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

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

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

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

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

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

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

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

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

Plasmid from clone 4 was taken, transformed into E.Coli DH5aand named piGEM-021.

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

A 1 mL preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL). After that the cultures were induced with 20 µL IPTG for 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 5 and 10 µL volume per sample.

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

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

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

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

The cultures were incubated overnight at 30°C.

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

The gel shows a high concentration of a specific protein.

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

piGEM-021 and pFlaiS were transformed into E.Coli BL21(DE3) and plated out on LB-Canamycin plates. The plate was incubated at 37°C overnight.

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

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

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

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

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

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

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

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

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

The cultures were incubated overnight at 30°C.

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

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

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

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

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

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

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

After the heat shock 100 µL dilutions from 10-4 to 10-6 of each culture were plated out on MLS-X-Gal so that 9 plates could be incubated overnight at 42°C.

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

The following steps were performed:

• equilibration with Buffer A 10 min
• taking 40 µL supernatant (load)+ 10 µL SDS-Buffer - L-Sample
• 50 mL load on column
• taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample
• first washing with 25ml Buffer A (half the load)
• taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample
• equilibrate with Buffer B (output pipe off the column into glas, input into B)
• hanging pipe on column, 6x2ml Evolutions - E 1-6
• taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-sample 1-6

regeneration of column:

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

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

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

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

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

The GeFi was started according to the following settings:

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

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

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

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

Core I and II were used for setting drops.

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

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

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

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

The following steps were performed:

• equilibration with Buffer A 10 min
• taking 40 µL supernatant (load)+ 10 µL SDS-Buffer - L-Sample
• 50 mL load on column
• taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample
• first washing with 25ml Buffer A (half the load)
• taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample
• equilibrate with Buffer B (output pipe off the column into glas, input into B)
• hanging pipe on column, 6x2ml Evolutions - E 1-6
• taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-sample 1-6

regeneration of column:

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

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

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

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

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

The GeFi was started according to the following settings:

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

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

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

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

Core I - IV were used for setting drops.

From the dilution plates was one WHITE clone picked and transferred on a Master X-Gal Plate as well as on a MLS plate so that 9clones were proven for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C overnight.

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

The cut vector was isolated via gel extraction.

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

The mix was incubated 1h at 50 °C , transformed into E. Coli XL1-Blue  and plated out on LB-Amp plates. Incubation overnight at 37°C.

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

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

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

Gibson reactions were transformed into E.Coli XL1-Blue after 1h incubation at 50°C and plated out on LB-Amp. Incubation was proceeded at 37°C overnight.

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

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

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

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

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

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

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

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

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

The cultures were incubated overnight at 30°C.

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

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

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

A new strategy has to be planned for further work.

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

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

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

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

WT3610 and WT-D23-mutant were used to inoculate 50 mL LB-medium for overnight culture incubated at 37°C.

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

The following steps were performed:

• equilibration with Buffer A 10 min
• taking 40 µL supernatant (load)+ 10 µL SDS-Buffer - L-Sample
• 50 mL load on column
• taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample
• first washing with 25ml Buffer A (half the load)
• taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample
• equilibrate with Buffer B (output pipe off the column into glas, input into B)
• hanging pipe on column, 6x2ml Evolutions - E 1-6
• taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-sample 1-6

regeneration of column:

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

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

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

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

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

The GeFi was started according to the following settings:

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

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

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

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

Core I - IV were used for setting drops.

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

Incubation at 37°C for 2h.

Meanwhile the DTs were generated by a PCR:

The DTs were integrated into the vector by gibson assembly:

The attempts were incubated at 50°C for an hour and transformed into E.Coli XLI-blue and plated out on LB-Amp. Incubation overnight.

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