Standard E. coli transformation protocol was followed and cells were plated on LB-CM plates.

The isolated plasmids from the positive clones were digested with NcoI/ XhoI in order to see if the 900 bp were flipping out of the pET16b backbone.

Incubation at 37°C for 40 min.

The gel showed that all picked clones were positive. Clone 17 was sent was sequencing and transformed into E. coli BL21(DE3).

In order to check the expression of the StrepDARPidin a colony containing piGEM-028 (pET16b-StrepDARPidin) was used to inoculate 20 mL of LB-Amp. The culture incubated at 37°C until an OD of 0,7.

A approx. 1 mL preinduction probe (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL). After that the cultures were induced with 20 µL IPTG for 3h.

An induction probe (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 probes were analysed on an SDS-PAGE gel with 10 µL volume per probe.

The gel showed a positive overexpression of a protein at approx. 30 kDa which fits the calculated molecular mass.

The amplification of KSII did not work: some unspecific bands were visible but not of the expected size. The amplification of flank 1 amyE fw, flank 3 lacA fw and flank 4 lacA rev was successful but yielded in a low amount of PCR product.

The plasmids of 13.78 were isolated at the weekend. Frozen and competent B. subtilis cells were thawed without ice. 15 µl of each plasmid were given to the cells that were incubated at 37°C for 0.5 hours. After regeneration the cells were plated on LB-Cm plates (5 g/µl) and were incubated at 30°C for several days.

Test digest with the isolated plasmid was performed with the corresponding enzymes (EcoRI and PstI) over night. Besides 600 ng of the already existing PCR fragments Hag-KpnI and StrepDARPidin were digested with the same enzymes.

Concentrations:

Hag-KpnI 130 ng/µl

Strep-DARPidin 55 ng/µl after purification

psB1C3 130 ng/µl after plasmid isolation

Digest:(20µl reaction mix)

The bands were at a height of approx. 1500 bp which is too small for the expected fragment of 1847 bp.

The reactions were incubated at 50°C for an hour and used to transform E. Coli XL1-Blue, plated out on LB-Amp. An additional reaction was started without pMAD in a lower (1 µl) and a higher (2 µl) amount of amyE flank I. These reactions were used to do a PCR reaction with the primers piGEM034 and piGEM037 to test if the assembled fragment is present in the reaction. The expected bands should have a size of 1847 bp but the fragment visible is only approx.1300 bp big. It seems like the flank I is missing but then the fragment should not have been amplified, since the primers were chosen to include both flanks.

Information: iBB8 has a size of 1461 bp, vector 2000bp, Inserts Hag-KpnI and StrepDARPidin 1000 bp

Image of the gelextraction, as a marker the 1kb gene ruler was used, 1 = StrepDARPidin, 2= Hag-KpnI, 3 and 4 = pSB1C3

Concentrations:

pSB1C3= 18 ng/µl

StrepDARPidin= 5 ng/µl

Hag-KpnI= 13 ng/µl

Ligation at RT for 90 minutes

After the ligation the whole reaction mix was used to transform chemically competent E. coli XL-1-blue cells (standard protocol). Cells were plated on LB-Cm plates and incubated at 37°C over night.

Since the Gibson assembly of the single parts without the vector (22.7) and the Fusion PCR yielded no positive results, the KSI should be amplified once again to assure usage of the correct and pure fragments for the FusionPCR or Gibson assembly.

The gel showed the expected band for the killswitch module 1 but only one band for flank 1 in the reaction with the annealing temperature of 58°C. The remaining reactions for KSI were pooled and purified with a Gel Ex kit. Since the only band for the flank 1 is visible at 58°C and not above another PCR was prepared with lower annealing temperatures but the same reaction mix as above.

All plasmids (from positive clones 13.78) were again used to transform B. subtilis PY79, since the former plates seemed to be contaminated by other organisms. E. coli DH5µ was transformed with the rest of the plasmids to multiply it for later purposes.

Three colonies were grown on the plate with the Gibson assembly transformed XLI-Blue cells. The control also contained three colonies. However, the colonies of the Gibson plate were tested by colony PCR with the primer surrounding the flanks of the connected KSI module, since no primer were available which target regions in the vector pMAD.

The Gibson assembly of KSI with flanks I and II did not work, but the flank I was amplified well.

Colonies could be detected on both transformation plates, whereas no colonies grew in the ligation control plate. Therefore a colony PCR will be performed in order to analyze the transformants for the insertion of the fragments into the plasmid. 5 clones from both plates were chosen for the colony PCR and inoculated in liquid LB-Cm for plasmid isolation in the evening (in case positive clones can be detected in the colony PCR).

Saved colony PCR program in the PCR cycler was used with 1 minute elongation time.

Colony PCR negative, therefore plasmids were isolated and digested with the EcoRI and PstI.

Restriction: 4 µl plasmid in a total 10 µl restriction mix

Restriction positive (2kb plasmid and 1kb insert), one clone each will be send for sequencing.

3 L LB medium were prepared with 1 mL ampicillin each and inoculated with some clones from the B21 transformation plate. The cultures were induced with 50 ml lactose solution (12,5g/ 50 mL) each and incubated at 30°C over night.

In order to check the expression of the StrepDARPidin a colony containing piGEM-028 (pET16b-StrepDARPidin) was used to inoculate 20 mL of LB-Amp.

The fragments were all amplified and purified. This time a gradient was performed with an initial step of 50°C for 0.5 hours (similar to a Gibson assembly)

The fusion-PCR worked and the bands had the expected size of approx.1800 bp.

iGEM Primer in the list of last year's team number 37 and 38 (Sample premix)

AGB0023-642= StrepDARPidinFw (Clone 1)

AGB0023-643=StrepDARPidinRv

AGB0023-644= Hag-KpnI Fw (Clone 4)

AGB0023-645= Hag-KpnI Rv

The 3 x 1 liter LB culture which were induced overnight with lactose were harvested and the pellets resuspended in 15 mL Buffer A. The cell suspension was cracked with the micro fluidizer. The lysate was centrifuged at 20000 rpm for 20 min and purified with a Ni-NTA Column.

The following steps were performed for the Ni-NTA Column:

• equilibration with Buffer A 10 min
• taking 40µL supernatant (load)+ 10 µL SDS-Buffer - L-Probe
• 50 mL load on column
• taking 40 µL of flow through + 10 µL SDS-buffer - FT-Probe
• first washing with 25 ml Buffer A (half the load)
• taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Probe
• equilibrate with Buffer B (output pipe off the column into glas, input into B)
• hanging pipe on column, 20 ml Evolution- E
• taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-probe
• SDS-PAGE analysis

regeneration of column:

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

Meanwhile the elution was transferred in a concentrator column which was centrifuged at 4000 rpm until the volume in the filter was 1.5 mL for injection into the gel filtration station.

The gel showed that there was not the desired StrepDARPidin loaded on the column. A SDS-PAGE probe from the pellet showed us that the pellet contained the protein.

We considered that the StrepDARPidin might be in inclusion bodies because of the Streptavidin part and the codon optimization. So we tried expression overnight with 0.1 mM IPTG of an 100 mL culture at OD 0,7. We induced as well with lactose overnight as a control.

The 4 cultures were incubated at 20°C and 30°C in the shaker overnight.

piGEM-026 and -027 were used as a PCR template to amplify Hag-D2 constructs for cloning into pET24d in order to express the flagellin modification for crystallization. Fragments the size of approx. 1450 for Hag-D2-Cup and 1350 for Hag-D2-Strep were expected.

The PCR Probes were purified and pooled with the Omega Gel Extraction Kit due to the enzymatic reaction purification protocol. Final concentrations:

Hag-D2-Cup: 41 ng/ µL

Hag-D2-Strep: 25 ng/ µL

The PCR products from 19.12 were digested with NcoI and BamHIHI to get the restriction sites for cloning into pET24d NcoI/BamHI cut.

Incubation overnight at room temperature.

The fragment of the fusion PCR (22.10) was separated on a 1% agarose gel, digested out and purified with a Gel Ex kit. The already digested pMAD plasmid was used to perform a Gibson assembly.

The protocol and the strains were provided by AG Bremer of the microbiology department of the Philipps Universität Marburg. All media were prepared as possible (methods) and the strains B. subtilis 168 and B. subtilis JH642 were streaked on LB-Agar and incubated at 37°C.

The cultures from the day before were spinned down at 4000 rpm for 20 min. The pellets were resuspended in 10 ml Buffer A and cracked with the micro fluidizer. The lysate was centrifuged at 20000 rpm for 20 min and purified with a Ni-NTA Beads from Qiagen according to the protocol. Probes from the pellet, supernatant and Elution wereanalysed on an SDS-PAGE gel:

The gels showed that the protein remains in the pellet no matter which attempt.

For further experiments for the isolation of the protein in inclusion bodies new expression cultures from a freshly transformed BL21(DE3) strain with piGEM-028 were used to inoculate 3 L expression culture with lactose overnight.3 L LB were prepared with 1 mL ampicillin each. The cultures were induced with 50 ml lactose solution (12,5g/ 50 mL) each and incubated at 30°C overnight.

The NcoI/BamHI digested PCR fragments from 19.13 were ligated into predigested pET24d. The 20 µL attempts were transformed into E. coli XL1-Blue after inactivation (10min at 65°C) and selected on LB- Amp plates incubated overnight at 37°C.

The control plate showed no colonies in comparison the ligation plates I and II over 15 clones. 6 Clones per plate were picked for a inoculation of minipreps for a test digest next day.

The cultures were centrifuged at 4000 rpm. The pellets were resuspended in 10 ml Buffer A and centrifuged down at 4000 rpm again. The supernatant was discarded and the pellets were frozen at -80°C.

Sequencing results -positive for both sent plasmids.

In order to rescue the StrepDARPidin from the inclusion bodies we tried different protocols. The pellet from 07.09.2014 was warmed up and the cells were cracked with the microfluidizer after resuspension in 15 mL Buffer A (lysate)).

The suspension was centrifuged at 20.000 x g for 15 min. The supernatant (S1) was discarded and the pellet (P1)resuspended in 5 mL 6 M guanidinium-HCL solution for solubilisation and unfolding of the StrepDARPidin.

After centrifugation at 4000 rpm for 10 min the supernatant (S2) was used for refolding. The membranous pellet (P2) was thrown away.

For refolding 100 mL Buffer A were filled into a Erlenmeyer-Flask on ice with a magnet stirrer inside on 600 rpm.

For refolding it was necessary to dilute the Guanidinium-HCL dilution very fast and softly. For that purpose rapid dilution was done by pipetting the supernatant drop by drop into the vortex of the Buffer A. After adding the whole Guanidinum-HCL/ Protein solution the suspension was observed and precipitation noticed. The suspension was centrifuged at 20.000 rpm (Pellet P3). The supernatant was loaded on a Ni-NTA Column from GE-Healthcare (L). Flow through (FT), Wash (W) in Buffer A and Elution (E) in Buffer B were kept for gel analysis. The probes lysat to Load were cooked for 5 min at 95 degrees.

From every fraction 40 µL were taken for a commassie SDS-PAGE and 10 µL 10x SDS Buffer added. The pellet probes were resuspended in 60 µL water and 40 µL SDS-Buffer.

The gel showed us that the StrepDARPidin is still in the pellet fraction but we were able to make some of it soluble as it can be seen in S2 and the load. The StrepDARPidin was thought to build tetramers with approx. 120 kDa size. The complex seems to be stabil enough to be seen on the gel above the marker which fits to the size of approx. 120 kDa. Boiling up would break the tetramer.

In order to get enough protein for a gel filtration run 4 L of BL21 with piGEM-028 were induced with lactose overnight.

The plasmids from 6 clones per Ligation plate were isolated and approx. 350 ng DNA were digested with NcoI/BamHI in order to check the right insertion of the 1350-1450 bp sized inserts.

Digest at 37°C for half an hour.

Upper half of the gel shows hag-D2 cup, lower half shows hag-D2-Strepdarp; 1kb gene ruler ladder was used. 3 clones seem to be positive for cup, but as an mixture occurred, the cloning procedure will be repeated from the PCR step on.

Template was diluted plasmid piGEM026/027

After the PCR the fragments were purified via gel extraction for further cloning steps. In the gel the digested vector pET24d was also purified.

Concentrations after clean up:

pET24d = 20 ng/µl, D2-Cup = 208 ng/µl, D2-Strep = 180 ng/µl

After the clean up the two fragments they had to be digested with NcoI and BamHI in order to clone them into the digested pET24d vecor.

The restriction was performed for 1h at 37°C (heating block).

After the plasmid and the two inserts have been digested, two ligations and a ligation control were performed in order to gain the final expression vectors.

For the optimal ligation results the ligation calculator of the iGEM team Dallas was used. Ligation was performed for 2h. Afterwards the ligase was inactivated at 65°C for 15 min in order to improve the ligation/transformation result.

After the ligase inactivation the whole reaction mix was used to transform E. coli XL-blue which seem to be the best cells for important transformations. Standard transformation protocol was used.

The Gibson assembly plate showed sixteen colonies. All colonies were picked, transferred onto a master plate and into a premixed colony PCR. The same colonies were used to inoculate a miniprep.

The colony PCR was negative, even the positive control with the amplified KS part I with the flanks.

The plasmids were isolated and digested with KpnI and SacI to test the insertion of the KS part I into pMAD. The expected sizes were 5054 bp and 6402 bp with the insert and pMAD only would yield in 4440 bp, 170 bp and 5054 bp fragments.

The clones 1, 2, 5, 7, 8, 9, 10, 11, 12 and 14 showed the expected results. The rest of the isolated plasmids were discarded.

The overnight cultures from 18.70 were harvested for 10 min at 4000 x g and the pellets were stored at -80°C until use.

A preculture of 10 ml LB was inoculated with B. subtilis WT3610 cells and incubated at 37°C over night.

The midi prep of the plasmids piGEM002 with the silver and copper promoters and the killswitch promoters was carried out according to the Qiagenmidiprep kit instructions. The isolated plasmids were tested via restriction with BamHI and SpeI.

Precultures of all four strains (JH642, PY79, 168 and WT3610) were inoculated in 3 ml HS-medium and 10 ml LB and were incubated over night at 37°C.

After the successful ligation (colonies in both plates), 5 clones per ligation have been inoculated in LB-Kan Medium for a later Mini-prep and test digest of the expression plasmids. Colonies with the numbers 6-10 were chosen as 1-5 did not grow over the day. 6-10 were incubated over night.

The only preculture, which was grown in HS was the JH642. Every other strain did not grow in High Salt medium. 20 ml of prewarmed (30°C) Low Salt medium were inoculated with 1 ml of the HS culture and incubated in a shaking waterbath at 30°C with 120 rpm for 3 hours. 5 µg of the plasmids were preset in Eppendorf cups and 1 ml of the LS culture was transferred into these cups. piGEM002 was transformed as a control. These culture/plasmid mixes were incubated at 37°C shaking at 200 rpm. After 2 hours of incubation the cells were centrifuged at 8000 rpm for 1.5 minutes, most of the medium was withdrawn,the cells were resuspended in the rest of the medium and plated out on LB-Cm plates that were incubated at 37°C over night. Precultures of all strains were again inoculated, since many were not grown.

The precultures in LB medium were all grown. 10 ml Spizizens minimal medium were inoculated with an OD₆₀₀ of 0.1. The cultures were incubated at 37°C until they reached an OD₆₀₀ of 1.5 µg of DNA were mixed with 1 ml of the culture and incubated for further 2 hours. 200 and 500 µl of each transformation sample were plated out on separate LB-Cm plates, which were incubated at 37°C over night.

10 ml of MNGE-medium were inoculated 1/100 with the preculture. This culture was grown at 37°C with an agitation of 200 rpm until it reached OD 1.4. 5 µg of he isolated plasmids pMAD-KSI from clone 1, 2, 10 and 11 (22.12) were used to perform four transformations. 400 µl of the cells were added to the plasmids in a test tube and were incubated at 37°C 200 rpm for 1 hour. 100 µl expression mix were added and the cells were incubated for another hour. Dilutions up to 10^-6 were carried out and the 10^-5 and 10^-6 dilutions were plated out on LB-MLS-X-Gal plates that were incubated at 30°C for several days.

Plasmids were extracted from the cells and a test restriction was performed with 300 ng plasmid. Gelfoto was unfortunately not saved but the bands were at the expected size (appr. 1500 bp). Therefore one plasmid each was sent for sequencing. Cup Number 6!

Due to the sequencing results pET24d with Hag-D2-Cup is perfect (the plasmid map was compared to the sequence delivered by eurofins).

pET24d with Hag-D2-Strep cannot be used, since the strep tag seems to be lost. Therefore this plasmid will be cloned again from the beginning in order to exclude any mistakes at any step.

The plates of the former transformations did not show any colonies, so the trafo was repeated with the precultures (13.82). Again the only strain which grew in HS-medium was JH642. The procedure was carried out like before with a higher agitation (200 rpm) in the waterbath.

The fragment with the size of approx. 1300 bp was successfully amplified.

After the purification via gel extraction the fragments had the following concentrations:

Attempt I = 27 ng/µl Attempt II = 35 ng/µl

20 µl reactions were prepared:

In order to gain the complete expression vector the digested insert had to be ligated into digested pET24d. 20 µl reaction mix was prepared, correct ratio of insert to vector was calculated with the iGEM ligation calculator.

Whole ligation mix was transformed into E. coli XL-1-Blue with the standard transformation protocol.

5 ml LB-MLS were inoculated with a blue colony of the trafo plates and incubated over night at 37°C. Colonies of clone 1, 2, 10 and 11 were picked.

The transformation of B. subtilis WT3610 with the plasmids piGEM026 and piGEM027was carried out. In order to check for positive clones a colony PCR was performed using the Hag primers.

No positive clone could be detected.

Standard E. coli transformation protocol was used. Cells were plated on LB-Cm, incubation over night at 37°C.

8 Clones from the ligation plates were inoculated in 5ml LB-Kan and incubated over night. Plasmids were extracted with the quiagen kit and digested with NcoI and BamHI in order to check if Hag-D2-strep is in pET24d.

Every picked clone was positive so that clone 1 was sent for sequencing. The gel photo got lost unfortunately. pET24d-Hag-D2-Strep was named piGEM-030.

4 ml of LB-MLS were inoculated with 100 µl of the precultures (in a test tube) and incubated at 30°C and 210 rpm for 2 hours before switching the temperature of the incubator to 42°C for 6 h. Dilutions up to 10^-6 were made and 10^-5 and 10^-6 were plated out on LB-MLS-XGal plates at 42°C.

The picked clones were boiled at 100°C in 1x PBS for 10 minutes and the the cell rests were centrifuged. The colony PCR was repeated as follows:

The size of the expected bands was approx.1400 bp. The negative clones with the wildtypeflagellin (approx.1 kb) were not further used. Clones D2-Strep 1, 10 and D2-Cup 1 were picked and streaked out separately on LB plates.

Incubation over night shaking at 37°C

4 ml of LB were inoculated (in a test tube) with a blue colony of the plates from the first heat shock. These cultures were incubated at 30°C at 210 rpm for 6 hours and afterwards the temperature was shifted to 42°C for 3 hours. The plating was performed as usual.

The colony PCR was repeated as listed above (19.26) and the fragments of the right size were purified by Gel Ex.

Additionally the positive clones were used to inoculate 100 mL LB with WT3610 as a control. The cultures were raised to an OD₆₀₀ of 0,7 and 2 mL of cultures were centrifuged before dissolving them in 60 µL water and 40 µL SDS-PAGE buffer. The samples were cooked for 10 min at 95°C and analyzed via SDS-PAGE with coomassie stain.

It is possible to see that the D2-Strep clone expresses the modified Hag-D2-Strep. The size between the WT flagellin the D2-3-Hag fits perfectly. Hag-D2-Cup seems to be not expressed by the clone. The sequencing results had to be expected.

The plates of the transformation with the Spizizens-medium were still stored at 37°C and none of the plates contained colonies except the plate of Bacillus subtilis 168. These clones were picked, transferred onto a new plate and into 1x PBS medium boiled at 100°C or 10 minutes.

The expected size of the bands was approx.750 bp, the negative control was genomic DNA of the wt3610, the positive control was the isolated plasmid piGEM037.

Plasmid isolation of the pSB1C3-Hag-KpnI plasmid from E. coli was performed with the Quiagen Mini-prep kit. Plasmid concentration was determined with Nanodrop (96 ng/µl).

Restriction of the plasmid with KpnI was performed in a duplicate for the creation of further biobricks.

The digest was incubated at 37°C for 210 minutes and subsequently analyzed on a 1% agarose gel.

The band of the digested plasmid pSB1C3-Hag-KpnI was digested out and purified with the Qiuagen gel extraction kit. Concentration after purification was 100 ng/µl.

For the creation of further biobricks three PCR fragments that were already present from previous PCR reactions have been inserted into flagellin:

• Cup 1-1 (amplified with primer iGEM24 and iGEM25) 16 ng/µl
• D2-Cup (amplified with primers flo96 and flo97) 189 ng/µl
• D2-Strep (mplified with primers flo96 and flo97) 233 ng/µl

For the Gibson assembly 4 Gibson assembly mixes (3 reactions and one control) have been thawed on ice. 2.5 µl of digested plasmid and the PCR fragment have been added respectively. The total mix of 20 µl was incubated at 50°C for one hour (PCR cycler). Afterwards the complete mix was used to transform competent E. coli XL-1-Blue. Cells were afterwards plated on LB-Cm plates.

Since we tried the Bacillus trafo with the ethanol free isolated nose plasmids (without degradation tags) as well and succeeded in nearly all cases except piGEM035, all strains are there for plate reader tests. Competent cells of PY79 were again transformed with piGEM035. The new promoters should also be tested with GFP containing degradation tags. In order to do that the plasmids piGEM007, piGEM008 and piGEM009 that contain the ssrA tags behind the GFP were digested with the digest enzymes NcoI and SacI.

The restriction mixes were incubated at 37°C for over 1 h, separated on an agarose gel and purified via Gel Ex.

To connect the linearized plasmids with the promoter fragments ligations and Gibson assemblies were performed similar to 13.76 and 13.77.

Ligation of piGEM007/8/9 with the killswitch promoters:

The reaction was incubated at room temperature for over 1 hour and afterwards used to transform XLIBlue cells, which were plated out on LB-Amp plates.

Gibson assembly of linearized piGEM007/8/9 with the new silver and copper promoters:

The Gibson reaction was incubated at 50°C for 1 hour and the whole sample was used to transform XLIBlue cells that were plated out on LB-Amp.

A colony PCR with the primers that bind to the ends of the amyE flanks was carried out but it was negative. Since it was not sure whether the primer would be able to bind the PCR was repeated with the primers surrounding the killswitch module I iGEM038 and iGEM039:

The expected bandsize was about 360 bp, which was visible in nearly all the samples. The positive control for the PCR reaction was isolated piGEM 031 and the negative control was chromosomal DNA of the unmodified wt3610.

In order to improve the rescue the StrepDARPidin from the inclusion bodies we tried an additional IB washing buffer. The pellet from last week at -80°C was warmed up and the cells were cracked with the microfluidizer after resuspension in 15 mL Buffer A (lysate)).

The suspension was centrifuged at 20.000 x g for 15 min. The supernatant (S1) was discarded and the pellet (P1) resuspended in 5 mL IB Wash Buffer. Then the suspension (SS1) was centrifuged at 27.000 x g for 10 min. The resulting pellet (P2) and the supernatant (S2) were used for taking probes for SDS-PAGE. The wash was repeated. 6M Guanidinium-HCL solution for solubilisation and unfolding of the StrepDARPidin was added.

After centrifugation at 4000 rpm for 10 min the supernatantwas used for refolding. The membranous pellet (P3) was thrown away.

For refolding 100 mL Buffer A were filled into a Erlenmeyer-Flask on ice with a magnet stirrer inside on 600 rpm.

For refolding it was necessary to dilute the Guanidinium-HCL dilution very fast and softly. For that purpose rapid dilution was done by pipetting the supernatant drop by drop into the vortex of the Buffer A. After adding the whole Guanidinum-HCL/ Protein solution the suspension was observed and precipitation noticed. The suspension was centrifuged at 20.000 rpm (Pellet P4) . The supernatant was loaded on a Ni-NTA Column from GE-Healthcare (L). Flow through (FT), Wash (20 mL W) in Buffer A and Elution (15 mL) (E) in Buffer B were kept for gel analysis. The samples lysate to Load were cooked for 5 min at 95°C.

From every fraction 40 µL were taken for a commassie SDS-PAGE and 10 µL 10x SDS Buffer added. The pellet probes were resuspended in 60 µL water and 40 µL SDS-Buffer.

The gel shows that the monomeric and tetramericStrepDARPidin can be found in Load, Flow through and elution. The forming of the tetramer tells us that the refolding had to be successful partially. The elution was concentrated with an Amicon to an endvolume of 1,5 mL. 75 µL glycerol were added and the concentrate was frozen away in liquid nitrogen and stored at -80°C for further use.

Colonies appeared on all plates. Control plate showed appr. 50 colonies whereas the 3 reactions showed uncountable colonies on the plates. 4 clones have been picked per reaction and incubated at 37°C over night for a test restriction of the correct insertion of the 3 inserts of interest.

The stored protein concentrate was thawn on ice and injected into the injection valve of the ÄtkaPurifier which was buffered with PBS+ 2,5% glycerin. A S200 SepharoseColumn was used for the gel filtration run.

40 µL of the Fraction C6. C10, C11(Peak) and D12 were picked for SDS-PAGE analysis. 10 µL SDS-Buffer were added and 20 µL loaded on the gel. Additionally the rest was cooked up and the same amount loaded onto the gel.

The gel shows us that the whole peak contains the tetramericStrepDARPidin. After cooking it up the tetramer dissolves into the monomeric StrepDARPidin.

The fractions covering the peak were concentrated with an amicon to an endvolume of 500 µL. 25 µL glycerin were added and the protein aliquot in 60 µL aliquods.

piGEM-029 and piGEM-030 were transformed together with FliS into E. Coli BL21 (DE3) and plated out on LB-Amp/Can plates for overnight incubation at 37°C.

The following entries of the nose plasmids will care all about the cloning, which involves creating the plasmid by Gibson/ligation checking by colony PCR, transformation of E. coli DH5α and Bacillus subtilis PY79 with the plasmids. Not every step of transformation will be mentioned, since they all were carried out according to the standard protocols. After each plasmid was checked by colony PCR and/or control restriction DH5α and Bacillus were transformed with it. DH5α were transformed with the plasmids piGEM034, 035, 036 and 037.

Plasmid isolation of the 12 cultures was performed with the Quiagen Mini Prep Kit. Concentration afterwards was about 250 ng/µl in all samples.

Test restriction of the previously isolated plasmids with EcoRI and PstI:

12 plasmids + control → 14x master mix for 20µl reaction mixes

Restriction was incubated at 37°C for 180 minutes and subsequently analyzed on a 1% agarose gel.

Result: All tested clones were negative, i.e. bands at the same size of the control occurred.

1500bp= Hag-KpnI; 2000bp= 13C plasmid backbone

Plan: inoculate 5 new clones each for plasmid isolation and restriction tomorrow in LB-Cm.

An overnight expression culture from piGEM-030/FliS was made by inoculating 2 x 1L LB-Amp/Can with clones from the transformation plates and inducing with 50 mL 25% lactose solution overnight at 30°C at 150 rpm.

Overnight cultures were inoculated of 5 mL LB in test tubes from WT3610 , Hag-D2-Cup & -Strep strains. Incubation was done at 37°C overnight.

The Bacillus subtilis PY79 cells were made competent according to the protocol using SPC and SPII medium. As a test the cells were transformed with piGEM038, which was transformed before.

The size of the expected band was depending on the promoter between 700 and 850 bp. As a positive control cells containing the piGEM002 vector with the different promoters were used. The clones that seemed positive were given in culture for a miniprep. More clones were picked from the plates to test them via colony PCR (same reaction as above).

Same procedure as yesterday (17.09)

Plasmid concentration after plasmid isolation was about 250 to 300 ng/µl. 2 µl of plasmid DNA were used for the restriction reaction.

Incubation for 240 min. digested plasmids were analyzed on a 1% agarose gel.

All tested clones were negative. In order to repeat the Gibson assembly pSB1C3 with Hag-KpnI will be digested with KpnI over night (37°C).

The OD₆₀₀ of the overnight cultures was measured and was approx. 5. 100 µL of culture were centrifuged and the pellet resuspended in 50 µL medium to get an OD of 10.

10 µL of the cultures was dropped on Swimming and Swarming plates (0,3% and 0,7% LB Agar plates) and incubated at 37°C.

The cellular diffusion was observed after 30-60 min and the distanced measured starting from the cell circle after letting the drop dry.

The measurements showed that Hag-D2-Strep is able to swim and swarm. In comparison to the wild type the strain is slower in swarming and swimming. Hag-D2-Strep did not swarm or swim as the commassie gel showing no flagellin suggested before. Electronmicroscopy and alternatives for Hag-D2-Strep were planned.

For reproduction of the results new precultures of Hag-D2-Strep & -Cup were inoculated and incubated at 30°C.

The cultures from yesterday were harvested at 4000 rpm at 4 °C and the pellets were resuspended in 10 mL Buffer A for microfluidizing. The lysate was centrifuged at 20000 rpm so that the clear lysat could be loaded on a 1 mL Ni-NTA column. Preinduction probe (PI), induction probe (I), Load (L), flow through (FT), wash (W) and elution with 15 mL Buffer B (E) were analysed on a SDS-PAGE gel commassie stained:

Meanwhile the elution was concentrated with an amicon at 4000 rpm until an endvolume of 1 mL. The ÄKTApurifier was buffered with GeFi Buffer and the concentrate was injected. Presets: 4 mL fraction size, 0,65 mPa pressure alarm, fractioning after 90 mL run, endvolme 400 mL. A Superdex S200 column was used.

FT and W contained much protein. The 1 mL Ni-NTA had not enough capacity to bind all the protein.

The Ni-NTA column seemed to be contaminated with left StrepDARPidin on the NI-NTA columns from previous use. We made the GeFi run to see if we culd purifier the proteins anyways.

The fractions building the peak were analyzed on a SDS-Gel to cover the peak. Then the fractions from were concentrated with an amicon to an endvolume of 500 µL. Aliquods of 50 µL were frozen in liquid nitrogen and stored at -80°C.

To be sure that the promoter sequences were inserted correctly the isolated plasmids were digested with the enzymes SpeI and BamHI. The difference between the negative and positive clones should be very few but be detectable on the gel: positive: 3426 and approx.3500 bp; negative: 3426 and 3276 bp.

The restriction reaction was incubated at 37°C for over one hour and analyzed with an agarose gel.

Although the colony PCR for most of the plasmids was like expected, some plasmids were not digested at all. The bands around 3000 bp are double bands of the two fragments. A difference between negative and positive plasmids could not be discovered since the amount of DNA was too high and the bands were not separated well enough. It was striking that every undigested plasmid was a plasmid with the piGEM007 backbone (strong degradation tag). For a better check a control PCR was performed with the 007-plasmids as template:

According to the control PCR on the plasmids the plasmids were ok and the promoters were inserted correctly. The copper sensitive promoter is slightly bigger than the used lac or constitutive promoters, which can be seen very well on the gel. The plasmids were assumed to be correct and were ready for further using (trafo of Bacillus and DH5α). However, a further Gibson assembly and ligation reaction (according to 13.85) was carried out with all promoters and plasmids that were negative (piGEM007 + Cu + Ag; 008 + Cu; 009 + Ag + Cu; 007 + const + tetO + lac; 008 + const; 009 + tetO + lac).

Lewis Lung cell Carcinoma Cell line and A549 Adenocarcinoma cell line were kindly provided by Dr. Jürgen Adamkiewicz and Margitta Alt (ZTI Marburg) and splitted 1:3 at 37°C in DMEM + 10% FCS and L-Glutamine. The cells were cultivated for further use.

Checked Test digestion on agarose gel, three bands were visible, which could not be. Only 2 bands were expected.

Check undigested plasmids on agarose gel. Result: Two bands

Retransformation of the already sequenced plasmid pSB1C3-Hag-KpnI for further cloning processes.

The OD of the overnight cultures was measured and was approx. OD 5. 100 µL of culture were centrifuged and the pellet resuspended in 50 µL medium to get an OD of 10.

10 µL of the cultures were spotted on Swimming and Swarming plates (0,3% and 0,7% LB Agar plates) and incubated at 37°C.

The cellular diffusion was observed after 30-60 min and the distanced measured starting from the cell circle after letting the drop dry.

The Hag-D2-Strep strain swarms and swims slower than the WT but is motile in comparison to the Hag-D2-Cup strain.

Plasmids were isolated from cultures inoculated yesterday. These plasmids were again digested with BamHI and SpeI to check for the insertion of the promoters.

The reactions were incubated at 37°C for over 1 hour.

All bands were in the expected height of approx. 3400 bp. The plasmids were used further for transformation of DH5α and Bacillus.

• Inoculation of positive clones of pSB1C3-Hag-KpnI for Mini-prep (3 clones) in the morning
• Plasmid isolation in the evening
• Overnight restriction of pSB1C3-Hag-KpnI for Gibson assembly.

Check initial plasmids for purity: an additional band was visible, which could probably be coiled plasmid.

The clones on the plates of the new ligation and transformation were screened for positive constructs by colony PCR.

As a positive control XLIBlue containing piGEM034 or piGEM035 were used, the negative control was an untransformed XLIBlue. None of the picked clones was positive for the lac and constitutive promoters + tetO. Some clones containing the plasmids piGEM009 + Ag promoter and piGEM009 + Cu promoter were positive. These positive clones were picked and used to inoculate a miniprep culture.

In order to check the interaction between the Strep-Tag in the D2-Flagellin and the Streptavidin in our StrepDARPidin we used an analytical Superdex S200 column. Probes from both proteins were injected into the ÄTKAPurifier and a separate run each done in order to see when the protein gets through the column.

In case of an interaction the complex of both proteins would come off early from the column because of its size. The column was buffered with PBS+2,5% glycerin.

Size and ratio were calculated with http://web.expasy.org/protparam/ and http://christoph-leidig.de/tprot.html for injecting adequate amounts of both proteins.

StrepDARPidin:

Hag-D2-Strep/FliS

88.57/ 122,19 = 0,7248

(1-0,7248)* 50 µL (StrepDARPidin) =13,76 µL(Hag-D2-Strep)

Analytical run StrepDARPidin 50 µL + 50 µL PBS+2,5% glycerin:

Analytical run Hag-D2-Strep/ FliS 13,7 µL+ 86,3 µL PBS+2,5% glycerin:

13,7 µL Hag-D2-Strep/FliS & 50 µL StrepDARPidin + 36,3 µL PBS+2,5% glycerin incubated together for 1h at room temperature:

The peaks showed no shift to the front and ran into each other. They seemed to show no interaction. In order to be sure we tried to make a pulldown with Strep-Beads.

Strep-Beads from Novagene were used to check the interactions between the Strep-Tag in our Flagellin with the StrepDARPidin and the Strep-Beads themselves.

Theoretically the flagellin should bind to the beads and should be found in the elution in contrast to the StrepDARPidin which should be washed off.

In the third elution there should be less Flagellin or even nothing in case of an interaction with the StrepDARPidin because of the competitive situation. The flagellin should bind totally to the StrepDARPidin and for that reason not/ less to the beads.

3 columns were prepaired and filled with 500 µL GeFi Buffer. 20µL Strep-Beads were added and the columns were spinned down at 4000 rpm for 1 min. Then another 500 µL GeFi buffer was added.

The first (1) probe of 3 µL Hag-D2-Strep was pipetted into the buffer. 20 µL StrepDARPidin were added to column 2. Column 3 was used for loading a mixture of 20 µL StrepDARPidin and 3 µL Flagellin onto the column. The three columns were spinned for 20 min at room temperature in a spinning wheel. After that the columns were washed twice with 500 µL GeFi buffer. For eluting 40 µL Elution Buffer from Novagene (with Desthiobiotin) was used to resuspend the beads. After centrifugation at 13000 rpm the elution was mixed with 10 µL SDS-Loading buffer and analyzed on a SDS-PAGE gel with commassie stain.

As a control 40 µL of a 1:10 dilution of the flagellin concentrate was used on the gel. Unfortunately there was not enough StrepDARPidin left for a second control.

Gel-Analysis:

The gel shows that a very tiny amount of the flagellin binds to the Strep-Beads (line 1). The StrepDARPidin does not (line 2). Surprisingly the mixture shows both proteins in the elution (line 3). Unfortunately this cannot be seen on the scanned picture . The control (line 4) shows us that the flagellin purification was not pure enough and the flagellin concentrate was contaminated with StrepDARPidin from the Ni-NTA purification.

The Strep-Beads might have been not efficient working enough because of their age and usage. The binding of the flagellin might tell us that the Strep-Tag is able to interact with the Streptavidin.

In order to get a better and reliable result we decided to purify the flagellin and the StrepDARPidin newly and repeat the pulldown.

New expression cultures of 1L volume each were induced with lactose and incubated overnight at 30°C and 150 rpm.

→ Templates piGEM021, piGEM 026 and piGEM 027.

1:30 min elongation times was changed in standard PCR program.

The digested vector (over night) and the PCR products were purified via gel extraction for further use in Gibson assembly reactions.

All fragments have been amplified in the correct size with some side products (probably partially coming from the plasmids used as a backbone).

The digested plasmid yielded in a similar pattern like the last time (2 bands). Therefore the gel was run for additional 20 minutes and a third band could be seen. After additional 40 min it became clear that the middle thin band resembles the digested plasmid. It was therefore digested out and purified together with the PCR products.

The ligation was carried out like in 13.85. The reaction was incubated at room temperature for over one hour and E. coli XLIBlue were transformed with the whole reaction mix.

Restriction results in the same pattern → idea cook DNA before restriction and shock on ice, restrict for appr. 3h afterwards

Digestion with EcoRI/PstI showed that the plasmid is pure and not contaminated with other plasmids. Restriction with less DNA and cooking DNA before yielded in a thicker band for the hopefully digested plasmid at 3 kb. Band was digested out and purified via gel extraction (Omega Kit) for further Gibson assembly and transformation.

The cultures from yesterday were harvested at 4000 rpm at 4 °C and the pellets were resuspended in 10 mL Buffer A for microfluidizing. The lysate was centrifuged at 20000 rpm so that the clear lysate (Load/L) could be loaded on a 5 mL Ni-NTA column. Load (L), flow through (FT), wash (W) and elution with 15 mL Buffer B (E) were analysed on a SDS-PAGE gel coomassie stained:

FT and W contain much protein. The 1 mL Ni-NTA had not enough capacity to bind all the protein.

Meanwhile the elution was concentrated with an amicon at 4000 rpm until an endvolume of 1 mL. The ÄKTAPrime was buffered with GeFi Buffer and the concentrate was injected. Presets: 4 mL fraction size, 0,65 mPa pressure alarm, fractioning after 90 mL run, endvolme 400 mL. A Superdex S200 column was used.

The Ni-NTA column seemed to be not contaminated with StrepDARPidin. So the last contamination came form the left StrepDARPidin on the NI-NTA columns from previous use.

Unfortunately the S200 column seemed to be very dirty so that it was not possible to separate the proteins properly.

The fractions were collected and concentrated again. After that the concentrate was injected into ÄktaPrime again with a different S200 with the same settings as before. The run was done overnight.

Additionally new expression cultures with E. Coli BL21(DE3) containing piGEM-030 were induced with lactose for a new purification run.

E. coli XLIBlue piGEM037 (002 + tetO) was used as a positive control. Clones 2 and 3 of piGEM007 + tetO were positive and cultures for a miniprep were inoculated with these clones.

Positive clones could only be detected for Cup-1. Clones 5 and 7 looked most promising and were inoculated in LB-Cm overnight for plasmid isolation and test-restriction. PCR for the other two modules has to be repeated or clones need to be inoculated for plasmid isolation and test-restriction.

The fractions building the peak were analyzed on a SDS-Gel to cover the peak.

The gel showed a degradation of our protein. For crystallization it is not adequate enough. A new purification with the expression cultures from yesterday was started.

The cultures from yesterday were harvested at 4000 rpm at 4 °C and the pellets were resuspended in 10 mL Buffer A for lysis by microfluidizer. The lysate was centrifuged at 20000 rpm so that the clear lysate (Load/L) could be loaded on a 5 mL Ni-NTA column. Load (L), flow through (FT), wash (W) and elution with 15 mL Buffer B (E) were analysed on a SDS-PAGE coommassie stained:

The gel showed no contamination with StrepDARPidin as well.

The elution was concentrated with an amicon at 4000 rpm until an endvolume of 1,5 mL. The ÄKTAPrime was buffered with GeFi Buffer and the concentrate was injected. Presets:

4 mL fraction size, 0,65 mPa pressure alarm, fractioning after 90 mL run, endvolme 400 mL. A Superdex S200 column was used.

The fractions building the peak were analyzed on a SDS-Gel to cover the peak which looked fine.

Then the fractions were concentrated with an amicon to an endvolume of 300 µL. The new robot for setting drops was used for crystallization. Cores I-III were set in full concentration (A280=25) and half concentration diluted with GeFi-Buffer. Left hag-d2-Strep was aliquoted in 50 µL and stored at -80°C.

2 L expression culture were made for new purification attempts and incubated overnight at 30°C after lactose induction.

Since some plasmids with the silver and copper sensitive promoters were still missing a new Gibson assembly was carried out with the

Nine reactions were carried out: piGEM 008 + Cu, piGEM009 + Ag and piGEM007 + Ag in three different variations in DNA concentration. The reactions were kept on room temperature for 30 seconds and incubated at 50°C for one hour. Afterwards the whole reaction mix was used to transform E. coli XLIBlue, which were incubated over night at 37°C.

Both prepared plasmids have been digested with EcoRI/PstI and compared with the digested 13C-Hag-KpnI. No significant difference could be detected. Therefore different Plasmids have been digested with EcoRI/PstI and will be compared for the different inserts.

New transformation with sequenced plasmids → strepDARP clone 1 and Hag-KpnI clone 4

10 L expression culture were inoculated with clones from a BL21(DE3) transformation plate and induced with lactose overnight at 30°C.

The cultures were harvested at 4000 rpm at 4°C and the pellet was frozen in liquid nitrogen and stored at -80°C for further use.

A colony PCR was carried out to screen the transformands for the correct plasmids. No transformands could be detected on the plate with piGEM008 + Cu.

As a positive control E. coli XLIBlue piGEM034 (02 + Ag) was used. Clone 4 was positive. A LB culture was inoculated with clone 4 for a miniprep. It was grown shaking at 37°C over night.

It was noticed that the IPTG inducible Gram positive (described as lac) promoter, the strong constitutive for gram positive promoter and the tetO including variant were designed falsely. Only a part (30 bp) of the sequence was taken and cloned. That means that all nose plasmids containing the lac, const. and const. + tetO promoters were disposed. Only the plasmids with the metal ion sensitive promoters were used further.

Since the last PY79 cells were not competent a new try of producing competent cells was carried out according to the protocol in the methods section using SPC and SPII medium. After making the cells competent they were stored in a -80°C freezer without shock freezing in liquid nitrogen. The protocol does not including shock freezing of the cells. For a test transformation four aliquots of cells were thawed on room temperature and 100 µl each were transferred into sterile test tubes and mixed with 7 µl of the plasmids piGEM034, piGEM035, piGEM044 and piGEM050. The test tubes were incubated shaking at 37°C for 0.5 hours. The whole transformation samples were plated out on LB-chloramphenicol (5 µg/ml) plates and were incubated at 30°C for several days.

Transformation from yesterday: inoculate clones at 8 in order to isolate the plasmids, perform Gibson assembly and transformation.

Plasmid isolation → To exclude a that samples were exchanged by mistake a test-restriction of the plasmids has to be performed with EcoRI/KpnI, since KpnI does not digested in pSB1C3-Strep DARP. When cutting Hag-KpnI a 600 bp fragment should occur in the gel whereas no fragment is expected when cutting pSB1C3-Strep DARP. According to the test restriction samples have not been switches, restrictions 1, 2 and 4 can be used for the Gibson assembly (and transformation). Restriction reaction 1 was used.

Restriction (25µl mix with complete plasmid)

The cells from yesterday were harvested and cracked with the microfluidizer after resuspension in 40 mL Buffer A (lysate)).

The suspension was centrifuged at 20.000 x g for 15 min. The supernatant (S1) was discarded and the pellet (P1) resuspended in 5 mL IB Wash Buffer. Then the suspension (SS1) was centrifuged at 27.000 x g for 10 min. The resulting pellet (P2) and the supernatant (S2) were used for taking probes for SDS-PAGE. The wash was repeated. 10 mL 6M Guanidinium-HCL solution for solubilisation and unfolding of the StrepDARPidin was added and the pellet resuspended. The suspension was incubated at room temperature permanently stirring with a magnet.

After centrifugation at 4000 rpm for 10 min the supernatant was used for refolding. The membranous pellet (P3) was thrown away.

For refolding 5 mL of the Guanidinium-HCL 100 mL Buffer A were filled into a Erlenmeyer-Flask on ice with a magnet stirrer inside on 600 rpm. The other 5 mL Guanidinium-HCL/ Pellet resuspension was stored in the fridge until further use.

For refolding it was necessary to dilute the Guanidinium-HCL dilution very fast and softly. For that purpose rapid dilution was done by pipetting the supernatant drop by drop into the vortex of the Buffer A. After adding the whole Guanidinum-HCL/ Protein solution the suspension was observed and precipitation noticed. The suspension was centrifuged at 20.000 rpm (Pellet P4) . The supernatant was loaded on a Ni-NTA Column from GE-Healthcare (L). Flow through (FT), Wash (20 mL W) in Buffer A and Elution (15 mL) (E) in Buffer B were kept for gel analysis. The samples lysate to Load were cooked for 5 min at 95 degrees.

From every fraction 40 µL were taken for a coommassie SDS-PAGE and 10 µL 10x SDS Buffer added. The pellet samples were resuspended in 60 µL water and 40 µL SDS-Buffer.

The gel shows that the monomeric and tetrameric StrepDARPidin can be found in Load, Flow through and elution. The forming of the tetramer tells us that the refolding had to be successful partially. The elution was concentrated with an Amicon to an endvolume of 1,5 mL. 75 µL glycerol were added and the concentrate was frozen away in liquid nitrogen and stored at -80°C for further use.

The plasmids with the ssrA-degradation tags were digested with NcoI and SacI to linearize them for further cloning of promoters in front of the GFP. Different concentrations of plasmid were digested with the enzymes to test the capacity of the restriction enzymes and to be sure that all plasmid was cut.

The reaction was incubated at 37°C for over one hour and afterwards separated on a gel. As a control the undigested piGEM008 was used.

No difference between the undigested and the digested plasmids could be detected, but a small band directly under the thick, visible one. The linearized plasmids should have a size of 6711 bp. It is possible that the undigested plasmids do rarely form supercoiled structures and mostly behave like the linear form, only a small part of it is supercoiled. Since the same plasmids were digested before with the same enzymes and looked the same on the gel but were linear (further cloning of promoter sequences otherwise not possible), the plasmids were assumed to be linearized. The three concentrations of each plasmid were pooled and were purified with an Omega Gel Ex kit.

No colonies on plates. New Miniprep with much higher concentrations of plasmid → yesterday mostly only 20 ng/µl. Band before gel extraction was very thin.

New Gibson assembly and Transformation. Clones will be inoculated tomorrow; spin down and pellets will be frozen at -20°C.

The stored protein concentrate was thawn up on ice and injected into the injection valve of the ÄktaPrime which was buffered with PBS+ 2,5% glycerin. A S200 Sepharose Column was used for the gel filtration run.

40 µL of the Fraction 31. 37, 38 and 44 were picked for SDS-PAGE analysis. 10 µL SDS-Buffer were added and 20 µL loaded on the gel. Additionally the rest was boiled and the same amount loaded onto the gel.

The gel shows us that the whole peak contains the tetrameric StrepDARPidin. After cooking it up the tetramer dissolves into the monomeric StrepDARPidin.

The fractions covering the peak were concentrated with an amicon to an endvolume of 500 µL. 25 µL glycerin were added and the protein aliquoted in 60 µL aliquods with an Absorption of 8 A₂₈₀ nm.

A new Gibson assembly was carried out to insert the missing promoters into the different nose plasmids. Every combination (007 + Ag, 008 + Cu, 009 + Cu) was carried out in different relations of vector to insert:

The reactions were kept on room temperature for 0.5 min and were incubated at 50°C for 1 h. Afterwards E. coli XLIBlue were transformed with the whole reaction mix, plated on LB-ampicillin and incubated over night at 37°C.

The existent nose plasmids (piGEM034/035/039/044/050) were previously transformed into DH5α cells, which were plated out new to inoculate cryo stocks of the cells.

To compare the competence of our PY79 cells with cells that are competent for sure, one aliquot of competent PY79 were borrowed together with three LB-chloramphenicol plates. The 300 µl aliquot was divided into 100 µl portions. One negative control was mixed with 10 µl water, the other two 100 µl portions were treated with 5 µl and 10 µl piGEM034 (approx.400 ng/µl), which has been shown that it could be transformed. The cell/DNA mixture was incubated in a test tube, shaking for 30 minutes before plating them out on the mentioned medium plates. This time the plates were incubated at 37°C.

KSII could not be amplified with the nesting primers which cover the whole KSII module, even with a variety of modified reaction conditions. Thus, primers were ordered to amplify the modules (holing, tetR, rplE (L5)) separately.

The sizes of the expected fragments were: holin: approx.700 bp, tetR: 684 bp and L5: 540 bp. The fragments of the expected size could be seen, it meets the eye that the unspecific bands that appeared in previous amplifications of KSII with the nesting primers were visible as well in the lane of tetR. However, tetR also showed an amplificate with the correct size.

Although the KSII parts were amplified successfully no further work will be done with KSII, because of the false promoter in front of these genes. Like mentioned before the IPTG inducible promoter for Gram positives and the strong constitutive promoter for Gram positives used in the killswitch and nose and taken from the iGEM registry were falsely designed. Only a short part of the real sequence was used.

For this reason the promoters were designed again correctly: the lac promoter (BBa_K090501; 107 bp) will be ordered in single stranded oligonucleotides from which one contains overhangs like it was digested with NcoI and SacI. These oligos will be annealed and phosphorylated. A ligation with the nose plasmids and KSI follows. It was only enough time left to build KSI anew and not the other killswitch modules. The primer to connect the new lac promoter with the rest of KSI also introduces a RBS.

The constitutive promoter (BBa_K090504) will be amplified from gDNA of Bacillus cereus, which was kindly provided by members of the Zentralinstitut für Ernährungs- und Lebensmittelforschung ZIEL, Technische Universität München. The amplificate will be used for ligation with the nose plasmids.

The left 5 mL Gua-suspension in the fridge were refolded with the same protocol from 18.72.

P1 shows a very weak line at the expected size which comes from picking just a membranous part on top of of the pellet. The line would have been more prominent if we had taken more of the pellet itself. The gel shows that the monomeric and tetrameric StrepDARPidin can be found in Load, Flow through and elution. The forming of the tetramer tells us that the refolding had to be successful partially. The elution was concentrated with an Amicon to an endvolume of 1,5 mL. 75 µL glycerol were added and the concentrate was frozen away in liquid nitrogen and stored at -80°C for further use.

Saturday clones could be observed on all plates (more than on the control plate). 5 clones have been inoculated in LB-Cm and were centrifuged and frozen in the evening. Plasmid isolation were performed in the evening and restriction was performed over night.

The stored protein concentrate was thawn up on ice and injected into the injection valve of the ÄktaPrime which was buffered with PBS+ 2,5% glycerin. A S200 Sepharose Column was used for the gel filtration run.

40 µL of the Fraction 32. 38, 39 and 45 were picked for SDS-PAGE analysis. 10 µL SDS-Buffer were added and 20 µL loaded on the gel. Additionally the rest was boiled and the same amount loaded onto the gel.

The gel shows us that the whole peak contains the tetrameric StrepDARPidin. After boiling it the tetramer dissolves into the monomeric StrepDARPidin.

The fractions covering the peak were concentrated with an amicon to an endvolume of 500 µL. 25 µL glycerin were added and the protein aliquoded in 60 µL aliquods with an Absorption of 7 A280 nm.

Presumably Cup-1 clones 3-5 are positive, all D2-Strep clones are positive but D2-Cup clones are negative. Therefore PCR reactions will be performed with the positive plasmids and colony PCR with 10 clones of D2-Cup will be added.

1:30 Elongation time.

Agarose gel: Colony PCR for Hag-KpnI-D2-Cup is negative, but all other results from the test restriction have been confirmed.

We decided to cultivate the LLCs for Fluorometer assays in 12-Well plates. The medium of the 25 mL culture flask was used to resuspend the cells carefully because of their sparsely adherent trait.

The cells were transferred into a 50 mL the culture was spinned down at 1500 rpm for 5 min at 4°C.

The supernatant was discarded and resuspended in 10 mL DMEM. The cells were counted with a Neubauer-chamber.

10 µL of the cell suspension was mixed with 200 µL Trypan blue and 85-100 cells were counted in the grids of the chamber.

Calculation:

90 (cells total in grid) / 4 (number of quadrants) *20 (dilution factor) * 10000 (Neubauer factor) * 10 mL (volume of suspension)= approx. 40 mio. Cells/ 10 mL

We calculated with 50 mio. Cells in total.

The suspension was filled up to 50 mL to get 1 mio cells/ mL. 2 mL/ well were pipetted into each well of the 12-well plate and incubated overnight at 37°C.

A Black Fluotrac600 96-well plate was kindly received from the immunology. Row H 1-12 was coated with 100000 cells/ well overnight at 37°C.

A colony PCR was carried out to screen the transformands for the correct plasmids. No transformands could be detected on the plate with piGEM008 + Cu.

As a positive control E. coli XLIBlue piGEM034 and piGEM035 were used. No positive clones could be seen. Indeed there was a band at the right size of clone 2 07 + Ag but there was a bigger and a smaller band as well.

The transformation of the previus batch of cells with the plasmid piGEM034 worked only in case of the higher concentration. Two colonies could be observed. For the next competent cells fructose will be used instead of glucose. For making of new competent PY79 cells the cells were plated out again on a new LB plate and were incubated at 37°C over night.

Better image, bands occur exactly at expected size. Clone 3 from the Cup-1 clones will be sent for sequencing today. Clone 1 - 4 will be used for mutagenesis, when mutagenesis primer arrives.

The flagella isolation was tried for the strain Hag-D2-Strep according to the protocol from ''Purification and Thermal Stability of Intact Bacillus subtilis Flagella''.

Because of the literature we found we decided to throw the LLCs for now into the waste. We did not find enough information about the EpCAM expression in that cell line.

Instead we prepared the A549 for a Fluophor-Assay tomorrow. The 25 mL flask was splitted 1:3. 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 4 mL 1x trypsin from the opposite of the adherent cells the flask was incubated for 5 min at 37°C depending on how fast the cells come off 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 5 mL DMEM.

10 µL of the cell suspension was mixed with 200 µL Trypan blue and approx. 140 cells were counted in the grids of the chamber.

Calculation:

140 (cells total in grid) / 4 (number of quadrants) *20 (dilution factor) * 10000 (Neubauer factor) * 5 mL (volume of suspension)= approx. 35 mio. Cells/ 10 mL

The cells were splitted. 2 mL :1 mL : 1 mL of cell suspension was filled into 3 new 15 mL flasks and filled up with 10 mL DMEM+10%FCS and L-Glutamin. The 2mL flask was planned for microscopy on Friday with the StrepDARPidin constructs. The other flasks are for backups and assays. Incubation was performed at 37°C.

Additionally row H1-12 of the Fluotrac600 was coated with 25 µL cell suspension and 100 µL DMEM +10%FCS+L-Glut. (incubation at 37°C).

As a backup Row F of a 6x8 was filled with 25 µL cell suspension as well and 300 µL DMEM +10%FCS+L-Glut. (incubation at 37°C).

The pellets from 23.09.14 were resuspended in 10 mL Buffer A for microfluidizing. The lysat was centrifuged at 20000 rpm so that the clear lysat could be loaded on a 5 mL Ni-NTA column. Load (L), flow through (FT), wash (W) and elution with 15 mL Buffer B (E) were analysed on a SDS-PAGE gel commassie stained:

Further purification was done. Meanwhile the elution was concentrated with an amicon at 4000 rpm until an endvolume of 1 mL. The ÄKTApurifier was buffered with GeFi Buffer and the concentrate was injected. Presets: 4 mL fraction size, 0,65 mPa pressure alarm, fractioning after 90 mL run, endvolume 400 mL. A Superdex S200 column was used.

Then the fractions building the peak were concentrated with an amicon to an endvolume of 500 µL which were used for setting dropes with core I and IV (QIAGEN cores). Aliquods of left flagellin of 50 µL were frozen in liquid nitrogen and stored at -80°C.

The SPC medium and the SPII medium were assembled. The media were prewarmed before the cells were transferred into the media. New LB-chloramphenicol plates were made. After making the cells competent their competence was tested by transformation of piGEM034.

The yield of transformands after ligations or Gibson assemblies with the plasmids piGEM007, piGEM008 and piGEm009 was erratically fewer than with the piGEM002, which was digested a few weeks ago. Since the only difference between these plasmids are the ssrA-degradation tags on the gfp-gene, there should be no difference in their restriction behavior. This is aggravated by the fact that all four plasmids contain the restriction sites for NcoI and SacI on the same positions. For this reason the plasmids were digested again, together with test plasmids to check the activity of the enzymes NcoI and SacI. The test plasmids were pMAD that contains two SacI sites, thus resulting in fragments of 4431 bp and 5235 bp size and piGEM002, digested by NcoI and EcoRI yielding in fragments of 476 bp and 6190 bp.

The reactions were incubated at 37°C for over one hour.

According to the bands NcoI was functional, the expected bands could be seen after the restriction of piGEM002. However, the functionality of SacI was not fully determined since there was no control with the undigested plasmid. A band in the height between 5000 and 6000 bp could be seen, another very slight band at approx.4 kb could also be noticed, what seems to be the other expected fragment. Yet it the bigger band was more intense than the smaller one. The digested plasmids piGEM007, 008 and 009 were in had the correct size and seemed to be digested. These bands were pooled and purified via a Gel Ex kit.