primer iGEM-34	1	-
Primer iGEM-37	1	-
Primer iGEM-40	-	1
Primer iGEM-43	-	1
Phusion Buffer 5x	4	4
Phusion DNA-polymerase	0,5	0,5
dNTPs	1	1
Water	9,5	9,5
Total Volume	20	20

Step	Temperature °C	Time
1	95	5 min
2	95	30 sec
3	52-62	30 sec
4	72	200 sec
5	Go To 2	32x
6	72	5 min
7	4	infinite

Again, no fragments could be noticed on the gel.

13.73 Amplification of new Cu- and Ag-Promotor sequences

Aim: Amplification of Cu- and Ag promoter sequences from the chromosomal DNA of Bacillus subtilis.

Content	Master mix (µL)	2x Attempt Cu (µL)	2x Attempt Ag (µL)
Template (gDNA B. subtilis)	4	-	-
Primer iGEM-55	-	-	1
Primer iGEM-56	-	-	1
Primer iGEM-57	-	1	-
Primer iGEM-58	-	1	-
Phusion Buffer 5x	40	-	-
Phusion DNA-polymerase	4	-	-
dNTPs	4	-	-
Water	144	-	-
Mastermix	-	48	48
Total Volume (µl)	196	50	50

Step	Temperature °C	Time
1	98	10 min
2	98	20 sec
3	55	20 sec
4	72	20 sec
5	Go To 2	30x
6	72	4 min
7	8	infinite

The gel shows positive bands at approx. 100 bp and 250 bp, which was like expected.

13.74 NcoI/SacI digest of piGEM-002

Aim: linearizing the plasmid backbone for Gibson Assembly and Ligation with Cu/Ag promotor sequences and IPTG/ Constitutive (/tetO) promotor sequences.

Component	Attempt I (µl)
piGEM-002 (476 ng/µL)	6,5
NcoI	0,25
SacI	0,25
CutSmart Buffer 10x	2
Water	11
Total Volume	20

Incubation at 37°C for 60 min and heat shocked at 85°C for 10 min.

13.75 Ligation of NcoI/Sac cut piGEM-002 with annealed oligos containing promoter sequences

Aim: insertion of a IPTG inducible/ constitutive and constitutive+tetO into piGEM-002

In order to test our nose system with new promoter sequences oligos were designed and annealed by heating up water and letting it cool down with the oligo pairs iGEM-59/60, -61/62, - 63/64 so that the individual fragments were able to anneal with overlaps to the NcoI/SacI sites.

Component	Ligation I (µl)	Ligation II (µl)	Ligation III (µl)
piGEM-002 (209 ng/µL)	5	5	5
iGEM-59/60	1	-	-
iGEM-61/62	-	1	-
iGEM-63/64	-	-	1
T4 Ligase	1	1	1
Ligation Buffer 10x	2	2	2
Water	11	11	11
Total Volume (µl]	20	20	20

The reactions were incubated at room temperature for an hour and transformed into E. coli XL1-Blue, plated out on LB-Amp.

13.76 Gibson assembly of NcoI/SacI piGEM-002 & Cu/ Ag promotor

Aim: assembling of Nco/Sac cut piGEM-002 with Cu/ ag promotor sequences

In order to test new Cu/ Ag promotor sequences the PCR fragments from 13.73 were used for a Gibson assembly.

Component	Gibson Cu I (µL)	Gibson Ag II (µL)	Control (µL)
piGEM-002 (209 ng/µL)	2,5	2,5	5
Cu promotor	2,5	-	-
Ag promotor	-	2,5	-
Gibson mix	15	15	15
Total Volume	20	20	20

The attempts were incubated at 50°C for an hour and transformed into E. coli XL1-Blue, plated out on LB-Amp.

27.08.2014

22.3b Repeated Amplification of Killswitch modules I and II

Aim: Increase the amount of modules for further purification, since Fusion PCR did not work 'quick and dirty'

This time the Phusion polymerase was used. The template was the already amplified module.

Mix	KS module I	KS module II
Template (already amplified modules)	0,5	0,5
Primer iGEM-38	1	-
Primer iGEM-39	1	-
Primer iGEM-44	-	1
Primer iGEM-45	-	1
5x HF buffer	10	10
dNTPs	1	1
Water	36	36
Phusion DNA-polymerase	0,5	0,5
Total Volume (µl)	50	50

Step	Temperature °C	KS I	KS II
1	98	5 min	5 min
2	98	30 sec	30 sec
3	55	30 sec	30 sec
4	72	120 sec	200 sec
5	Go To 2	34x	34x
6	72	5 min	5 min
7	8	infinite	infinite

The gel can be seen under the next topic

22.3c Repeated amplification of killswitch flanks

Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the amyE and the lacA locus of Bacillus subtilis after cloning into pMAD, yield higher amount of fragments for purification and FusionPCR.

The Phusion polymerase was used.

Mix	Master Mix	amyE FlankI	amyE FlankII	lacA FlankI	lacA FlankII	amyE-yvyD FlankII
Template		0,5	-	-	-	-
Primer iGEM-34	-	1	-	-	-	-
Primer iGEM-35	-	1	-	-	-	-
Primer iGEM-36	-	-	1	-	-	-
Primer iGEM-37	-	-	1	-	-	-
Primer iGEM-40	-	-	-	1	-	-
Primer iGEM-41	-	-	-	1	-	-
Primer iGEM-42	-	-	-	-	1	-
Primer iGEM-43	-	-	-	-	1	-
Primer iGEM-48	-	-	-	-	-	1
Primer iGEM-37	-	-	-	-	-	1
dNTPs	11	1	-	-	-	-
DMSO	3,3	0,3
MgCl2	2,2	0,2
5x HF-buffer	110	10
Phusion DNA-polymerase	5,5	0,5
Water	390,5	35,5
Mastermix	-	47,5	47,5	47,5	47,5	47,5
Total Volume (µl)	522,5	50	50	50	50	50

With exception of KSI and StrepDARP the PCR was negative for all other samples. Even for the positive reactions the bands were very thin.

18.57 repeated PCR amplification of Hag-KpnI StrepDARPidin

Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in E. coli BL21(DE3)

The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged and resuspended in 20 µL millipore water to get an end concentration of 10 ng/µL as a stock solution.

0,5 µL were used as a PCR template. The PCR fragment should be approx. 900 bp long.

Content (µl)	2x Attempt I (µL)	2x Attempt II(µL)
Template 1: StrepDARPidin (38 ng/ µL)	-	1
Template 2: Hag-KpnI (10 ng/ µL)	1	-
Primer iGEM-51	-	1
Primer iGEM-52	-	1
Primer iGEM-53	1	-
Primer iGEM-54	1	-
Phusion Buffer 5x	10	10
Phusion DNA-polymerase	1	1
dNTPs	1	1
Water	35	35
Total Volume	50	50

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	60 sec
5	Go To 2	31x
6	72	4min
7	8	infinite

The PCR was clearly positive for StrepDARP, Hag-KpnI could be seen as a very slight band at 900 bp, but more unspecific bands could be noticed.

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

Aim: Checking the overexpression of pET24d-StrepDARPidin

In order to check the expression of the proteins 5 clones (I6, II3, III3, III4, III6) containing piGEM-028 (pet24d-StrepDARPidin) were used to inoculate 20 mL of LB-Can. 5 cultures were incubated at 37°C until an OD of 0,7.

A ca. 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 (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 10 µL volume per sample.

The gel did not show any signs of over expression so that lactose was chosen as an alternative inducer to test the expression. New test expression cultures (I6, II3, III3, III4, III6) were made with 100 mL LB-Can and 5 mL lactose (6,25 g/ 25 mL) per culture. Incubation was performed over night at 30°C.

28.08.2014

Sequencing of constructs from 28.08.2014

Premix	Label-Nr.	Construct	Primer
1	AGB0023467	piGEM-028 I6(pET24d-StrepDARPidin)	T7 turn
2	AGB0023468	piGEM-028 III3 (pET24d-StrepDARPidin)	T7 turn
3	AGB0023469	piGEM-026 (pMAD-D2-Cup)	Flo D2_3 fw
4	AGB0023470	piGEM-027.1 (pMAD-D2-Strep)	Flo D2_3 fw
5	AGB0023471	piGEM-027.2 (pMAD-D2-Strep)	Flo D2_3 fw

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

Aim: Checking the overexpression of pET24d-StrepDARPidin

No sign of an overexpression could be noticed.

18.57 His-bead purification of pellet from E. coli BL21(DE3)

Aim: Checking the overexpression of pET24d-StrepDARPidin

The 100 mL culture from I6 & III3 were centrifuged at 4000 rpm for 10 min in 2x50 mL falcons. The pellets were washed in 10 mL buffer A and cracked with the microfluidizer. After that the suspension was centrifuged at 20000 rpm for 20 min/ 4°C. The supernatant was transferred into a 50 mL falcon.

200 µL Ni-NTA beads were added to the suspension, inverted and incubated on ice for half an hour.

After that the suspension was centrifuged or 5 min at 4000 rpm so that the pellet could be resuspended in 500 µL Buffer A after discarding the supernatant. The 500 µL were transferred into a 1,5 mL tube and centrifuged at 4000 rpm for washing. After discarding the supernatant the pellet was washed again with 500 µL Buffer A. After a second centrifugation at 4000 rpm the supernatant was discarded, the pellet resuspended in 200 µL Buffer A and centrifuged at 13000 rpm to destroy the Ni-bead – protein interaction.

In the end the supernatant was transferred into a new 1,5 mL tube. The Ni-beads were mixed with 100 µL 20% ethanol.

40 µL of the supernatant from clone I6 and III3 were added with 10 µL SDS-PAGE Buffer and analysed on the SDS-Gel (visible at 18.56).

22.3d Repeated amplification of killswitch flanks

Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the amyE and the lacA locus of Bacillus subtilis after cloning into pMAD, yield higher amount of fragments for purification and FusionPCR.

Mix [µl]	Master Mix 6x	amyE FlankI	amyE FlankII	lacA FlankI	lacA FlankII	amyE-yvyD FlankII
Template (B. subtilis gDNA)	6	-	-	-	-	-
Primer iGEM-34	-	1	-	-	-	-
Primer iGEM-35	-	1	-	-	-	-
Primer iGEM-36	-	-	1	-	-	-
Primer iGEM-37	-	-	1	-	-	-
Primer iGEM-40	-	-	-	1	-	-
Primer iGEM-41	-	-	-	1	-	-
Primer iGEM-42	-	-	-	-	1	-
Primer iGEM-43	-	-	-	-	1	-
Primer iGEM-48	-	-	-	-	-	1
Primer iGEM-37	-	-	-	-	-	1
Q5-Master mix	150	-	-	-	-	-
Water	132	-	-	-	-	-
Mastermix	-	48	48	48	48	48
Total Volume	288	50	50	50	50	50

Step	Temperature °C	Time
1	98	5 min
2	98	25 sec
3	55	25 sec
4	72	60 sec
5	Go To 2	31x
6	72	4 min
7	8	infinite

The gel is visible under 22.3e

22.3e Repeated Amplification of Killswitch modules I and II

Aim: Increase the amount of modules for further purification, since Fusion PCR did not work 'quick and dirty'. Q5 was used and gDNA since the previously amplified modules did not work as template yesterday.

Mix [µl]	KS module I	KS module II	KS module III
Template (gBlock DNA)	0,5	0,5	0,5
Primer iGEM-38	1	-	-
Primer iGEM-39	1	-	-
Primer iGEM-44	-	1	-
Primer iGEM-45	-	1	-
Primer iGEM-49	-	-	1
Primer iGEM-50	-	-	1
Q5-Master mix	25	25	25
Water	2,5	2,5	2,5
Total Volume	50	50	50

Step	Temperature °C	KS I & III	KS II
1	98	5 min	5 min
2	98	25 sec	25 sec
3	55	25 sec	25 sec
4	72	60 sec	120 sec
5	Go To 2	31x	31x
6	72	4 min	4 min
7	8	infinite	infinite

The expected bands could be seen for the flank I of amyE and flank I and II of lacA. These bands were cut out and purified via a Gel Extraction kit, but the concentration was too low to work further.

13.75 NcoI/SacI digest of piGEM-002

Aim: linearizing the plasmid backbone for Gibson Assembly and Ligation with Cu/Ag promotor sequences and IPTG/ Constitutive (/tetO) promotor sequences.

The already digested piGEM002 of 26.08. was nearly empty. To have some digested plasmid as backup a new digestion was carried out. piGEM002 was isolated and used for the restriction.

Component	Attempt (µl)
piGEM-002 (476 ng/µL)	10
NcoI	0,25
SacI	0,25
CutSmart Buffer 10x	2
Water	7,5
Total Volume	20

An agarose gel of the previous restriction was done with unrestricted piGEM002 as reference. A band at ca between 6000 and 8000 bp could be seen, which indicates a successful restriction (expected size: ca 6600 bp).

13.76 Repeated Gibson assembly of NcoI/SacI piGEM-002 & Cu/ Ag promotor

Aim: assembling of NcoI/SacI cut piGEM-002 with Cu/Ag promoter sequences

In order to test new Cu/ Ag promoter sequences the PCR fragments from 13.73 were used for a Gibson assembly.

Component	Gibson Cu I (µL)	Gibson Ag II (µL)	Control (µL)
piGEM-002 (ca. 40ng/µL)	2,5	2,5	2,5
Cu promotor (ca 4 ng/µl)	2,5	-	-
Ag promotor (ca 4 ng/µl)	-	2,5	-
H2O	-	-	-
Gibson Mix	15	15	15
Total Volume	20	20	20

The attempts were incubated at 50°C for an hour and used to transform E. coli XL1-Blue, plated out on LB-Amp.

13.77 Repeated ligation of NcoI/SacI cut piGEM-002 with annealed oligos containing promoter sequences

Aim: insertion of an IPTG inducible/ constitutive and constitutive+tetO into piGEM-002

The ligation was prepared to contain ca 100 ng of the restricted piGEM002 with an appropriate amount of insert (much fewer).

Component	Ligation I (µL)	Ligation II (µL)	Ligation III (µL)
piGEM-002 (ca 40 ng/µL)	2,5	2,5	2,5
iGEM-59/60 (ca 4 ng/µl)	0,8	-	-
iGEM-61/62 (ca 4 ng/µl)	-	0,8	-
iGEM-63/64 (ca 4 ng/µl)	-	-	0,8
Polynucleotide kinase	1	1	1
T4 Ligase	1	1	1
Ligation Buffer 10x	2	2	2
Water	12,7	12,7	12,7
Total Volume	20	20	20

The reactions were incubated at room temperature for an hour and transformed into E. coli XL1-Blue, plated out on LB-Amp.

29.08.2014

Sequencing results of constructs from 28.08.2014

Premix	Label-Nr.	Construct	Results
1	AGB0023467	piGEM-028 I6(pET24d-StrepDARPidin)	No results
2	AGB0023468	piGEM-028 III3 (pET24d-StrepDARPidin)	No alignment
3	AGB0023469	piGEM-026 (pMAD-D2-Cup)	Good
4	AGB0023470	piGEM-027.1 (pMAD-D2-Strep)	Point mutation
5	AGB0023471	piGEM-027.2 (pMAD-D2-Strep)	Point mutation

The sequencing showed that pET24d did not contain any known insert.

In order to check the correct synthesis of the templates purified PCR products were sent for sequencing again. In the meanwhile the constructs were cloned again.

Sequencing of constructs from 29.08.2014

Premix	Label-Nr.	Construct	Primer
1	AGB0023512	PCR Fragment StrepDARPidin I	piGEM-032 fw
2	AGB0023513	PCR Fragment StrepDARPidin II	piGEM-032 fw
3	AGB0023514	PCR Fragment D2-StrepII	Flo96 D2_3 fw
4	AGB0023515	PCR Fragment StrepDARPidin I	piGEM-033 rv
5	AGB0023516	PCR Fragment StrepDARPidin II	piGEM-033 rv
6	AGB0023517	PCR Fragment D2-StrepII	Flo97 D2_3 rv

18.58 New PCR amplification of StrepDARPidin and D2-Cup

Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in E. coli BL21(DE3) & amplification of D2-StrepII for Gibson Assembly into piGEM-005

The synthesized fragments were supposed to be sequenced in order to check the right synthesis. 10 ng/µL as a Stock solution and a purified PCR Product were used as template for each reaction.

0,5 µL were used as a PCR template. The PCR fragment should be ca. 900 bp long for StrepDARPidin and ca. 450 bp for D2-StrepII.

Mix (µl)	PCR StrepDARPidin I	PCR StrepDARPidin II	PCR StrepDARPidin III	PCR D2-Strep I	PCR D2-Strep II
Pur. StrepDARP. (1:10- 10 ng/µL)	1	-	-	-	-
StrepDARP. Stock (20 ng/µL)	-	0,25	0,25	-	-
D2-StrepII Stock 20 ng/µL)	-	-	-	0,25	0,25
Primer iGEM-32	1	1	1	-	-
Primer iGEM-33	1	1	1	-	-
Flo96 D2_3 fw	-	-	-	1	1
Flo97 D2_3 rv	-	-	-	1	1
Q5-Master mix	25	25	25	25	25
Water	22	22	22	22	22
Total Volume (µl)	50	50	50	50	50

Step	Temperature °C	Time
1	98	5 min
2	98	30 sec
3	55	30 sec
4	72	60sec
5	Go To 2	31x
6	72	4min
7	8	infinite

The PCR samples were purified with the Omega Gel Extraction Kit according to the enzymatic reaction purification protocol.

18.59 Digestion of amplified of StrepDARPidin for cloning into pET16b

Aim: digest with NcoI and XhoI of the StrepDARPidin PCR product for cloning into the expression vector pET16b & digest of pET16b

The PCR products from 15.58 were cut with NcoI and XhoI to get the restriction sites for cloning into the NcoI/XhoI digested pET16b. This vector was used because of its Amp resistance for fast transformation.

Component	PCR StrepDARPidin I (µL)	PCR StrepDARPidin II(µL)	PCR StrepDARPidin III (µL)	Pet16b(µL)	Mastermix
StrepDARP. I ( 45 ng/µL)	10	-	-	-	-
StrepDARP. II (70 ng/µL)	-	15	-	-	-
StrepDARP. III (45 ng/µL)	-	-	10	-	-
pET16b ( 58 ng/µL)	-	-	-	17	-
CutSmart 10x	-	-	-	-	8
NcoI	-	-	-	-	0,5
XhoI	-	-	-	-	0,5
Water	-	-	-	-	17
Mastermix	10	5	10	-	-
Total Volume	20	20	20	20	28

Incubation for 1h at 37°C with heat shock at 85°C for 10 min.

18.60 Ligation of digested StrepDARPidin for cloning into pET16b

Aim: ligation of StrepDARPidin PCR product with the expression vector pET16b

The PCR product was ligated into the NcoI/XhoI digested pET16b. Two attempts were made for a transformation into E. Coli XL1-Blue and BL21.

Component	Ligation I (µL)	Ligation II (µL)	Ligation III (µL)	Control
NcoI/XhoI StrepDARPidin (ca. 40 ng/ µL) I/II/III	1	1	1	-
pET16b NcoI/XhoI (45 ng/µL)	5	5	5	5
Ligation Buffer 10x	2	2	2	2
Ligase	1	1	1	1
Water	11	11	11	11
Total Volume	20	20	20	20

Incubation for 1,5 hours at rom temperature with heat shock at 85°C for 10 min. The 20 µL from ligation I were used to transform E. coli XLI-Blue, which was plated out on LB-Amp. Incubation was done overnight at 37°C.

18.61 Gibson assembly of piGEM-005 & D2-StrepII

Aim: Insertion of Hag-D2-Strep into piGEM-005

Component	Hag-D2-Strep I(µL)	Hag-D2-Strep II (µL)
Gibson-mix	15	15
Insert I (Hag-D2-Strep I 60 ng/µL)	2,5	-
Insert II (Hag-D2-Strep II 70 ng/µL)	-	2,5
piGEM-005 SpeI (50 ng/ µL)	2,5	2,5
Total Volume	20	20

Incubation was done at 50°C for 1 hour with following transformation into E. coli XL1-Blue, plated out on LB-Amp plates and incubated overnight at 37°C.

13.78 colony PCR of colonies of Gibson and ligation (13.76 and 13.77)

Aim: check for correct ligation/Gibson assembly of the oligos with the piGEM002 plasmid

Several clones were picked and transferred onto another plate, the same colony was used to inoculate a PCR reaction to check the insertion of the oligos.

Mix [µl]	Master mix for 45 reactions (µl)	Const. promoter	Cont. + tetO	Mod. Lac promoter	Ag promoter new	Cu promoter new
Template	-	Small part	Small part	Small part	Small part	Small part
Primer iGEM006	45	-	-	-	-	-
Primer iGEM061	-	1	-	-	-	-
Primer iGEM063	-	-	1	-	-	-
Primer iGEM059	-	-	-	1	-	-
Primer iGEM057	-	-	-	-	1	-
Primer iGEM055	-	-	-	-	-	1
5x HF buffer	180	-	-	-	-	-
Phusion DNA polymerase 1:10	45	-	-	-	-	-
Water	585	-	-	-	-	-
Master Mix	-	19	19	19	19	19
Total Volume	855	20	20	20	20	20

Step	Temperature °C	Time
1	95	10 min
2	98	30 sec
3	55	30 sec
4	72	60 sec
5	Go To 2	34x
6	72	5 min
7	8	infinite

The positive clones (band at ca 1000 bp) were transferred into LB-amp medium and were incubated at 37°C over night for a miniprep. A new colony PCR was carried out with six colonies of the XLIBlue piGEM002+Ag-promoter.

22.4 Repeated amplification of KS module II

Aim: amplify module II for further purification and fusion PCR with flanks of lacA

The PCR for the KSII was repeated with the Phusion DNA-polymerase. A gradient was carried out from 52°C to 62°C.

Content	Master mix	KSII (µl)
Template: (gBlock KSII)	4,5	0,5
Primer iGEM044	9	1
Primer iGEM045	9	1
HF Buffer 5x	90	10
Phusion DNA-polymerase	9	1
dNTPs	9	1
Water	319,5	35
Total Volume (µl)	450	50

Step	Temperature °C	Time	KS II
1	95	5 min	5 min
2	95	30 sec	20 sec
3	52-62	30 sec	20 sec
4	72	1 min 45 sec	120 sec
5	Go To 2	30x	33x
6	72	4min	4 min
7	8	infinite	infinite

The gel showed many unspecific bands of fewer basepairs than expected. The expected band of ca 2000 bp was very weak but got stronger the higher the annealing temperature was.

22.4 repeated fusion-PCR of the Killswitch modules with the flanks

Aim: fuse the modules with the flanks to integrate them into the B. subtilis genome

For this fusion-PCR the flanks of 22.08.14 and the amplified and purified modules I and III from 28.08.14 were used. The module II from 24.08.14 was separated on an agarose gel, cut out and purified as well.

Content	KS I (µl)	KS II (µl)
Template KSI (ca 5 ng/µl)	1	-
Template KSII (7 ng/µl)	-	1
amyE flank I (1.5 ng/µl)	3	-
amyE flank II (ca. 6.5 ng/µl)	1	-
lacA flank I (ca. 5.5 ng/µl)	-	1
lacA flank II (7 ng/µl)	-	1
Primer piGEM034	1	-
Primer piGEM037	1	-
Primer piGEM040	-	1
Primer pIGEM043	-	1
HF Buffer 5x	10	10
Phusion DNA-polymerase	1	1
dNTPs	1	1
Water	31	31
Total Volume	50	50

Step	Temperature °C	Time (min/sec) KSI	Time (min/sec) KSII
1	95	5 min	5 min
2	95	30 sec	30 sec
3	55	30 sec	30 sec
4	72	2 min	3 min 10 sec
5	Go To 2	34x	34x
6	72	5 min	5 min
7	8	infinite	infinite

The fusion PCR was negative, expected bands would be around 1881 bp for KSI and around 3000 for KSII.

30.08.2014

18.62 Gibson assembly of piGEM-005 & D2-StrepII/ Ligation StrepDARPidin-pET16b

The transformation plates were taken out of the incubator and put in the fridge at 4°C.

31.08.2014

18.63 cPCR with clones from Ligation pET16b & StrepDARPidin

Aim: checking the success of the ligation

The grown clones from the ligation from 18.60 were checked via cPCR with primers iGEM-032 and -033. 40 clones from Ligation plates I-III were picked for cPCR and transferred on a plate before.

Content	Master mix for 40 clones (µL)	Clones from Ligation I (µL)	Clones from Ligation II(µL)	Clones from Ligation III(µL)
Template (colony)	-	1	1	1
Primer iGEM-32	20	-	-	-
Primer iGEM-33	20	-	-	-
Phusion Buffer 5x	160	-	-	-
Phusion DNA-polymerase	20	-	-	-
dNTPs	20	-	-	-
Water	560	-	-	-
Master mix	-	19	19	19
Total Volume	800	20	20	20

Step	Temperature °C	Time
1	98	10 min
2	98	20 sec
3	55	20 sec
4	72	60 sec
5	Go To 2	33x
6	72	4 min
7	8	infinite

The positive clones show a band at ca. 900 bp and were used for inoculation minipreps so that a test digest could confirm the insertion of StrepDARPidin.

18.64 cPCR with XLI-Blue clones containing piGEM005 Hag-D2-Strep or Hag-D2-Cup

Aim: Checking insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005

In order to check the successful integration of D2-Cup and D2-Strep clones were picked for cPCR and transferred on a LB-Amp master plate. As primers Flo96 (D23-fw) and Flo97 (D23-v) were used. 12 clones were screened.

Mix	Master-Mix (14x)	D2-Strep
Template	-	Colony
Primer Flo96 D23-fw	7	-
Primer Flo96 D23-rv	7	-
dNTPs	7	-
5x Phusion Buffer	56	-
Phusion DNA-polymerase	7	-
Water	196	-
Mastermix	-	20
Total Volume (µl)	280	20

Step	Temperature °C	Time
1	98	10 min
2	98	30 sec
3	55	30 sec
4	72	40 sec
5	Go To 2	33x
6	72	4 min
7	8	infinite

The clones I1, I3, I6, II2 and II4 were picked for plasmid isolation and sent for sequencing the next day.

@@ Line 1: / Line 1: @@
 {{Team:Marburg/Template:Header}}
-{{Team:Marburg/Template:NavigationProject}}
+{{Team:Marburg/Template:Navigation:Project:Notebook}}
+{{Team:Marburg/Template:Start}}
+Notebook: August
+{{Team:Marburg/Template:StartContinue}}
+{{Team:Marburg/Template:Submenu:Notebook:August}}
 <html>
-<div class="mr_content">
-<br /><br /><br />
-<!--main content -->
-<h1>Notebook</h1>
+<!-- 01.08.14 -->
-<h3>August</h3>
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="01.08.2014">01.08.2014</a>
+</h2>
+<fieldset class="exp18">
+    <legend><a name="exp18.50">18.50 Design of a Strep-Tag/ StrepDARPidin system</a></legend>
+    <div class="aim">
+    <p>Aim: Designing a new system for using the Flagellin-DARPin-Konstrukt</p>
+    </div>
+    <div class="exp-content">
+    <p>A new plan was made to obtain the flagellas multi-functionality, using an affinity tag. We want to integrate a Strep-Tag into the flagellin so that the DARPin domain can be fused to the flagellum via streptavidin. </p>
+    <p>The D2 domain of <i>Salmonella typhimurium</i>'s flagellin was used as a linker between the hag of <i>Bacillus subtilis</i> and the Strep-Tag, furthermore GSGS linkers were used between the D2 and the StrepTag.</p>
+    <p>Additionally  the DARPin was designed with an N-terminal His-Tag and C-Terminal streptavidin, so a chimeric protein would be generated.</p>
+    <p>The designed  structures were synthesized by Integrated DNA Technologies.</p>
 </div>
+</fieldset>
+<fieldset class="exp15">
+    <legend><a name="exp15.61">15.61 Design of Hag-D2-Cup</a></legend>
+    <div class="aim">
+    <p>Aim: Designing a new domain construct for expression of cup1-1-Hag</p>
+    </div>
+    <div class="exp-content">
+    <p>We decided to design the flagellin as well with the D2 domain and the cup1-1.</p>
+    <p>The designed structures were synthesized by Integrated  DNA Technologies</p>
+    </div>
+</fieldset>
+</div>
+<!-- ROMAN -->
+<!-- 04.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="04.08.2014">04.08.2014</a>
+</h2>
+<fieldset class="exp14">
+ <legend><a name="exp14.68">14.68 Crystallization of Arc1p-C</a></legend>
+    <div class="aim">
+    <p>Aim: Crystallize the Arc1p-C-single domain</p>
+    </div>
+    <div class="exp-content">
+    <p>For the experiments the sitting drop method in SWISSCI MRC 2 well crystallization plates with a drop size of 1 &micro;L was used (1:1 mixture of protein and crystallization solution) with an reservoir volume of 50 &micro;L. Crystals of Arc1p-C-single were obtained
+by room temperature and at 6 mg/mL protein concentration after 1 week in 0.1 M MES, pH 6.0, 10% (v/v) glycerol, 30% (w/v) PEG 600 and 5% (w/v) PEG 1000. To collect data the crystals
+were flash frozen in liquid nitrogen and measured at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France at beamline ID23-1. The structure of Arc1p-C-single
+was determined by molecular replacement (MR) using the crystal structure of human Arc1p-C (PDB ID: 1FL0).</p>
+    </div>
+</fieldset>
+</div>
+<!-- ROMAN -->
+<!-- 05.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="05.08.2014">05.08.2014</a>
+</h2>
+<fieldset class="exp18">
+    <legend><a name="exp18.51">18.51 Isolation of flagella from <i>Bacillus subtilis</i> WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: isolation of filaments of WT3610 as negative control for e.g. FACS analysis & microscopy</p>
+    </div>
+    <div class="exp-content">
+    <p>1 L LB was inoculated with WT 3610 and incubated over night at 37&deg;C.</p>
+</div>
+</fieldset>
+<fieldset class="exp15">
+    <legend><a name="exp15.62">15.62 Restriction digest of piGEM-005 SpeI</a></legend>
+    <div class="aim">
+    <p>Aim: linearizing vector for Gibson assembly with Hag-D2-Cup and -D2-Strep</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">piGEM-005 (100 ng/&micro;L</th>
+    <td>20</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart 10x</th>
+    <td>2,5</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Spe</i>I</th>
+    <td>0,2</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>2,3</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>25</td>
+  </tr>
+</table>
+	<p>Incubation at 37&deg;C for 2 hours &ndash; Inactivation by heat shock at 85&deg; for 10  min.</p>
+    <p>End concentration: 50 ng/&micro;L</p>
+</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.51">18.51 <i>Nco</i>I/<i>Bam</i>HI & <i>Nco</i>I/<i>Xho</i>I digest of pET24d</a></legend>
+    <div class="aim">
+    <p>Aim: purification of vector for ligation with StrepDARPidin gene</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Volume [&micro;l]</th>
+    <th scope="col">Volume [&micro;l]</th>
+  </tr>
+  <tr>
+    <th scope="row">pET24d (80 ng/&micro;L)</th>
+    <td>30</td>
+    <td>30</td>
+    </tr>
+  <tr>
+    <th scope="row">CutSmart 10x</th>
+    <td>3,5</td>
+    <td>3,5</td>
+    </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I</th>
+    <td>0,25</td>
+    <td>0,25</td>
+    </tr>
+  <tr>
+    <th scope="row"><i>Bam</i>HI</th>
+    <td>0,25</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row"><i>Xho</i>I</th>
+    <td>-</td>
+    <td>0,25</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row"><strong>Total</strong> Volume</th>
+    <td>35</td>
+    <td>35</td>
+    </tr>
+</table>
+	<p>Incubation at 37&deg;C for 2h .</p>
+	<p>The fragments were purified via gel extraction.</p>
+    <p>End concentration: 25 ng/&micro;L pET24d-N/B & 15 ng/&micro;L-N/X</p>
+</div>
+</fieldset>
+</div>
+<!-- 06.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="06.08.2014">06.08.2014</a>
+</h2>
+<fieldset class="exp18">
+    <legend><a name="exp18.52">18.52 Isolation of flagella from <i>Bacillus subtilis</i> WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: isolation of filaments of WT3610 as negative control for e.g. FACS analysis & microscopy</p>
+    </div>
+    <div class="exp-content">
+    <p>The preculture  was centrifuged at 4000 rpm for 15 minutes and the pellet was resuspended in 33 mL TRIS/HCl buffer  (pH 8.0) with 0,5% Brij-58. A stock solution of lysozyme with 10 mg/mL was made.  330 &micro;L were added to the TRIS/HCl buffer mix. The lysis was performed at 4&deg;C while rotating the tubes until the pellet was completely solved which took approx. 2 - 3 hours. DNase (5 &micro;g/ mL) in presence of 0,01 M magnesium chloride was added and incubated for  half an hour at 4&deg;C in the rotator as well.</p>
+    <p>After that the  suspension was centrifuged at 10000x g for 10 min. 80 &micro;L of the supernatant were  transferred into a 1,5 mL tube. The supernatant was then centrifuged at 87000xg  for 90min.</p>
+    <p>The pellet was  resuspended in 10 mL standard saline citrate (0,01M trisodium citrate &amp;  0,1M sodium chloride, pH 7,3) on ice and 80 &micro;L were taken as well. For fractioning, 2  g of ammonium sulfate (20%) were added slowly until precipitation could be seen.</p>
+    <p>After some  minutes of resting the suspension was centrifuged at 20000 rpm for 15 min. The  pellet was then resuspended in 2 mL of standard saline citrate buffer. After  taking a sample of 80 &micro;L the 4 samples ware mixed with 20 &micro;L SDS-loading buffer  each and analysed on an SDS-PAGE.</p>
+    <p>The purified  filaments were frozen in liquid nitrogen and then at -80&deg;C.</p>
+    <img src="https://static.igem.org/mediawiki/2014/c/cc/MR_2014-08-06_18.52.jpg" width="30%" />
+    <p>The flagella purification was successful. Besides the flagellin the samples also contain the hook proteins, the smaller proteins were removed from the purified pellet.</p>
+    <img src="https://static.igem.org/mediawiki/2014/b/bb/MR_2014-08-06_18.52_2.jpg" width="30%" />
+</div>
+</fieldset>
+<!-- ROMAN -->
+<fieldset class="exp14">
+ <legend><a name="exp14.69">14.69 tRNA<sup>Phe</sup> assay</a></legend>
+    <div class="aim">
+    <p>Aim: Measure aminoacylation levels</p>
+    </div>
+    <div class="exp-content">
+    <p>To measure aminoacylation of tRNA<sup>Phe</sup> the components were mixed.</p>
+	<table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Final concentration</th>
+  </tr>
+  <tr>
+    <th scope="row">Dialysis-buffer</th>
+    <td>ad 250 &micro;L</td>
+  </tr>
+  <tr>
+    <th scope="row">DTT</th>
+    <td>1 mM</td>
+  </tr>
+  <tr>
+    <th scope="row">MgCl<sub>2</sub></th>
+    <td>20 mM</td>
+  </tr>
+  <tr>
+    <th scope="row">KCl</th>
+    <td>10 mM</td>
+  </tr>
+  <tr>
+    <th scope="row">tRNA<sup>Phe</sup></th>
+    <td>10 &micro;M</td>
+  </tr>
+  <tr>
+    <th scope="row">L- or D-Phenylalanine</th>
+    <td>1 mM</td>
+  </tr>
+  <tr>
+    <th scope="row">catalytic fusion Protein</th>
+    <td>10 &micro;M</td>
+  </tr>
+</table>
+<p>After mixing, reaction was initiated by the addition of 2 mM ATP and then incubated for 1 to 60 min by 37 &deg;C and 350 rpm. Assays were stopped by the addition of 25 &micro;L 3 M sodium acetate solution. Afterwards tRNAs were precipitated with ethanol. Dissolved pellets were further purified using size-exclusion columns. The elution fraction was split into two aliquots. One samples was treated with 0.2 M NaOH (final concentration: 10 mM) and incubated at 50 &deg;C for 10 min, while the other aliquot was kept on ice. The base treated sample was again acidified by the addition of 3 M sodium acetate (pH 5.2, final concentration: 300 mM). The volume of the untreated sample was adjusted using 2 mM sodium acetate (pH 5.2). Subsequently, both samples were subjected to LCMS analysis using 100 &micro;L for injection.</p>
+    </div>
+</fieldset>
+<!-- ROMAN -->
+</div>
+<!-- 11.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="11.08.2014">11.08.2014</a>
+</h2>
+<fieldset class="exp22">
+    <legend><a name="exp25.0">25. Cell culture assays</a></legend>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp25.1">25.1 Splitting of Caco-2-cells</a></legend>
+    <div class="aim">
+    <p>Aim: Splitting cell culture for growing</p>
+    </div>
+    <div class="exp-content">
+    <p>The Caco-2-cells were kindly provided by Dr. Hartmann Reifert (BMFZ). In order to let them grow, the cells had to be  splitted according to a specific protocol. Caco-2-cells require DMEM (Dulbecco&rsquo;s  Modified Eagle Medium) with 20% FCS and L-Glutamine.</p>
+    <p>A centrifuge was precooled to 4&deg;C.</p>
+    <p>The medium of the culture flask was discarded  from the side opposite to the adherent cells and washed with 1X PBS from the  opposite site as well.</p>
+    <p>The PBS was aspirated with a glass pipette  until the supernatant was clear. After adding 1 - 2 mL 1x trypsin from the  opposite of the adherent cells the flask was incubated for 5 - 10 min at 37&deg;C  depending on how fast the cells unfastened of from the ground. Under the microscope  the free floating cells were checked.</p>
+    <p>The cells were transferred into a 15 mL falcon  and rinsed with 10 mL DMEM to collect all remaining cells in the flask. The 15  mL falcon was filled with DMEM and the culture centrifuged at 1500 rpm&nbsp; for 5 min at 4&deg;C.</p>
+    <p>The supernatant was discarded and resuspended  in 2 mL DMEM. The cells were splitted 1:3 which means that 666 &micro;L were taken  from the suspension and transferred into a new culture flask. After adding  20-25 mL DMEM the flask was incubated at 37&deg;C and checked on the 3rd  / 4th day under the microscope.</p>
+    <p>On the medium flask were the following notes:<br />
+      Date, Name, Cell Line, Medium, Ratio of splitting, passage number</p>
+    </div>
+</fieldset>
+<fieldset class="exp15">
+    <legend><a name="exp15.63">15.63 PCR Amplification of Hag-D2-Cup</a></legend>
+    <div class="aim">
+    <p>Aim: Amplification of insert for Gibson assembly into piGEM-005</p>
+    </div>
+    <div class="exp-content">
+    <p>The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was  centrifuged and resuspended in 20 &micro;L Millipore water to get an end concentration  of 10 ng/&micro;L as a Stock solution.</p>
+    <p>0,5 &micro;L were used as a PCR  template.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix (&micro;l)</th>
+    <th scope="col">Mastermix</th>
+    <th scope="col">D2-Cup</th>
+    <th scope="col">D2-Strep</th>
+  </tr>
+  <tr>
+    <th scope="row">Template</th>
+    <td>-</td>
+    <td>2</td>
+    <td>2</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  Flo96 D23-fw</th>
+    <td>3</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  Flo97 D23-rv</th>
+    <td>3</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5-Master  mix</th>
+    <td>75</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>63</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>48</td>
+    <td>48</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>144</td>
+    <td>50</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>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</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/2014/5/56/MR_2014-08-11_15.63.jpg" width="30%" />
+		<br />
+		<p>The expected bands could be seen.
+	</div>
+</fieldset>
+<fieldset class="exp15">
+    <legend><a name="exp15.64a">15.64 Gibson assembly with linearized piGEM-005 & PCR products from 15.63</a></legend>
+    <div class="aim">
+    <p>Aim: Insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005</p></div>
+    <div class="exp-content">
+	<table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Hag-D2-Cup(&micro;L)</th>
+    <th scope="col">Hag-D2-Strep (&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">Gibson-mix</th>
+    <td>15</td>
+    <td>15</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert I:<br />      &nbsp;(Hag-D2-Cup 431 ng/&micro;L)</th>
+    <td>2,5</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert II:<br />
+      (Hag-D2-Strep 423 ng/&micro;L)</th>
+    <td>-</td>
+    <td>2,5</td>2
+  </tr>
+  <tr>
+    <th scope="row">piGEM-005 <i>Spe</i>I<br />
+      &nbsp;(50 ng/  &micro;L)</th>
+    <td>2,5</td>
+    <td>2,5</td>
+  </tr>
+  <tr>
+    <th scope="row"><strong>Total</strong></th>
+    <td>20</td>
+    <td>20</td>
+  </tr>
+</table>
+	<p>Incubation was done &nbsp;at 50&deg;C for  1 hour with following transformation into <em>E. coli</em>  XL1-Blue plated out on LB-Amp plates and incubated overnight at 37&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 12.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="12.08.2014">12.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.64b">15.64 Screening clones from 15.63</a></legend>
+    <div class="aim">
+    <p>Aim: Checking insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005</p>
+    </div>
+    <div class="exp-content">
+    <p>In order to check the successful integration of D2-Cup and D2-Strep, clones were picked for cPCR and transferred onto a LB-Amp plate. The primers Flo96 (D23-fw) and Flo97 (D23-v) were used.</p>
+       <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix (&micro;l)</th>
+    <th scope="col">Mastermix</th>
+    <th scope="col">D2-Cup</th>
+    <th scope="col">D2-Strep</th>
+  </tr>
+  <tr>
+    <th scope="row">Template</th>
+    <td>-</td>
+    <td>Colony</td>
+    <td>Colony</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  Flo96 D23-fw</th>
+    <td>4,5</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  Flo97 D23-rv</th>
+    <td>4,5</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTP</th>
+    <td>4,5</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">5x Phusion Buffer</th>
+    <td>36</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>4,5</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>126</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>20</td>
+    <td>20</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>180</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>10 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>40 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>33x</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/c/c3/MR_2014-08-12_15.64.jpg" width="20%" />
+        <p>The gel shows that clone 2 from the Gibson Hag-D2-Cup clones and clones  3 &amp; 4 from the Gibson Hag-D2-Strep clones contain the integrated plasmid  constructs. All 3 clones were picked for a miniprep and used for the inoculation of  10 mL LB-Amp. Incubation was performed over night at 37&deg;C. </p>
+        <p><strong>The new  constructs were labelled with piGEM-026 (pMAD-Hag-D2-Cup) and piGEM-027  (pMAD-Hag-D2-Strep).</strong></p>
+</div>
+</fieldset>
+<fieldset class="exp15">
+    <legend><a name="exp15.65">15.65 Overnight culture for WT3610</a></legend>
+    <div class="aim">
+    <p>Aim:Preparation for mainculture the next day</p>
+    </div>
+    <div class="exp-content">
+    <p>5 mL of LB  were inoculated with <i>Bacillus subtilis</i> WT3610 from an LB plate and incubated at 37&deg;C  over night.</p>
+</div>
+</fieldset>
+</div>
+<!-- 13.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="13.08.2014">13.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.66">15.66 Transformation of competent <i>Bacillus subtilis</i> WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: transformation of plasmid into <i>Bacillus subtilis</i> WT3610</p>
+    </div>
+    <div class="exp-content">
+    <p>100 &micro;L  preculture were added to 10 mL of MNGE-Medium and  incubated to an OD<sub>600</sub> of 1,1-1,3 at 37&deg;C which could take 4-5h.</p>
+    <p>After reaching an OD<sub>600</sub> of 1,1-1,3 2 x 400 &micro;L of the culture were transformed  with 1,5 &micro;g plasmid (piGEM-026 &amp; -027). After 1 hour of incubation at 37&deg;C 100 &micro;L  expression mix were added and incubated for 1 hour as well.</p>
+    <p>In the end the 500 &micro;L attempt was 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_20">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>AGB0023458</td>
+    <td>piGEM-023</td>
+    <td>TW260</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023459</td>
+    <td>piGEM-024</td>
+    <td>TW260</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023460</td>
+    <td>piGEM-025</td>
+    <td>TW260</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB0023461</td>
+    <td>piGEM-026 D2-Cup cl.2</td>
+    <td>Flo96-D2 3 for</td>
+  </tr>
+  <tr>
+    <th scope="row">5</th>
+    <td>AGB0023462</td>
+    <td>piGEM-026 D2-Cup cl.2</td>
+    <td>Flo56-Hag rv</td>
+  </tr>
+  <tr>
+    <th scope="row">6</th>
+    <td>AGB0023463</td>
+    <td>piGEM-027 D2-Strep cl.3</td>
+    <td>Flo96-D2 3 for</td>
+  </tr>
+  <tr>
+    <th scope="row">7</th>
+    <td>AGB0023464</td>
+    <td>piGEM-027 D2-Strep cl.3</td>
+    <td>Flo56-Hag rv</td>
+  </tr>
+  <tr>
+    <th scope="row">8</th>
+    <td>AGB0023465</td>
+    <td>piGEM-027 D2-Strep cl.4</td>
+    <td>Flo96-D2 3 for</td>
+  </tr>
+  <tr>
+    <th scope="row">9</th>
+    <td>AGB0023466</td>
+    <td>piGEM-027 D2-Strep cl.4</td>
+    <td>Flo56-Hag rv</td>
+  </tr>
+</table>
+    </div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.72">13.72 Restriction of Nose plasmids with <i>Eco</i>RV</a></legend>
+    <div class="aim">
+    <p>Aim: check if <i>amyE</i> and <i>cat</i> are still in plasmid, since <i>B. subtilis</i> could not successfully be transformed with the plasmids yet</p>
+    </div>
+    <div class="exp-content">
+    <p>All Nose-plasmids (iGEM002-004 and 007-015) were digested with <i>Eco</i>RV to check if <i>amyE</i> and <i>cat</i> are still present in these plasmids, since <i>B. subtilis</i> could not be transformed successfully.</p>
+    <p>Per sample 1 &micro;l 2x CutSmart Buffer, 7.9 &micro;l H2O,  0.1 &micro;l <em>EcoR</em>V and 2 &micro;l of plasmids  were added. The reaction was incubated over night at RT.</p>
+</div>
+</fieldset>
+</div>
+<!-- 14.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="14.08.2014">14.08.2014</a>
+</h2>
+<fieldset class="exp13">
+    <legend><a name="exp13.72">13.72 Restriction of Nose plasmids with <i>Eco</i>RV - continuation</a></legend>
+    <div class="aim">
+    <p>Aim: Analysis of the restriction on an agarose gel</p>
+    </div>
+    <div class="exp-content">
+    <p>The samples were mixed with 2 &micro;l of 5x Loading dye and 8 &micro;l of it was loaded on a 1% agarose gel.</p>
+    <img src="https://static.igem.org/mediawiki/2014/e/e5/MR_2014-08-14_13.72.jpg" width="30%" />
+    <p>All plasmids but piGEM004 and piGEM011 show the expected bands of ca 2000 and 4000 bp.</p>
+    <p>The plasmids were used to transform  <i>Bacillus  subtilis</i>, which was incubated at 30&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 18.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="18.08.2014">18.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.67">15.67 Transformation of competent WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: <i>Bacillus subtilis</i> pMad Trafo mit D2-Strep and D2-Cup</p>
+    </div>
+    <div class="exp-content">
+    <p>Next step: inoculate overnight culture from the X-gal plate with 3 ml LB-MLS.</p>
+    <p>As the cultures were  recognizable blue (thin or old plate) wildtype cultures were inoculated for a new  transformation. Therefore the plasmids piGEM-026 and -027 were isolated from frozen pellets. </p>
+    </div>
+</fieldset>
+</div>
+<!-- 19.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="19.08.2014">19.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.68">15.68 Transformation of competent WT3610</a></legend>
+    <div class="exp">
+    <p><i>Bacillus subtilis</i> pMad trafo mit D2-Strep and D2-Cup</p>
+    </div>
+    <div class="exp-content">
+    <p>For the experimental procedure see the Materials section. Anyway, two transformations were running currently that will be referred to as 'old' and 'new' transformation from now on.</p>
+    <p>For the old transformation the first temperature  shift was performed in LB-MLS medium. The culture was plated on LB-MLS-X-Gal plates  and incubated at 42&deg;C over night.</p>
+    <p>The new transformation was started with a culture  grown in MNGE medium, inoculated with a wildtype overnight culture. The culture  was grown until an optical density of 1,2 that is normally reached after 4-5 h.  Strangely the culture reached this OD after almost 7 h today. Plasmids (1.5&micro;g) piGEM026 and -027 were  transformed and the protocol was followed as described below.</p>
+</div>
+</fieldset>
+</div>
+<!-- 20.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="20.08.2014">20.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.69">15.69 Transformation of competent WT3610</a></legend>
+    <div class="exp">
+    <p><i>Bacillus subtilis</i> pMad Trafo mit D2-Strep and D2-Cup</p>
+    </div>
+    <div class="exp-content">
+    <p>New trafo showed no colonies so far. It was further incubated.</p>
+    <p>For the old trafo 2nd temperature shift of positive colonies (only light blue).</p>
+    </div>
+</fieldset>
+</div>
+<!-- 21.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="21.08.2014">21.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.70a">15.70 Transformation of competent WT3610</a></legend>
+    <div class="exp">
+    <p><i>Bacillus subtilis</i> pMad Trafo mit D2-Strep and D2-Cup</p>
+    </div>
+    <div class="exp-content">
+    <p>Old trafo: white colonies were re-streaked on x-Gal plates and on MLS plates to check for MLS sensitivity. Incubation at 42&deg;C.</p>
+    </div>
+</fieldset>
+</div>
+<!-- 22.08.2014  -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="22.08.2014">22.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.70ab">15.70 Transformation of competent WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: integration of  pMAD-Insert into Bacillus chromosome via flanks</p>
+    </div>
+    <div class="exp-content">
+    <p>The blue/ white screening showed positive transformed blue clones from  the new transformation.&nbsp; Three piGEM026 and -027 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 over night at 30&deg;C  with the cultures.</p>
+</div>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.0">22. The Killswitch</a></legend>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.1">22.1 Amplification of flanks</a></legend>
+    <div class="aim">
+    <p>Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the <i>amyE</i> and the <i>lacA</i>/<i>ganA</i> locus of <i>Bacillus subtilis</i> after cloning into pMAD</p>
+    </div>
+    <div class="exp-content">
+    <p>Primers arrived and have been dissolved in millipore water, dilution 1:10. Q5 Master Mix was used for the following PCRs to create the flanks of the killswitch parts I-III:</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Fragment</th>
+    <th scope="col">Fw primer (iGEM-0xx)</th>
+    <th scope="col">Rv primer (iGEM-0xx)</th>
+  </tr>
+  <tr>
+    <th scope="row">AmyE flankI</th>
+    <td>iGEM-034</td>
+    <td>iGEM-035</td>
+  </tr>
+  <tr>
+    <th scope="row">AmyE flankII</th>
+    <td>iGEM-036</td>
+    <td>iGEM-037</td>
+  </tr>
+  <tr>
+    <th scope="row">lacA flankI</th>
+    <td>iGEM-040</td>
+    <td>iGEM-041</td>
+  </tr>
+  <tr>
+    <th scope="row">lacA flankII</th>
+    <td>iGEM-042</td>
+    <td>iGEM-043</td>
+  </tr>
+  <tr>
+    <th scope="row">KSIII AmyE-yvyd  FlankII</th>
+    <td>iGEM-048</td>
+    <td>-</td>
+  </tr>
+</table>
+<br />
+<table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">Master Mix</th>
+    <th scope="col"><i>amyE</i> FlankI</th>
+    <th scope="col"><i>amyE</i> FlankII</th>
+    <th scope="col"><i>lacA</i> FlankI</th>
+    <th scope="col"><i>lacA</i> FlankII</th>
+    <th scope="col"><i>amyE</i>-<i>yvyD</i> FlankII</th>
+  </tr>
+  <tr>
+    <th scope="row">Template  (<i>B. subtilis</i>  gDNA)</th>
+    <td>11</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-34</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-35</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-36</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-37</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-40</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-41</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-42</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-43</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-48</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-37</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5-Master  mix</th>
+    <td>165</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>132</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>28</td>
+    <td>28</td>
+    <td>28</td>
+    <td>28</td>
+    <td>28</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume [&micro;l]</th>
+    <td>308</td>
+    <td>30</td>
+    <td>30</td>
+    <td>30</td>
+    <td>30</td>
+    <td>30</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>60 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/2014/9/92/MR_2014-08-22_22.1.jpg" width="30%" />
+        <p>All bands had the expected size (500 /1000 bp)</p>
+</div>
+</fieldset>
+</div>
+<!-- 23.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="23.08.2014">23.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.71a">15.71 Transformation of competent WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: First heat shock - integration of  pMAD-Insert into Bacillus chromosome via flanks</p>
+    </div>
+    <div classs="exp-content">
+    <p>The&nbsp; overnight cultures were used to  inoculate 10 mL LB MLS until&nbsp; 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 dilutions from 10-5 to 10-6 of  each culture were plated out on MLS-X-Gal so that plates could be incubated  overnight at 42&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 24.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="24.08.2014">24.08.2014</a>
+</h2>
+<fieldset class="exp22">
+    <legend><a name="exp22.2">22.2 Amplification of Killswitch modules I-III </a></legend>
+    <div class="aim">
+    <p>Aim: The modules had to be amplified via PCR in order to proceed with a fusion PCR linking the modules with the flanks for integration into the Bacillus Loci.</p>
+    </div>
+    <div class="exp-content">
+    <p>The synthesized fragments by Integrated DNA technologies arrived as a dried 200 ng DNA pellet. In order to use it as a PCR template the pellet was  centrifuged down and resuspended in 20 &micro;L millipore water to get an end concentration of 10 ng/&micro;L as a stock solution.</p>
+    <p>0,5&nbsp; &micro;L were used as a PCR  template. The PCR fragments should be ca. 350, 650 &amp; 2000 bp long. </p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">KS module I</th>
+    <th scope="col">KS module II</th>
+    <th scope="col">KS module III</th>
+  </tr>
+  <tr>
+    <th scope="row">Template (gBlock DNA)</th>
+    <td>0,5</td>
+    <td>0,5</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-38</th>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-39</th>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-44</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-45</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-49</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-50</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5-Mastermix</th>
+    <td>25</td>
+    <td>25</td>
+    <td>25</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>22,5</td>
+    <td>22,5</td>
+    <td>22,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l)</th>
+    <td>50</td>
+    <td>50</td>
+    <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<br />KSI & III</th>
+            <th scope="col">Time<br />
+            KSII</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>3 min</td>
+            <td>3 min</td>
+            </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>20 sec</td>
+            <td>30 sec</td>
+            </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>20 sec</td>
+            <td>20 sec</td>
+            </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>60 sec</td>
+            <td>120 sec</td>
+            </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</td>
+            <td>31x</td>
+            </tr>
+          <tr>
+            <th scope="row">6</th>q
+            <td>72</td>
+            <td>4min</td>
+            <td>4min</td>
+            </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+            <td>infinite</td>
+            </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/7/7d/MR_2014-08-23_22.2.jpg" width="30%" />
+		<br />
+		<p>The modules I and III could be amplified without problems, but KSII showed no distinct band and many unspecific fragments.</p>
+		</div>
+</fieldset>
+<fieldset class="exp15">
+    <legend><a name="15.7b">15.71 Transformation of competent WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: Second heat shock -flip out of the pMAD backbone</p>
+    </div>
+    <div class="exp-content">
+    <p>One blue colony per diluted piGEM026  and -027 clone was used to inoculate 4 mL LB. The 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>-5</sup> to 10<sup>-6</sup> were plated out on&nbsp; 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. Some clones  (piGEM027.1 and one of 27.2) did not grow and will be inoculated again. They  will be inoculated in LB-MLS for the first temperature shift tomorrow.</p>
+</div>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.3a">22.3 Transformation of competent DH5&alpha; with pMAD</a></legend>
+    <div class="aim">
+    <p>Aim: Gain <i>E.coli</i> on plate for mini preps of pMAD</p>
+    </div>
+    <div class="exp-content">
+    <p>Standard heat shock transformation protocol was  followed, incubation over night at 37&deg;C. pMAD from the plasmid box was used for  transformation.</p>
+</div>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.4">22.4 Restriction of pMAD with <i>Nco</i>I and <i>Bam</i>HI over night</a></legend>
+    <div class="aim">
+    <p>Aim: Linearizing Vector for Gibson Assembly with KS modules</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">pMAD (108 ng/&micro;L)</th>
+    <td>9</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart 10x</th>
+    <td>2</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I</th>
+    <td>0,2</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Bam</i>HI</th>
+    <td>0,2</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>8,6</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+	<p>Incubation over night at room temperature.</p>
+    </div>
+</fieldset>
+</div>
+<!-- 25.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="25.08.2014">25.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.72">15.72 Transformation of competent WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: checking the correct flip out of the pMAD backbone and MLS sensitivity</p>
+    </div>
+    <div class="exp-content">
+    <p>From the dilution plates was one white clone picked and transferred on a  X-Gal plate as well as on a MLS plate so that 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="exp22">
+    <legend><a name="exp22.5a">22.5a Fusion PCR </a></legend>
+    <div class="aim">
+    <p>Aim: Assembly of Killswitch modules I & II with <i>amyE</i> and <i>lacA</i> flanks</p>
+    </div>
+    <div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Segment</th>
+    <th scope="col"><strong>Concentraction  (ng/&micro;L)</strong></th>
+  </tr>
+  <tr>
+    <th scope="row">KS module I</th>
+    <td>401</td>
+  </tr>
+  <tr>
+    <th scope="row">KS module II</th>
+    <td>414</td>
+  </tr>
+  <tr>
+    <th scope="row">KS module III</th>
+    <td>369</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i> flank I</th>
+    <td>446</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i> flank II</th>
+    <td>403</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i> flank I</th>
+    <td>368</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i> flank II</th>
+    <td>369</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i> <i>yvyD</i> flank II</th>
+    <td>372</td>
+  </tr>
+</table>
+	<br />
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col"><i>amyE</i> KS I</th>
+    <th scope="col"><i>lacA</i> KS II</th>
+  </tr>
+  <tr>
+    <th scope="row">KS  module I (40,1 ng/&micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">KS  module II (41,4 ng/&micro;L)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i>  flank I ( 44,6 ng/&micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i>  flank II (40,3 ng/&micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i>  flank I (36,8 ng/&micro;L)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i>  flank II (39,6 ng/&micro;L)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-34</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-37</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-40</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer  iGEM-43</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion  Buffer 5x</th>
+    <td>10</td>
+    <td>10</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>33</td>
+    <td>33</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume [&micro;l]</th>
+    <td>50</td>
+    <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<br />
+            amyE KSI</th>
+            <th scope="col">Time<br />
+            amyE KSII</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>3 min</td>
+            <td>3 min</td>
+            </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>20 sec</td>
+            <td>30 sec</td>
+            </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>20 sec</td>
+            <td>20 sec</td>
+            </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>120 sec</td>
+            <td>190 sec</td>
+            </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</td>
+            <td>31x</td>
+            </tr>
+          <tr>
+            <th scope="row">6</th>q
+            <td>72</td>
+            <td>4min</td>
+            <td>4min</td>
+            </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+            <td>infinite</td>
+            </tr>
+        </table>
+        <p>No PCR amplificates could be seen on the gel.</p>
+    </div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.53">18.53 PCR amplification of StrepDARPidin</a></legend>
+    <div class="aim">
+    <p>Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in <i>E. coli</i> BL21(DE3)</p>
+    </div>
+    <div class="exp-content">
+    <p>The synthesized fragments by Integrated DNA technologies arrived as a dried  200n g DNA pellet. In order to use it as a PCR template the pellet was  centrifuged and resuspended in 20 &micro;L millipore water to get an end concentration  of 10 ng/&micro;L as a stock solution.</p>
+    <p>0,5&nbsp; &micro;L were used as a PCR  template. The PCR fragment should be ca. 900 bp long. </p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">PCR attempt</th>
+  </tr>
+  <tr>
+    <th scope="row">Template</th>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-33</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-34</th>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5-Mastermix</th>
+    <td>25</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>22,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</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</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>60 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</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/a/a7/MR_2014-08-25_18.53.jpg" width="30%" />
+        <p>Both samples had a  concentration of 380 ng/&micro;L.</p>
+		</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.54">18.54 Digestion of amplified of StrepDARPidin for cloning into pET24d</a></legend>
+    <div class="aim">
+    <p>Aim: digest with <i>Nco</i>I and <i>Xho</i>I of the StrepDARPidin PCR product for cloning into the expression vector pet24d </p>
+    </div>
+    <div class="exp-content">
+    <p>The PCR product was cut with <i>Nco</i>I and <i>Xho</i>I to get the restriction sites for cloning into pET24d <i>Nco</i>I/<i>Xho</i>I cut.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">StrepDARPidin (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">PCR product (380 ng/&micro;L)</th>
+    <td>6</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart 10x</th>
+    <td>2</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I</th>
+    <td>0,25</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Xho</i>I</th>
+    <td>0,25</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>11,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+	<p>Incubation for 1h at  37&deg;C with heat shock at 85&deg;C for 10 min.</p>
+</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.55">18.55 Ligation of digested StrepDARPidin for cloning into pET24d</a></legend>
+    <div class="aim">
+    <p>Aim: ligation of StrepDARPidin PCR product with the expression vector pET24d </p>
+    </div>
+    <div class="exp-content">
+    <p>The PCR product was ligated into digested pET24d <i>Nco</i>I/<i>Xho</i>I. Two samples were made for a transformation of <i>E. coli</i> XL1-Blue and BL21.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Ligation I (&micro;l)</th>
+    <th scope="col">Ligation II (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I and <i>Xho</i>I StrepDARPidin (64 ng/&micro;L)</th>
+    <td>2</td>
+    <td>2</td>
+    </tr>
+  <tr>
+    <th scope="row">pET24d <i>Nco</i>I and <i>Xho</i>I (15  ng/&micro;L)</th>
+    <td>5</td>
+    <td>5</td>
+    </tr>
+  <tr>
+    <th scope="row">Ligation Buffer 10x</th>
+    <td>2</td>
+    <td>2</td>
+    </tr>
+  <tr>
+    <th scope="row">Ligase</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>10</td>
+    <td>10</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+    </tr>
+</table>
+	<p>Incubation for 1,5h at room temperature with heat shock at 85&deg;C for 10 min. The 20 &micro;L from ligation I were used to transform <i>E. coli</i> XLI-Blue, 10 &micro;L from Ligation II were used to transform <em>E. coli</em> BL21(DE3) and the rest of ligation II into  Xl1-Blue as well and selected on  LB-Canamycin plates.</p>
+</div>
+</fieldset>
+</div>
+<!-- 26.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="26.08.2014">26.08.2014</a>
+</h2>
+<fieldset class="exp15">
+    <legend><a name="exp15.73">15.73 Transformation of competent WT3610</a></legend>
+    <div class="aim">
+    <p>Aim: checking the correct flip out of the pMAD backbone and MLS sensitivity </p>
+    </div>
+    <div class="exp-content">
+    <p>All clones on the master plate grew on x-gal  plates but were MLS sensitive and could be seen as positive. In order to check  the correct insertion of D2-Cup an D2-Strep into the <i>Bacillus subtilis</i> genome  the clones were cooked in 100 &micro;L 1x PBS at 95&deg;C for 10 min and used for colony  PCR. Because of the repetition of the temperature shifts with clones 27.1.1,  27.1.2, 27.1.3 yesterday and today and 27.2.3, 6 clones from 26.1 -26.3, 27.2.1  &amp; 27.2.2 were picked for the cPCR (30 reactions).</p>
+           <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix (&micro;l)</th>
+    <th scope="col">Master-Mix for 30 attempts (&micro;L)</th>
+    <th scope="col">Mix for PCR attempt (&micro;L)</th>
+    </tr>
+  <tr>
+    <th scope="row">Colony PBS suspension</th>
+    <td>-</td>
+    <td>2</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer Flo54 Hag-fw</th>
+    <td>15</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer  Flo56 Hag-fw</th>
+    <td>15</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>15</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>120</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTP-mix</th>
+    <td>15</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>410</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">DMSO</th>
+    <td>10</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>18</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>600</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 30 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>There were no bands visible on the gel.</p>
+</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.56a">18.56 cPCR with plates from ligation</a></legend>
+	<div class="aim">
+	<p>Aim: checking the success of the ligation </p>
+	</div>
+	<div class="exp-content">
+	<p>The grown clones from the ligation the day before were inoculated for plasmid isolation and then checked via cPCR with primers iGEM-032 and -033.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content (&micro;l)</th>
+    <th scope="col">Master mix&nbsp; for  20 clones (&micro;L)</th>
+    <th scope="col">Clones from Ligation I (&micro;L)</th>
+    <th scope="col">Clones from Ligation II (&micro;L)</th>
+    <th scope="col">Clones from Ligation III (&micro;L)</th>
+    </tr>
+  <tr>
+    <th scope="row">Template (colony)</th>
+    <td>-</td>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-32</th>
+    <td>10</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-32</th>
+    <td>10</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>80</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase </th>
+    <td>10</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTP-mix</th>
+    <td>10</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>280</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>19</td>
+    <td>19</td>
+    <td>19</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>400</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>10 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>60 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>33x</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/a/a3/MR_2014-08-26_18.56.jpg" width="50%" />
+        <p>The gel shows that the clones I  2-6, II 1-6 and III 2, 3, 4 and 6 are positive due to the band at approx. 900 bp.  The other bands might be plasmid which was used as template.</p>
+        <p>The clones I6, II3, III3 were used to transform <i> E. coli</i> BL21(DE3) and selected on LB-Can plates. III4 and III6  were already on a plate. The clones were inoculated for plasmid isolation and sent for sequencing.</p>
+</div>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.5b">22.5b Fusion PCR Attempt 3</a></legend>
+	<div class="aim">
+	<p>Aim: Fusion of <i>amyE</i> and <i>lacA</i> flanks to the killswitch fragments</p>
+	</div>
+	<div class="exp-content">
+	<p>The Fusion PCR was repeated since  it did not work the first two times. The fragments of the killswitch I and II  were diluted 10fold. A gradient was carried out from 52&deg;C to 62&deg;C.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix (&micro;l)</th>
+    <th scope="col">amyE KSI</th>
+    <th scope="col">lacA KS II</th>
+  </tr>
+  <tr>
+    <th scope="row">KS module I (40,1 ng/&micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">KS module II (41,4 ng/&micro;L)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i> flank I ( 44,6 ng/&micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i> flank II (40,3 ng/&micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i> flank I (36,8 ng/&micro;L)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i> flank II (39,6 ng/&micro;L)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-34</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-37</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-40</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-43</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>4</td>
+    <td>4</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>0,5</td>
+    <td>0,5</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>9,5</td>
+    <td>9,5</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <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>95</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>95</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>52-62</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>200 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>32x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>4</td>
+            <td>infinite</td>
+          </tr>
+      </table>
+   	<p>Again, no fragments could be noticed on the gel.</p>
+</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.73">13.73 Amplification of new Cu- and Ag-Promotor sequences</a></legend>
+	<div class="aim">
+	<p>Aim: Amplification of Cu- and Ag promoter sequences from the chromosomal DNA of <i>Bacillus subtilis</i>.</p>
+	</div>
+	<div class="exp-content">
+	<table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Master mix &nbsp;(&micro;L)</th>
+    <th scope="col">2x Attempt Cu (&micro;L)</th>
+    <th scope="col">2x Attempt Ag (&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">Template (gDNA <i>B. subtilis</i>)</th>
+    <td>4</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-55</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-56</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-57</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-58</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>40</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>4</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>4</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>144</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>48</td>
+    <td>48</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l)</th>
+    <td>196</td>
+    <td>50</td>
+    <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</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>10 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>20 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>4 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/0/04/MR_2014-08-26_13.73.jpg" width="30%" />
+        <p>The gel shows positive bands at approx. 100 bp and 250 bp, which was like expected.</p>
+	</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.74">13.74 <i>Nco</i>I/<i>Sac</i>I digest of piGEM-002</a></legend>
+	<div class="aim">
+	<p>Aim: linearizing the plasmid backbone for Gibson Assembly and Ligation with Cu/Ag promotor sequences and IPTG/ Constitutive (/tetO) promotor sequences.</p>
+	</div>
+	<div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Attempt I (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 (476 ng/&micro;L)</th>
+    <td>6,5</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I</th>
+    <td>0,25</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Sac</i>I</th>
+    <td>0,25</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart Buffer 10x</th>
+    <td>2</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>11</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+	<p>Incubation at 37&deg;C for  60 min and heat shocked at 85&deg;C for 10 min.</p>
+	</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.75">13.75 Ligation of <i>Nco</i>I/<i>Sac</i> cut piGEM-002 with annealed oligos containing promoter sequences</a></legend>
+	<div class="aim">
+	<p>Aim: insertion of a IPTG inducible/ constitutive and constitutive+tetO into piGEM-002</p>
+	</div>
+	<div class="exp-content">
+	<p>In order to test our nose system with new promoter sequences oligos were designed and annealed by heating up water and letting it cool down with the oligo pairs iGEM-59/60, -61/62, - 63/64 so that the individual fragments were able to anneal with overlaps to the <i>Nco</i>I/<i>Sac</i>I sites.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Ligation I (&micro;l)</th>
+    <th scope="col">Ligation II (&micro;l)</th>
+    <th scope="col">Ligation III (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 (209 ng/&micro;L)</th>
+    <td>5</td>
+    <td>5</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">iGEM-59/60</th>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">iGEM-61/62</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">iGEM-63/64</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">T4 Ligase</th>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Ligation Buffer 10x</th>
+    <td>2</td>
+    <td>2</td>
+    <td>2</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>11</td>
+    <td>11</td>
+    <td>11</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l]</th>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    </tr>
+</table>
+	<p>The reactions were  incubated at room temperature for an hour and transformed into <em>E. coli</em> XL1-Blue, plated out on LB-Amp.</p>
+</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.76">13.76 Gibson assembly of <i>Nco</i>I/<i>Sac</i>I piGEM-002 & Cu/ Ag promotor</a></legend>
+	<div class="aim">
+	<p>Aim: assembling of Nco/Sac cut piGEM-002 with Cu/ ag promotor sequences</p>
+	</div>
+	<div class="exp-content">
+	<p>In order to test new Cu/ Ag promotor sequences the PCR fragments from 13.73 were used for a Gibson assembly.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Gibson Cu I (&micro;L)</th>
+    <th scope="col">Gibson Ag II (&micro;L)</th>
+    <th scope="col">Control &nbsp;(&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 (209 ng/&micro;L)</th>
+    <td>2,5</td>
+    <td>2,5</td>
+    <td>5</td>
+  </tr>
+  <tr>
+    <th scope="row">Cu promotor</th>
+    <td>2,5</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Ag promotor</th>
+    <td>-</td>
+    <td>2,5</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Gibson mix</th>
+    <td>15</td>
+    <td>15</td>
+    <td>15</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+  </tr>
+</table>
+	<p>The attempts were  incubated at 50&deg;C for an hour and transformed into <em>E. coli</em> XL1-Blue, plated out on LB-Amp.</p>
+</div>
+</fieldset>
+</div>
+<!-- 27.08.2014  -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="27.08.2014">27.08.2014</a>
+</h2>
+<fieldset class="exp22">
+    <legend><a name="exp22.3b">22.3b Repeated Amplification of Killswitch modules I and II</a></legend>
+	<div class="aim">
+	<p>Aim: Increase the amount of modules for further purification, since Fusion PCR did not work 'quick and dirty'</p>
+	</div>
+	<div class="exp-content">
+	<p>This time the Phusion polymerase was used. The template was the already amplified module.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">KS module I</th>
+    <th scope="col">KS module II</th>
+  </tr>
+  <tr>
+    <th scope="row">Template (already amplified modules)</th>
+    <td>0,5</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-38</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-39</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-44</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-45</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">5x HF buffer</th>
+    <td>10</td>
+    <td>10</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>36</td>
+    <td>36</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>0,5</td>
+    <td>0,5</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l)</th>
+    <td>50</td>
+    <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">&nbsp;KS I</th>
+            <th scope="col">KS II</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>5 min</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>30 sec</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>30 sec</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>120 sec</td>
+            <td>200 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>34x</td>
+            <td>34x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td> 5 min</td>
+            <td> 5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+		<br />
+		<p>The gel can be seen under the next topic</p>
+	</div>
+</fieldset>
+<fieldset class="exp..">
+    <legend><a name="exp22.3c">22.3c Repeated amplification of killswitch flanks</a></legend>
+	<div class="aim">
+	<p>Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the amyE and the lacA locus of Bacillus subtilis after cloning into pMAD, yield higher amount of fragments for purification and FusionPCR.</p>
+	</div>
+	<div class="exp-content">
+	<p>The Phusion polymerase was used. </p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">Master Mix</th>
+    <th scope="col"><i>amyE</i> FlankI</th>
+    <th scope="col"><i>amyE</i> FlankII</th>
+    <th scope="col"><i>lacA</i> FlankI</th>
+    <th scope="col"><i>lacA</i> FlankII</th>
+    <th scope="col"><i>amyE</i>-<i>yvyD</i> FlankII</th>
+  </tr>
+  <tr>
+    <th scope="row">Template </th>
+    <td>&nbsp;</td>
+    <td>0,5</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-34</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-35</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-36</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-37</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-40</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-41</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-42</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-43</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-48</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-37</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>11</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">DMSO</th>
+    <td>3,3</td>
+    <td>0,3</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">MgCl2</th>
+    <td>2,2</td>
+    <td>0,2</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">5x HF-buffer</th>
+    <td>110</td>
+    <td>10</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>5,5</td>
+    <td>0,5</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>390,5</td>
+    <td>35,5</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+    <td>&nbsp;</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>47,5</td>
+    <td>47,5</td>
+    <td>47,5</td>
+    <td>47,5</td>
+    <td>47,5</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l)</th>
+    <td>522,5</td>
+    <td>50</td>
+    <td>50</td>
+    <td>50</td>
+    <td>50</td>
+    <td>50</td>
+    </tr>
+</table>
+	<br />
+    <img src="https://static.igem.org/mediawiki/2014/0/07/MR-2014-08-27_22.3.jpg" width="50%" />
+	<br />
+	<p>With exception of KSI and StrepDARP the PCR was negative for all other samples. Even for the positive reactions the bands were very thin.</p>
+	</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.1">18.57 repeated PCR amplification of Hag-<i>Kpn</i>I StrepDARPidin</a></legend>
+	<div class="aim">
+	<p>Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in <i>E. coli</i> BL21(DE3)</p>
+	</div>
+	<div class="exp-content">
+	<p>The synthesized fragments by Integrated DNA technologies arrived as a dried  200 ng DNA pellet. In order to use it as a PCR template the pellet was  centrifuged and resuspended in 20 &micro;L millipore water to get an end concentration  of 10 ng/&micro;L as a stock solution.</p>
+	<p>0,5&nbsp; &micro;L were used as a PCR  template. The PCR fragment should be approx. 900 bp long. </p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content (&micro;l)</th>
+    <th scope="col">2x Attempt I (&micro;L)</th>
+    <th scope="col">2x Attempt II(&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">Template 1: StrepDARPidin (38 ng/ &micro;L)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Template 2: Hag-<i>Kpn</i>I (10 ng/ &micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-51</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-52</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-53</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-54</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>10</td>
+    <td>10</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>35</td>
+    <td>35</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>50</td>
+    <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</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>60 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</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/f/fb/MR-2014-08-27_18.57.jpg" width="30%" />
+		<br />
+		<p>The PCR was clearly positive for StrepDARP, Hag-<i>Kpn</i>I could be seen as a very slight band at 900 bp, but more unspecific bands could be noticed.</p>
+		</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.56b">18.56 Expression-Test with transformed <i>E. coli</i> BL21(DE3)</a></legend>
+	<div class="aim">
+	<p>Aim: Checking the overexpression of pET24d-StrepDARPidin</p>
+	</div>
+	<div class="exp-content">
+	<p>In  order to check the expression of the proteins 5 clones (I6, II3, III3, III4,  III6) containing piGEM-028 (pet24d-StrepDARPidin) were used to inoculate 20 mL  of LB-Can. 5 cultures were incubated at 37&deg;C until an OD of 0,7.</p>
+	<p>A ca. 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 (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 10 &micro;L volume per sample.</p>
+    <img src="https://static.igem.org/mediawiki/2014/e/ea/MR-2014-08-27_18.56.jpg" width="30%" />
+    <p>The gel  did not show any signs of over expression so that lactose was chosen as an alternative inducer to test  the expression. New test expression cultures (I6, II3, III3, III4, III6)&nbsp; were made with 100 mL LB-Can and 5 mL lactose  (6,25 g/ 25 mL) per culture. Incubation was performed over night at 30&deg;C.</p>
+</div>
+</fieldset>
+</div>
+<!-- 28.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="28.08.2014">28.08.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_21">Sequencing of constructs from 28.08.2014</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">Primer</th>
+  </tr>
+  <tr>
+    <th scope="row">1</th>
+    <td>AGB0023467</td>
+    <td>piGEM-028 I6(pET24d-StrepDARPidin)</td>
+    <td>T7 turn</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023468</td>
+    <td>piGEM-028 III3  (pET24d-StrepDARPidin)</td>
+    <td>T7 turn</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023469</td>
+    <td>piGEM-026  (pMAD-D2-Cup)</td>
+    <td>Flo D2_3 fw</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB0023470</td>
+    <td>piGEM-027.1  (pMAD-D2-Strep)</td>
+    <td>Flo D2_3 fw</td>
+  </tr>
+  <tr>
+    <th scope="row">5</th>
+    <td>AGB0023471</td>
+    <td>piGEM-027.2  (pMAD-D2-Strep)</td>
+    <td>Flo D2_3 fw</td>
+  </tr>
+</table>
+	</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.561">18.56 Expression-Test with transformed <i>E. coli</i> BL21(DE3)</a></legend>
+	<div class="aim">
+	<p>Aim: Checking the overexpression of pET24d-StrepDARPidin</p>
+	</div>
+	<div class="exp-content">
+	<img src="https://static.igem.org/mediawiki/2014/d/df/MR_2014-08-27_18.56.jpg" width="50%" />
+	<br />
+	<p>No sign of an overexpression could be noticed.</p>
+	</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.57">18.57 His-bead purification of pellet from <i>E. coli</i> BL21(DE3)</a></legend>
+	<div class="aim">
+	<p>Aim: Checking the overexpression of pET24d-StrepDARPidin</p>
+	</div>
+	<div class="exp-content">
+	<p>The 100 mL culture from I6 &amp;  III3 were centrifuged at 4000 rpm for 10 min in 2x50 mL falcons. The pellets  were washed in 10 mL buffer A and cracked with the microfluidizer. After that  the suspension was centrifuged at 20000 rpm for 20 min/ 4&deg;C. The supernatant  was transferred into a 50 mL falcon.</p>
+	<p>200 &micro;L Ni-NTA beads were added to  the suspension, inverted and incubated on ice for half an hour.</p>
+	<p>After that the suspension was  centrifuged or 5 min at 4000 rpm so that the pellet could be resuspended in 500  &micro;L Buffer A after discarding the supernatant. The 500 &micro;L were transferred into a 1,5 mL tube and centrifuged at 4000 rpm for washing. After discarding the  supernatant the pellet was washed again with 500 &micro;L Buffer A. After a second  centrifugation at 4000 rpm the supernatant was discarded, the pellet  resuspended in 200 &micro;L Buffer A and centrifuged at 13000 rpm to destroy the  Ni-bead &ndash; protein interaction.</p>
+	<p>In the end the supernatant was  transferred into a new 1,5 mL tube. The Ni-beads were mixed with 100 &micro;L 20%  ethanol.</p>
+	<p>40 &micro;L of the supernatant from  clone I6 and III3 were added with 10 &micro;L SDS-PAGE Buffer and analysed on the  SDS-Gel (visible at 18.56).</p>
+</div>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.3d">22.3d Repeated amplification of killswitch flanks</a></legend>
+	<div class="aim">
+	<p>Aim: The flanks have to be amplified for a fusion PCR with the Killswitch modules in order to integrate them into the <i>amyE</i> and the <i>lacA</i> locus of Bacillus subtilis after cloning into pMAD, yield higher amount of fragments for purification and FusionPCR.</p>
+	</div>
+	<div class="exp-content">
+	<table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix [&micro;l]</th>
+    <th scope="col">Master Mix 6x</th>
+    <th scope="col"><i>amyE</i> FlankI</th>
+    <th scope="col"><i>amyE</i> FlankII</th>
+    <th scope="col"><i>lacA</i> FlankI</th>
+    <th scope="col"><i>lacA</i> FlankII</th>
+    <th scope="col"><i>amyE</i>-<i>yvyD</i> FlankII</th>
+  </tr>
+  <tr>
+    <th scope="row">Template (<i>B. subtilis</i>  gDNA)</th>
+    <td>6</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-34</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-35</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-36</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-37</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-40</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-41</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-42</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-43</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-48</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-37</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5-Master mix</th>
+    <td>150</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>132</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>48</td>
+    <td>48</td>
+    <td>48</td>
+    <td>48</td>
+    <td>48</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>288</td>
+    <td>50</td>
+    <td>50</td>
+    <td>50</td>
+    <td>50</td>
+    <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</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>25 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>25 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>60 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>4 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+		<br />
+		<p>The gel is visible under 22.3e</p>
+	</div>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.3e">22.3e Repeated Amplification of Killswitch modules I and II</a></legend>
+	<div class="aim">
+	<p>Aim: Increase the amount of modules for further purification, since Fusion PCR did not work 'quick and dirty'. Q5 was used and gDNA since the previously amplified modules did not work as template yesterday.</p>
+	</div>
+	<div class="exp-content">
+	<table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix [&micro;l]</th>
+    <th scope="col">KS module I</th>
+    <th scope="col">KS module II</th>
+    <th scope="col">KS module III</th>
+  </tr>
+  <tr>
+    <th scope="row">Template (gBlock DNA)</th>
+    <td>0,5</td>
+    <td>0,5</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-38</th>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-39</th>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-44</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-45</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-49</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-50</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5-Master mix</th>
+    <td>25</td>
+    <td>25</td>
+    <td>25</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>2,5</td>
+    <td>2,5</td>
+    <td>2,5</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>50</td>
+    <td>50</td>
+    <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">&nbsp;KS I &amp; III</th>
+            <th scope="col">KS II</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>98</td>
+            <td>5 min</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>25 sec</td>
+            <td>25 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>25 sec</td>
+            <td>25 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>60 sec</td>
+            <td>120 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</td>
+            <td>31x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td> 4 min</td>
+            <td> 4 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/5/5b/MR_2014-08-27_22.3_2.jpg" width="30%" />
+        <p>The expected bands could be seen for the flank I of <i>amyE</i> and flank I and II of <i>lacA</i>. These bands were cut out and purified via a Gel Extraction kit, but the concentration was too low to work further.</p>
+	</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.75">13.75 <i>Nco</i>I/<i>Sac</i>I digest of piGEM-002</a></legend>
+	<div class="aim">
+	<p>Aim: linearizing the plasmid backbone for Gibson Assembly and Ligation with Cu/Ag promotor sequences and IPTG/ Constitutive (/tetO) promotor sequences.</p>
+	</div>
+	<div class="exp-content">
+	<p>The already digested piGEM002 of 26.08. was nearly empty. To have some digested plasmid as backup a new digestion was carried out. piGEM002 was isolated and used for the restriction.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Attempt (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 (476 ng/&micro;L)</th>
+    <td>10</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I</th>
+    <td>0,25</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Sac</i>I</th>
+    <td>0,25</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart Buffer 10x</th>
+    <td>2</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>7,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+  </tr>
+</table>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/8/8f/MR_20140828_piGEM002_NcoI_SacI.jpg" width="20%" />
+	<br />
+	<p>An agarose gel of the previous restriction was done with unrestricted piGEM002 as reference. A band at ca between 6000 and 8000 bp could be seen, which indicates a successful restriction (expected size: ca 6600 bp).</p>
+	</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.76">13.76 Repeated Gibson assembly of <i>Nco</i>I/<i>Sac</i>I piGEM-002 & Cu/ Ag promotor</a></legend>
+	<div class="aim">
+	<p>Aim: assembling of <i>Nco</i>I/<i>Sac</i>I cut piGEM-002 with Cu/Ag promoter sequences</p>
+	</div>
+	<div class="exp-content">
+	<p>In order to test new Cu/ Ag promoter sequences the PCR fragments from 13.73 were used for a Gibson assembly.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Gibson Cu I (&micro;L)</th>
+    <th scope="col">Gibson Ag II (&micro;L)</th>
+    <th scope="col">Control&nbsp; (&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 (ca.  40ng/&micro;L)</th>
+    <td>2,5</td>
+    <td>2,5</td>
+    <td>2,5</td>
+    </tr>
+  <tr>
+    <th scope="row">Cu promotor (ca 4 ng/&micro;l)</th>
+    <td>2,5</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Ag promotor (ca 4 ng/&micro;l)</th>
+    <td>-</td>
+    <td>2,5</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">H2O</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Gibson Mix</th>
+    <td>15</td>
+    <td>15</td>
+    <td>15</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    </tr>
+</table>
+	<p>The attempts were  incubated at 50&deg;C for an hour and used to transform <em>E. coli</em> XL1-Blue, plated out on LB-Amp.</p>
+</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.77">13.77 Repeated ligation of <i>Nco</i>I/<i>Sac</i>I cut piGEM-002 with annealed oligos containing promoter sequences</a></legend>
+	<div class="aim">
+	<p>Aim: insertion of an IPTG inducible/ constitutive and constitutive+tetO into piGEM-002</p>
+	</div>
+	<div class="exp-content">
+	<p>The ligation was prepared to contain ca 100 ng of the restricted piGEM002 with an appropriate amount of insert (much fewer).</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Ligation I (&micro;L)</th>
+    <th scope="col">Ligation II (&micro;L)</th>
+    <th scope="col">Ligation III (&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-002 (ca 40  ng/&micro;L)</th>
+    <td>2,5</td>
+    <td>2,5</td>
+    <td>2,5</td>
+    </tr>
+  <tr>
+    <th scope="row">iGEM-59/60 (ca 4 ng/&micro;l)</th>
+    <td>0,8</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">iGEM-61/62 (ca 4 ng/&micro;l)</th>
+    <td>-</td>
+    <td>0,8</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">iGEM-63/64 (ca 4 ng/&micro;l)</th>
+    <td>-</td>
+    <td>-</td>
+    <td>0,8</td>
+    </tr>
+  <tr>
+    <th scope="row">Polynucleotide kinase</th>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">T4 Ligase</th>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Ligation Buffer 10x</th>
+    <td>2</td>
+    <td>2</td>
+    <td>2</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>12,7</td>
+    <td>12,7</td>
+    <td>12,7</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    </tr>
+</table>
+	<p>The reactions were  incubated at room temperature for an hour and transformed into <em>E. coli</em> XL1-Blue, plated out on LB-Amp.</p>
+</div>
+</fieldset>
+</div>
+<!-- 29.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="29.08.2014">29.08.2014</a>
+</h2>
+<fieldset class="exp">
+    <legend><a name="exp_22">Sequencing results of constructs from 28.08.2014</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">Results</th>
+	    </tr>
+	  <tr>
+	    <th scope="row">1</th>
+	    <td>AGB0023467</td>
+	    <td>piGEM-028  I6(pET24d-StrepDARPidin)</td>
+	    <td>No results</td>
+	    </tr>
+	  <tr>
+	    <th scope="row">2</th>
+	    <td>AGB0023468</td>
+	    <td>piGEM-028 III3  (pET24d-StrepDARPidin)</td>
+	    <td>No alignment</td>
+	    </tr>
+	  <tr>
+	    <th scope="row">3</th>
+	    <td>AGB0023469</td>
+	    <td>piGEM-026  (pMAD-D2-Cup)</td>
+	    <td>Good</td>
+	    </tr>
+	  <tr>
+	    <th scope="row">4</th>
+	    <td>AGB0023470</td>
+	    <td>piGEM-027.1  (pMAD-D2-Strep)</td>
+	    <td>Point mutation</td>
+	    </tr>
+	  <tr>
+	    <th scope="row">5</th>
+	    <td>AGB0023471</td>
+	    <td>piGEM-027.2  (pMAD-D2-Strep)</td>
+	    <td>Point mutation</td>
+	    </tr>
+	  </table>
+	<p>The sequencing showed that pET24d did not contain any known insert.</p>
+	<p>In  order to check the correct synthesis of the templates purified PCR products  were sent for sequencing again. In the meanwhile the constructs were cloned again.</p>
+</div>
+</fieldset>
+<fieldset class="exp">
+    <legend><a name="exp_23">Sequencing of constructs from 29.08.2014</a></legend>
+	<div class="aim">
+	<p></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>AGB0023512</td>
+    <td>PCR Fragment  StrepDARPidin I</td>
+    <td>piGEM-032 fw</td>
+  </tr>
+  <tr>
+    <th scope="row">2</th>
+    <td>AGB0023513</td>
+    <td>PCR Fragment  StrepDARPidin II</td>
+    <td>piGEM-032 fw</td>
+  </tr>
+  <tr>
+    <th scope="row">3</th>
+    <td>AGB0023514</td>
+    <td>PCR Fragment D2-StrepII </td>
+    <td>Flo96 D2_3 fw</td>
+  </tr>
+  <tr>
+    <th scope="row">4</th>
+    <td>AGB0023515</td>
+    <td>PCR Fragment  StrepDARPidin I</td>
+    <td>piGEM-033 rv</td>
+  </tr>
+  <tr>
+    <th scope="row">5</th>
+    <td>AGB0023516</td>
+    <td>PCR Fragment  StrepDARPidin II</td>
+    <td>piGEM-033 rv</td>
+  </tr>
+  <tr>
+    <th scope="row">6</th>
+    <td>AGB0023517</td>
+    <td>PCR Fragment D2-StrepII</td>
+    <td>Flo97 D2_3 rv</td>
+  </tr>
+</table>
+	</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.58">18.58 New PCR amplification of StrepDARPidin and D2-Cup</a></legend>
+	<div class="aim">
+	<p>Aim: amplification of the StrepDARPidin for cloning into the expression vector pET24d for protein purification and overproduction in <i>E. coli</i> BL21(DE3) & amplification of D2-StrepII for Gibson Assembly into piGEM-005</p>
+	</div>
+	<div class="exp-content">
+	<p>The synthesized fragments were supposed to be sequenced in order to  check the right synthesis.&nbsp; 10 ng/&micro;L as a  Stock solution and a purified PCR Product were used as template for each  reaction.</p>
+	<p>0,5&nbsp; &micro;L were used as a PCR  template. The PCR fragment should be ca. 900 bp long for StrepDARPidin and ca.  450 bp for D2-StrepII.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix (&micro;l)</th>
+    <th scope="col">PCR StrepDARPidin I</th>
+    <th scope="col">PCR StrepDARPidin II</th>
+    <th scope="col">PCR StrepDARPidin III</th>
+    <th scope="col">PCR D2-Strep I</th>
+    <th scope="col">PCR D2-Strep II</th>
+  </tr>
+  <tr>
+    <th scope="row">Pur. StrepDARP. (1:10- 10 ng/&micro;L)</th>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">StrepDARP. Stock (20 ng/&micro;L)</th>
+    <td>-</td>
+    <td>0,25</td>
+    <td>0,25</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">D2-StrepII Stock 20 ng/&micro;L)</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>0,25</td>
+    <td>0,25</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-32</th>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM-33</th>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Flo96 D2_3 fw</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Flo97 D2_3 rv</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Q5-Master mix</th>
+    <td>25</td>
+    <td>25</td>
+    <td>25</td>
+    <td>25</td>
+    <td>25</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>22</td>
+    <td>22</td>
+    <td>22</td>
+    <td>22</td>
+    <td>22</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l)</th>
+    <td>50</td>
+    <td>50</td>
+    <td>50</td>
+    <td>50</td>
+    <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</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>60sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>31x</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/b/bf/MR_2014-08-28_18.58.jpg" width="30%" />
+      <p>The PCR samples were purified with the Omega Gel Extraction Kit according to the enzymatic reaction purification protocol.</p>
+</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.59">18.59 Digestion of amplified of StrepDARPidin for cloning into pET16b</a></legend>
+	<div class="aim">
+	<p>Aim: digest with <i>Nco</i>I and <i>Xho</i>I of the StrepDARPidin PCR product for cloning into the expression vector pET16b & digest of pET16b</p>
+	</div>
+	<div class="exp-content">
+	<p>The PCR products from 15.58 were cut with <i>Nco</i>I and <i>Xho</i>I to get the restriction sites for cloning into the <i>Nco</i>I/<i>Xho</i>I  digested pET16b. This vector was used because of its Amp resistance for fast transformation.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">PCR StrepDARPidin I (&micro;L)</th>
+    <th scope="col">PCR StrepDARPidin II(&micro;L)</th>
+    <th scope="col">PCR StrepDARPidin III (&micro;L)</th>
+    <th scope="col">Pet16b(&micro;L) </th>
+    <th scope="col">Mastermix</th>
+  </tr>
+  <tr>
+    <th scope="row">StrepDARP. I ( 45 ng/&micro;L)</th>
+    <td>10</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">StrepDARP. II (70 ng/&micro;L)</th>
+    <td>-</td>
+    <td>15</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">StrepDARP. III (45 ng/&micro;L)</th>
+    <td>-</td>
+    <td>-</td>
+    <td>10</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">pET16b ( 58 ng/&micro;L)</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>17</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">CutSmart 10x</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>8</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>Xho</i>I</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>17</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>10</td>
+    <td>5</td>
+    <td>10</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    <td>28</td>
+  </tr>
+</table>
+	<p>Incubation for 1h at  37&deg;C with heat shock at 85&deg;C for 10 min.</p>
+</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.60">18.60 Ligation of digested StrepDARPidin for cloning into pET16b</a></legend>
+	<div class="aim">
+	<p>Aim: ligation of StrepDARPidin PCR product with the expression vector pET16b</p>
+	</div>
+	<div class="exp-content">
+	<p>The PCR product was ligated into the <i>Nco</i>I/<i>Xho</i>I digested pET16b. Two attempts were made for a transformation into E. Coli  XL1-Blue and BL21. </p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Ligation I (&micro;L)</th>
+    <th scope="col">Ligation II (&micro;L)</th>
+    <th scope="col">Ligation III (&micro;L)</th>
+    <th scope="col">Control</th>
+  </tr>
+  <tr>
+    <th scope="row"><i>Nco</i>I/<i>Xho</i>I StrepDARPidin  (ca. 40&nbsp; ng/ &micro;L) I/II/III</th>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">pET16b <i>Nco</i>I/<i>Xho</i>I (45 ng/&micro;L)</th>
+    <td>5</td>
+    <td>5</td>
+    <td>5</td>
+    <td>5</td>
+    </tr>
+  <tr>
+    <th scope="row">Ligation Buffer 10x</th>
+    <td>2</td>
+    <td>2</td>
+    <td>2</td>
+    <td>2</td>
+    </tr>
+  <tr>
+    <th scope="row">Ligase</th>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>11</td>
+    <td>11</td>
+    <td>11</td>
+    <td>11</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    <td>20</td>
+    </tr>
+</table>
+	<p>Incubation for 1,5 hours at  rom temperature with heat shock at 85&deg;C for 10 min. The 20 &micro;L from ligation I  were used to transform <i>E. coli</i> XLI-Blue, which was plated out on LB-Amp. Incubation was  done overnight at 37&deg;C.</p>
+</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.61">18.61 Gibson assembly of piGEM-005 & D2-StrepII</a></legend>
+	<div class="aim">
+	<p>Aim: Insertion of Hag-D2-Strep into piGEM-005</p>
+	</div>
+	<div class="exp-content">
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Component</th>
+    <th scope="col">Hag-D2-Strep I(&micro;L)</th>
+    <th scope="col">Hag-D2-Strep II (&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">Gibson-mix</th>
+    <td>15</td>
+    <td>15</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert I<br />
+      (Hag-D2-Strep I 60  ng/&micro;L)</th>
+    <td>2,5</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Insert II<br />
+      (Hag-D2-Strep II 70  ng/&micro;L)</th>
+    <td>-</td>
+    <td>2,5</td>
+  </tr>
+  <tr>
+    <th scope="row">piGEM-005 SpeI&nbsp;(50 ng/ &micro;L)</th>
+    <td>2,5</td>
+    <td>2,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>20</td>
+    <td>20</td>
+  </tr>
+</table>
+	<p>Incubation was done&nbsp; at 50&deg;C for  1 hour with following transformation into <em>E. coli</em>  XL1-Blue, plated out on LB-Amp plates and incubated overnight at 37&deg;C.</p>
+</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.78">13.78 colony PCR of colonies of Gibson and ligation (13.76 and 13.77)</a></legend>
+	<div class="aim">
+	<p>Aim: check for correct ligation/Gibson assembly of the oligos with the piGEM002 plasmid</p>
+	</div>
+	<div class="exp-content">
+	<p>Several clones were picked and transferred onto another plate, the same colony was used to inoculate a PCR reaction to check the insertion of the oligos.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix [&micro;l]</th>
+    <th scope="col">Master mix for 45 reactions  (&micro;l)</th>
+    <th scope="col">Const. promoter</th>
+    <th scope="col">Cont. + tetO</th>
+    <th scope="col">Mod. Lac promoter</th>
+    <th scope="col">Ag promoter new</th>
+    <th scope="col">Cu promoter new</th>
+  </tr>
+  <tr>
+    <th scope="row">Template</th>
+    <td>-</td>
+    <td>Small part</td>
+    <td>Small part</td>
+    <td>Small part</td>
+    <td>Small part</td>
+    <td>Small part</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM006</th>
+    <td>45</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM061</th>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM063</th>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM059</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM057</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM055</th>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">5x HF buffer</th>
+    <td>180</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA polymerase 1:10</th>
+    <td>45</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>585</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Master Mix</th>
+    <td>-</td>
+    <td>19</td>
+    <td>19</td>
+    <td>19</td>
+    <td>19</td>
+    <td>19</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>855</td>
+    <td>20</td>
+    <td>20</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>95</td>
+            <td>10 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>60 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>34x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>5 min</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/b/b6/MR_2014-08-27_13.78_1.jpg" width="40%" />
+        <p>The positive clones (band at ca 1000 bp) were  transferred into LB-amp medium and were incubated at 37&deg;C over night for a  miniprep. A new colony PCR was carried out with six colonies of the XLIBlue piGEM002+Ag-promoter.</p>
+        <img src="https://static.igem.org/mediawiki/2014/8/83/MR_2014-08-27_13.78_2.jpg" width="30%" />
+</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp22.4">22.4 Repeated amplification of KS module II</a></legend>
+	<div class="aim">
+	<p>Aim: amplify module II for further purification and fusion PCR with flanks of <i>lacA</i></p>
+	</div>
+	<div class="exp-content">
+	<p>The PCR for the KSII was repeated  with the Phusion DNA-polymerase. A gradient was carried out from 52&deg;C to 62&deg;C.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Master mix</th>
+    <th scope="col">KSII (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">Template: (gBlock KSII)</th>
+    <td>4,5</td>
+    <td>0,5</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM044</th>
+    <td>9</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer iGEM045</th>
+    <td>9</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">HF Buffer 5x</th>
+    <td>90</td>
+    <td>10</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>9</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>9</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>319,5</td>
+    <td>35</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l)</th>
+    <td>450</td>
+    <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</th>
+            <th scope="col">KS II</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>95</td>
+            <td>5 min</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>95</td>
+            <td>30 sec</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>52-62</td>
+            <td>30 sec</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>1 min 45 sec</td>
+            <td>120 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>30x</td>
+            <td>33x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>4min</td>
+            <td>4 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/e/e4/MR_2014-08-2_22.4_1.jpg" width="30%" />
+        <p>The gel showed many unspecific bands of fewer basepairs than expected. The expected band of ca 2000 bp was very weak but got stronger the higher the annealing temperature was. </p>
+</div>
+</fieldset>
+<fieldset class="exp22">
+    <legend><a name="exp22.4">22.4 repeated fusion-PCR of the Killswitch modules with the flanks</a></legend>
+	<div class="aim">
+	<p>Aim: fuse the modules with the flanks to integrate them into the <i>B. subtilis</i> genome</p>
+	</div>
+	<div class="exp-content">
+	<p>For this fusion-PCR the flanks of  22.08.14 and the amplified and purified modules I and III from 28.08.14 were  used. The module II from 24.08.14 was separated on an agarose gel, cut out and  purified as well.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">KS I (&micro;l)</th>
+    <th scope="col">KS II (&micro;l)</th>
+  </tr>
+  <tr>
+    <th scope="row">Template KSI (ca 5 ng/&micro;l)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Template KSII (7 ng/&micro;l)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i> flank I (1.5 ng/&micro;l)</th>
+    <td>3</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>amyE</i>  flank II (ca. 6.5 ng/&micro;l)</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i> flank I (ca. 5.5 ng/&micro;l)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row"><i>lacA</i> flank II (7 ng/&micro;l)</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer piGEM034</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer piGEM037</th>
+    <td>1</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer piGEM040</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer pIGEM043</th>
+    <td>-</td>
+    <td>1</td>
+  </tr>
+  <tr>
+    <th scope="row">HF Buffer 5x</th>
+    <td>10</td>
+    <td>10</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>31</td>
+    <td>31</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>50</td>
+    <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) KSI</th>
+            <th scope="col">Time (min/sec) KSII</th>
+          </tr>
+          <tr>
+            <th scope="row">1</th>
+            <td>95</td>
+            <td>5 min</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>95</td>
+            <td>30 sec</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>30 sec</td>
+            <td>30 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>2 min</td>
+            <td>3 min 10 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>34x</td>
+            <td>34x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>5 min</td>
+            <td>5 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/a/a5/MR_2014-08-29_22.4_b.jpg" width="20%" />
+        <p>The fusion PCR was negative, expected bands would be around 1881 bp for KSI and around 3000 for KSII.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 30.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="30.08.2014">30.08.2014</a>
+</h2>
+<fieldset class="exp18">
+    <legend><a name="exp18.62">18.62 Gibson assembly of piGEM-005 & D2-StrepII/ Ligation StrepDARPidin-pET16b</a></legend>
+	<div class="exp-content">
+	<p>The transformation plates were taken out of the incubator and put in the fridge at 4&deg;C.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 31.08.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="31.08.2014">31.08.2014</a>
+</h2>
+<fieldset class="exp18">
+    <legend><a name="exp18.63">18.63 cPCR with clones from Ligation pET16b & StrepDARPidin</a></legend>
+	<div class="aim">
+	<p>Aim: checking the success of the ligation </p>
+	</div>
+	<div class="exp-content">
+	<p>The grown clones from the ligation from 18.60 were checked via cPCR with  primers iGEM-032 and -033. 40 clones from Ligation plates I-III were picked for  cPCR and transferred on a plate before.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Master mix&nbsp; for  40 clones (&micro;L)</th>
+    <th scope="col">Clones from Ligation I (&micro;L)</th>
+    <th scope="col">Clones from Ligation II(&micro;L)</th>
+    <th scope="col">Clones from Ligation III(&micro;L)</th>
+  </tr>
+  <tr>
+    <th scope="row">Template (colony)</th>
+    <td>-</td>
+    <td>1</td>
+    <td>1</td>
+    <td>1</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-32</th>
+    <td>20</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Primer iGEM-33</th>
+    <td>20</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion Buffer 5x</th>
+    <td>160</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>20</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>20</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>560</td>
+    <td>-</td>
+    <td>-</td>
+    <td>-</td>
+    </tr>
+  <tr>
+    <th scope="row">Master mix</th>
+    <td>-</td>
+    <td>19</td>
+    <td>19</td>
+    <td>19</td>
+    </tr>
+  <tr>
+    <th scope="row">Total Volume</th>
+    <td>800</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>10 min</td>
+          </tr>
+          <tr>
+            <th scope="row">2</th>
+            <td>98</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">3</th>
+            <td>55</td>
+            <td>20 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">4</th>
+            <td>72</td>
+            <td>60  sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>33x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>4 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+          </tr>
+      </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/9/92/MR_2014-08-31_18.63.jpg" width="30%" />
+        <p>The positive clones show a band at ca. 900 bp  and were used for inoculation minipreps so that a test digest could confirm the  insertion of StrepDARPidin.</p>
+</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.64">18.64 cPCR with XLI-Blue clones containing piGEM005 Hag-D2-Strep or Hag-D2-Cup</a></legend>
+	<div class="aim">
+	<p>Aim: Checking insertion of Hag-D2-Cup and Hag-D2-Strep into piGEM-005</p>
+	</div>
+	<div class="exp-content">
+	<p>In order to check the successful integration of D2-Cup and D2-Strep clones were picked for cPCR and transferred on a LB-Amp master plate. As primers Flo96 (D23-fw) and Flo97 (D23-v) were used. 12 clones were screened.</p>
+    <table width="100%" border="1">
+  <tr>
+    <th scope="col">Mix</th>
+    <th scope="col">Master-Mix (14x) </th>
+    <th scope="col">D2-Strep</th>
+  </tr>
+  <tr>
+    <th scope="row">Template</th>
+    <td>-</td>
+    <td>Colony</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer Flo96 D23-fw</th>
+    <td>7</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Primer Flo96 D23-rv</th>
+    <td>7</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">dNTPs</th>
+    <td>7</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">5x Phusion Buffer</th>
+    <td>56</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Phusion DNA-polymerase</th>
+    <td>7</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Water</th>
+    <td>196</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">Mastermix</th>
+    <td>-</td>
+    <td>20</td>
+  </tr>
+  <tr>
+    <th scope="row">Total Volume (&micro;l)</th>
+    <td>280</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>10 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>40 sec</td>
+          </tr>
+          <tr>
+            <th scope="row">5</th>
+            <td>Go To 2</td>
+            <td>33x</td>
+          </tr>
+          <tr>
+            <th scope="row">6</th>
+            <td>72</td>
+            <td>4 min</td>
+          </tr>
+          <tr>
+            <th scope="row">7</th>
+            <td>8</td>
+            <td>infinite</td>
+          </tr>
+        </table>
+        <br />
+        <img src="https://static.igem.org/mediawiki/2014/6/68/MR_2014-08-31_18.64.jpg" width="30%" />
+        <p>The clones I1, I3, I6, II2 and II4 were picked for plasmid isolation and sent for sequencing the next day.</p>
+	</div>
+</fieldset>
+</div>
 </html>
+{{Team:Marburg/Template:ToTop}}
+{{Team:Marburg/Template:End}}

Team:Marburg:Project:Notebook:August

From 2014.igem.org

Latest revision as of 15:45, 17 October 2014

Notebook: August