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

Experiments from Florian Altegoer have shown that mutants with D23-domain from Salmonella integrated into Hag are expressing the modified flagellin and are motile. So the Strep-Tag was designed into Hag with D2 as a linker domain with additional GSGS linkers.

Additionally the DARPin was designed with an N-terminal His-Tag and C-Terminal Streptavidin.

The designed structures were synthesized by Intefrated DNA Technologies.

Based on the experiments from Florian Altegoer we decided to design Hag-D2-Cup.

The designed structures were synthesized by Intefrated DNA Technologies

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).

1 LB was inoculated with WT 3610 and incubated overnight at 37°C.

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

End concentration: 50 ng/ µL

Incubation at 37°C for 2h .

The fragments were purified via gel extraction.

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

The preculture was centrifuged at 4000 rpm and the pellet was resuspended in 33 mL Tris buffer (pH 8.0) with 0,5% Brij-58. A stock solution of lysozyme with10 mg/mL was made. 330 µL were added to the Tris buffer/ Buffer mix. The lysis was performed at 4°C with a rotator until the pellet was completely solved which ca. 2-3h. DNase (5 µg/ mL) in presence of 0,01M magnesium chloride were added and incubated for half an hour at 4°C in the rotator as well.

After that the suspension was centrifuged at 10000xg 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 taken as well. For fractioning 2 g of ammonium sulfate (20%) were added 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 gel.

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

The flagella purification was successful. Besides the flagellin the hook proteins are also in the samples but the smaller proteins were removed from the purified pellet.

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

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.

A culture flask of Caco-2-cells were received from Dr. Hartmann Reifert (BMFZ). In order to let them grow the cells had to be splitted due to a specific protocol. Caco-2-cells require DMEM (Dulbecco’s Modified Eagle Medium) with 20% FCS and L-Glutamine.

A centrifuge was set on 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 come 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 spinned down 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

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

0,5  µL were used as a PCR template.

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

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.

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 in used for inoculation of 10 mL LB-Amp. Incubation was done overnight at 37°C.

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

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

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

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

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

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

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

The plasmids were transformed into Bacillus subtilis and incubated at 30°C.

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

As the cultures were not that blue (thin or old plate) wildtype cultures were inoculated for a new transformation tomorrow. Therefore the plasmids piGEM-026 and -027 were prepped from frozen pellets.

For the experimental procedure see the Materials section below. Anyway, two transformations are running currently that will be refered to as ‘old’ and ‘new’ transformation from now on.

For the old transformation the first temperature shift was performed in LB-MLS medium. Culture was plated in 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 4-5h. Strangly the culture reached this OD after almost 7h today. Plasmids (1.5µg) piGEM026 and -027 were transformed and the protocol was followed as described below.

New trafo, no colonies so far, further incubation.

Old trafo 2nd temperature shift of hopefully positive colonies (only light blue).

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

The blue/ white screening showed positive transformed blue clones from the new transformation.  3 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 overnight at 30°C with the cultures.

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:

AmyE Flank 1 (Primer 46/34 and 47/35)
AmyE Flank2 (Primer 48/36 and 49/37)     
LacA Flank 1 (Primer 52/40 and 53/41)
LacA Flank 2 (Primer 54/42 and 55/43)
KSIII AmyE Flank2 (Primer 60/48  and 49/ 37)

Gel photo: all bands like expected (500 /1000 bp)

The synthesized fragments by Integrated DNA technologies came as a dried 200ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged down and resuspended in 20 µL Millipore 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.

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

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

Incubation over night at room temperature.

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 clones were proven for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C overnight.

Gel analysis:

no bands

The synthesized fragments by Integrated DNA technologies came as a dried 200ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged down and resuspended in 20 µL Millipore 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.

Both attempts had a concentration of 380 ng/ µL.

The PCR product was cut with NcoI and XhoI to get the restriction sites for cloning into pet24d Nco/Xho cut.

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

The PCR product was ligated into cut pet24d Nco/Xho. Two attempts were made for a transformation into E. Coli XL1-Blue and BL21.

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

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 attempts).

Gel analysis:
no bands

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

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

The clones I6, II3, III3 were transformed into E.Coli BL21(DE3) and selected on LB-Can plates. III4 and III6 were already on a master plate. The clones were inoculated for miniprep and sent for sequencing.

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.

Gel analysis: no bands

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

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

In order to test our nose system with new promotor 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 Nco/Sac sites.

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

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

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

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

The synthesized fragments by Integrated DNA technologies came as a dried 200ng DNA pellet. In order to use it as a PCR template the pellet was centrifuged down and resuspended in 20 µL Millipore 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.

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 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 overnight at 30°C.

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 ransferred 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 a1,5 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.

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.

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

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).

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

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

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

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

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.

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

The PCR products from 15.58 were cut with NcoI and XhoI to get the restriction sites for cloning into pet16b Nco/Xho cut. Pet16b was used because of his Amp resistance for fast transformation. It was given us from Florian Altegoer.

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

The PCR product was ligated into cut pet16b Nco/Xho. Two attempts were made for a transformation into E. Coli XL1-Blue and BL21.

Incubation for 1,5h at room temperature with heat shock at 85°C for 10 min. The 20 µL from ligation I were transformed into E.Coli XLI-Blue and plated out on LB-Amp. Incubation was done overnight at 37°C.

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

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.

The positive clones (band at ca 1000 bp) were transferred into LBamp 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.

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

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.

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.

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

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

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 Master Plate before.

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.

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.

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