After the purification steps until the ammonium sulphate precipitation a SDS-gel with coomassie staining was done and WT3610 as well as Hag-D2_3 were used as a control.

The gel shows that there is a band after the ammonium sulfate precipitation which is running to low to be flagellin. The isolation was repeated several times with the same result so that we think of unstable flagellins. Additionally, a B. subtilis PY79 strain with a Strep-Tag integrated into the normal Hag-gene was used for isolation of flagella. They might be more stable than Hag-D2-Strep filaments.

In order to check the interaction of StrepDARPidin with the EpCAM on A549 Lung Carcinoma cells we planned an ELISA-Like assay. Yesterday row H 1-12 of a black Fluotrac600 96-well plate was coated with 100000 cells A549/ well (counted with Neubauer-Chamber) overnight at 37°C.

We intended to incubate them with different concentrations of StrepDARPidin and then target the N-terminal His-Tag with and Anti-His antibody Alexa488 conjugated (1:50 endvolumen). In the end the signal in comparison to blank and negative control would be generated in an ELISA reader by stimulation of the ALexa488 at 480 nm and measurement at 520 nm.

In well H1-6 gel filtration purified StrepDARPidin with 87 µM was tested, in well H 7-12 Ni-NTA purified StrepDARPidin with 280 µM.

Following concentrations of StrepDARPidin dissolved in PBS (pH 7,4) +2,5 % glycerin were used:

The 96-well plate was taken out of the incubator and the medium was carefully discarded. 100 µL of the dilutions were transferred into the wells and incubated for 45 min. at room temperature under the Flow hood.

An Anti-His-Alexa488 conjugate (1 mg/mL stock) with 5 µL concentrate was dissolved in 40 µL  PBS. 1 µL of the antibody was added after washing to each well and incubated for 20 min at room temperature in the dark.

As a negative control t-cells clones were used and incubated the same way with 1 µM StrepDARPidin and 1 µL antibody (Well G2). Well G1 was left empty, well G3 was filled with 100 µL PBS+Glycerin and G4 with PBS+2,5 % glycerin with 1 µL Antibody.

After 20 min incubation the wells were washed carefully with 100 µL PBS to wash out unbound elements.

The plate was measured in the ELISA reader:

We are able to see that the fluorescence signal decreases with increasing dilution. Unfortunately the negative control was very high as well. The t-cells were incubated in Eppis because of their non-adherent features. They were incubated with StrepDARPidin and Antibody before. The supernatant was discarded after centrifugation at 1500x g but the cells did not stay at the pellet’s position so that it was not possible to remove the whole preincubated material which might be left when measuring the control.

Additionally it is noticeable that there is the same trend for both purification methods. The lower signal from NTA-purified StrepDARPidin can be explained by the fact that the concentration was higher but the purity also lower so that even less StrepDARPidin was found in the protein aliquods. Unfortunately the negative control had a high signal as well. The T-cell clones derive from human t-cells which carry the genes for  almost every protein and marker for human cells. In the process of immortalization it is possible that genes are expressed which do not belong to typical t-cells. A different negative control has to be used. In order to increase the signal more protein with a different serial dilution has to be used for reproducing the conditions.

Mutagenesis Primer arrived → PCR with pSB1C3-Hag-KpnI-Strep clone 1 and 2 (appr. 20 ng/µl) in 30 µl reaction

1 min elongation time in standard protocol was used. Whole PCR product was loaded on an agarose gel and purified via gel extraction with resulting concentrations of 16 and 17 ng/µl.

5 µl of the purified plasmid were used for a Gibson assembly reaction (total DNA amount appr. 80-90 ng). The complete Gibson mix was later transformed into chemically competent E. coli XL-1-blue cells.

The truncated cup1-1 gene should be amplified with overhangs to the streptavidin-gene and an overhang with a restriction site for NcoI. The used template was piGEM029.

The Streptavidin was amplified from the plasmid piGEM028, the StrepDARPidin plasmids, with an overhang to cup and an overhang with an XhoI restriction site.

The amplification of cup yielded in 3 different bands from which the middle, most severe band had the expected size (170 bp). The rest of the PCR reaction was separated on a 1% agarose gel, the band in the correct size was cut out and purified via a Gel Ex kit. Streptavidin with the size of 415 bp was amplified correctly and the DNA fragment was purified via a Gel Ex kit.

The annealed oligos were not phosphorylated. Thus additional ATP and a polynucleotide kinase (PNK) were given into the ligase reaction of the NcoI and SacI digested plasmids piGEM002, piGEM007, piGEM008 and piGEM009 with the annealed oligos iGEM071/iGEM072 that form the IPTG inducible promoter. The correct relations between vector and insert were calculated according to the formula, where V(T) is the total volume of added DNA, L(P) is the length of the plasmid, L(I) is the length of the insert, C(x) for the concentration of the plasmid and the insert and R is the ratio between vector and insert, which was set as 3 in this case.

The reaction was incubated for one hour at room temperature and subsequently used for the transformation of competent E. coli XLIBlue. These cells were plated out on LB-ampicillin and incubated at 37°C over night.

To introduce the IPTG inducible promoter into the killswitch module I it had to be amplified again with a new primer, which created an overhang of the antiholin gene to the new promoter. The already built KSI with the too short promoter was used as a template for the PCR.

The expected band in the size of ca 1350 bp was visible. The whole reaction was separated on a 1% agarose gel, the right band was cut out and the fragment was purified with a Gel Ex kit.

The purified Hag-D2-Strep which was left from crystallization and the purified Hag-Strep were used to make a pulldown with Streptavidin beads. We wanted to see if the Strep-Tag in the flagellin monomers can interact with the Streptavidin beads.

• Pd-column + bead-slurry/ 1 min. 4000 rpm, RT
• Add 400 µL GeFi(old) - wash/ 1 min. 4000 rpm, RT
• Discard Flow through, add 400 µL GeFi(old) + Protein → 20 min on Turning wheel at RT
• 1 min at 4000 rpm, RT
• 2 x 400 µL "Strep-wash"
• Elution with 40 µL 'Strep-Elution'
• 5 min. incubation at RT
• 1 min. 4000 rpm at RT, Elution in 10 µL Loading Buffer in 1,5 mL Eppi
• Remaining on filter resuspended in 40 µL water + 10 µL SDS loading buffer
  ( 5µL unstained protein ladder was used)

The Strep-pulldown showed us that the Strep-Tag interacts fine with the Streptavidin-beads. The next step would be to make a competitive pulldown to test the interaction between Hag-(D2-)Strep and StrepDARPidin.

Added two bricks to the registry to get numbers for the shipment of the samples.

Fill 20µl in PCR Cup, add lab tape with Biobrick number, wrap in parafilm, place in 50 ml falcon.

Sequencing of 1C3-Hag-KpnI-Cup-1 clone 3 is positive, can also be sent to the registry.

Inoculate 16 clones from the Gibson assembly (1.10.) in Lb-Cm for plasmid preparation and test restriction.

Small colonies on the Gibson transformation plates are visible in the morning; further incubation until colonies can be inoculated in LB-Cm. Plasmids will then be isolated from the cultures and digested with PstI in order to analyze the removal of the PstI restriction site in the insert.

The only plate that was covered with transformands was XLIBlue piGEM002+lac. The ligation with piGEM007+lac yielded in no transformands at all, piGEM008+lac showed two colonies and piGEM009+lac only one. The low number of transformands probably correlated with the low amount of vector used for the ligation. For the further ligations the vectors were used directly after the restriction and heat inactivation of the enzymes, without further purification with a Gel Ex kit.

A colony PCR was carried out.

The colony PCR for positive clones showed no results. It was repeated for piGEM002+lac and nine further clones were picked.

This time bands of the expected size (745 bp) could be noticed. Clone 14 was picked and restreaked on a LB-Amp plate.

Bacillus cereus gDNA was received from ZIEL, TU München. Based on this genomic DNA the promoter should be amplified with primers that lead to overhangs containing a SacI and a NcoI restriction site.

The amplification of the constitutive promoter worked (gel photo at 13.103). The fragment was in the size between 200 and 300 bp like expected (260 bp).

In order to fuse the genes for streptavidin and cup, which both had overhangs for each other, a Gibson assembly was carried out. Two ratios of DNA addition were tested: equal volumes of streptavidin and cup were added to the Gibson mix and the other ratio was calculated according to the formula in 13.102 with R = 1. A third Gibson reaction was prepared with the digested vector pET16b, since it was thought, the primers for strep-cup were designed with overhangs to this vector. Although the vector maps show only the overhangs with restriction sites for NcoI and SacI the reaction was prepared with the vector as well.

The Gibson assembly was separated on a 1 % agarose gel and in addition to the bands of the single parts streptavidin (cup went out of the gel due to its small size) a very light band in the right size of 561 bp was visible. Since the yield of correct product was so low, the Gibson reaction was repeated double, separated on an agarose gel, cut out and purified via Gel Extraction.

In order to check the interaction of StrepDARPidin with the EpCAM on A549 Lung Carcinoma cells we planned an ELISA-Like assay again. Yesterday row F 1-12 of a black Fluotrac600 96-well plate was coated with 100000 cells A549/ well (counted with Neubauer-Chamber) overnight at 37°C. 100000 cells of 3T3 fibroblasts were used as negative control and as well used to coat well G6.

We intended to incubate them with different concentrations of StrepDARPidin and then target the N-terminal His-Tag with and Anti-His antibody Alexa488 conjugated (1:50 endvolumen).

In wells H1-6 gel filtration purified StrepDARPidin with 87 µM was tested, in well H 7-12 Ni-NTA purified StrepDARPidin with 280 µM.

Following concentrations of StrepDARPidin dissolved in PBS (pH 7,4) +2,5 % glycerin were used:

The 96-well plate was taken out of the incubator and the medium was carefully discarded. 100 µL of the dilutions were transferred into the wells and incubated for 45 min. at room temperature under the Flow hood.

An Anti-His-Alexa488 conjugate (1 mg/mL stock) with 5 µL concentrate was dissolved in 40 µL  PBS. 1 µL of the antibody was added after washing to each well and incubated for 20 min at room temperature in the dark.

As a negative control t-cells clones were used and incubated the same way with 1 µM StrepDARPidin and 1 µL antibody (Well G8). Well G5 was left empty, well G7 was filled with 100 µL PBS+Glycerin and G4 with PBS+2,5 % glycerin with 1 µL Antibody. As negative Bacillus Subtilis WT culture was used which was already lysed causing the huge signal. A different EpCAM-negative cell line has to be used.

After 20 min incubation the wells were washed carefully with 100 µL PBS to wash out unbound elements.

The plate was measured in the ELISA reader:

We are able to see that the fluorescence signal decreases with increasing dilution. Unfortunately the negative control was very high as well. The t-cells were incubated in Eppis because of their non-adherent features. They were incubated with StrepDARPidin and Antibody before. The supernatant was discarded after centrifugation at 1500x g but the cells did not stay at the pellet’s position so that it was not possible to remove the whole preincubated material which might be left when measuring the control.

Additionally it is noticeable that there is the same trend for both purification methods. The lower signal from NTA-purified StrepDARPidin can be explained by the fact that the concentration was higher but the purity also lower so that even less StrepDARPidin was found in the protein aliquods. As negative Bacillus Subtilis WT culture was used which was already lysed causing the huge signal. A different EpCAM-negative cell line has to be used.

For the next measurement row C of the plate was coated with 100000 A549 cells & Caco-2 cells per well overnight and the same amount of 3T3 fibroblasts as negative control.

The purified Hag-D2-Strep which was left from crystallization and the purified Hag-Strep were used to make a pulldown with purified StrepDARPidin. We wanted to see if the Strep-Tag in the flagellin monomers can interact with the StrepDARPidin, especially with the Streptavidin part.

• 40 µL (105,5 µM) StrepDARPidin + Hag-(D2-)Strep à PRECIPITATES!
• Hag-(D2-)Strep & StrepDARPidin in 1xPBS+2,5% glycerine stable.
• 15 min, 13000 RPM-1min
• Supernatant 1:2 and 1:10 on SDS-PAGE, samples heated 5 min at 95°C
• Pellet resuspended in 10 µL LB

The competitive pulldown shows that just a small amount of protein remains in the supernatant. Hag-D2-Strep seems to be more stable than Hag-Strep. The highest amount of protein precipitates. We have assumed that the Strep-Tag/ Streptavidin-binding is very strong and causes maybe misfolding of the flagellin after binding which causes the precipitation. We plan to isolate the flagella in order to see if the same reaction takes place.

To 2ml of culture of Bacillus subtilis (OD=1.01) 120µl H20 and 80 µl were added for lysation. 10 µl of the lysate supplied with Roti-Load (Roth, K 929.1) were loaded on a 12% polyacrylamide gel. The electrophoresis ran at max. 130 V. After finishing, the gel was blotted onto PVDF membrane (6 cm * 9 cm) at 4°C at 250mA for about 1.5 hours. Then, the membrane was washed in BSA solution (3% BSA in TBS/Tween-20, Roth, 9127.1) over night at 4°C, before it was treated with the strep-antibody (Precision Protein StrepTactin-HRP Conjugate (Bio Rad, #161-0381): 1:5000) for 1 hr at RT. The membrane was washed 3 times for 10 minutes in TBS/Tween-20. The membrane was rinsed for 1 minute with ECL solution with a chemiluminescent substrate, which was detected by a Fusion Fx Vilber Lourmat imaging system.

It could be seen that in comparison to the wildtype the Hag-D2-Strep strain gave a positive signal for the StrepTag, which was incorporated into the flagellin.

Plasmid preparation of the inoculated E. coli cultures was performed after over night incubation. 500-600 ng of all plasmids have been digested with PstI for 2h (37°C). In case the PstI restriction site is removed from the insert, only one fragment (linearized plasmid) should occur in the gel. If the mutagenesis PCR failed and the restriction site is still inside the plasmid 2 fragments (3000bp + 470 bp) should occur in the gel.

The result of the restriction was analyzed on a 1% agarose gel:

Negative clones: 1.2, 1.4, 2.3 and 2.9

Only one fragment should occur in the gel in case of PstI linearization.

The purified Gibson assembly product was used as a template for further amplification of the StrepCup construct.

In addition to the expected band at 541 bp a smaller on was visible. The fragments of the right size were cut out, pooled and purified with a Gel Ex kit.

Since the last ligation yielded in barely any colonies on the plates, the vectors were not further purified after restriction with NcoI and SacI, which would result in a high loss of DNA. The ligation of piGEM007, piGEM008, piGEM009 with the IPTG inducible and the strong constitutive promoter and piGEM002 with the strong constitutive promoter was carried out. The DNA ratios were calculated according to the previously used formula (13.102).

The reactions were incubated at room temperature for one hour and subsequently used to transform E. coli XLIBlue cells that were plated out on LB-ampicillin plates and incubated at 37°C.

The dried samples were analysed under a Laser scanning microscope Zeiss LSM Series.

The dried samples were analysed under a Laser scanning microscope Zeiss LSM Series.

The microscopy pictures show that there is a colocalization of StrepDARPidin and membrane-associated F-actin. The Anti-His-Alexa488 antibody (green) stains the StrepDARPidin incubated A540 and Caco-2 cells generating a high intensity fluorescence signal at the cell membrane. Unspecific His-staining was detected in the nucleus of all cell lines both in presence and absence of StrepDARPidin. The negative control 3T3 fibroblasts stay unstained at the membrane.

We were able to prove that the StrepDARPidin is binding specifically to EpCAM-positive cells.

Eight colonies were visible on the trafo plate. The clones were screened for the positive plasmid construct by a colony PCR.

Fragments of the expected size (561 bp) could be seen for nearly all clones except clone 7 (see gel at 22.19). These clones were assumed to be positive. For further work clone 6 was chosen.

This time, a simple fusion PCR was performed to fuse the separate parts of the killswitch module I together, the primers were included directly and the PCR reaction was started as usual.

The fusion PCR of KSI did not work. The reaction just containing the primer iGEM034 and 037, which anneal to the outer sides of the amyE flanks yielded in no amplificate at all. The addition of primers iGEM075 and 076 that are the overhangs between the separate modules resulted in a band in the height of ca. 500 bp, which probably was the amplificated amyE flank I_lac fragment.

New test restriction with only 200 ng plasmid DNA since the last restriction showed a lot of undigested DNA. In order to test for the removal of the PstI restriction site 8 clones will be digested with PstI and PstI/EcoRI.

PstI restriction should lead to a linearized plasmid, i>EcoRI/PstI shows the expected size of 1500 and 2000 bp. Additionally some undigested plasmid is left in the PstI restriction.

• Contamination of plasmid? New digestion with new batch of PstI and EcoRI.
• PstI restriction lead to a linearized plasmid and PstI/EcoRI double digest lead to two fragments of a size of 1400 and 2000 bp
• Due to limited time clones 1.1 and 2.1 will be sent for sequencin

Result: PstI is contaminated with i>EcoRI.

On the plate with the transformed PY79 was a colony visible, which probably was tansformed with piGEM035 (nose plasmid without ssrA-tag and Cu sensitive promoter). The colony was restraked on LB-chloramphenicol and used for a colony PCR to check the correct transformation. For checking the insertion into the amyE locus, the colony was streaked onto LB-starch agar together with the wildtype.

The wildtype PY79 was taken as a negative control, the PY79 that had already been transformed with piGEM034 as a positive control with the primer iGEM057 for the Ag promoter. Since not even the positive control was positive, the reaction should be defective. The PCR was repeated and the amount of colony taken to boil it in 1x PBS was slightly lower, so there was no turbidity visible in the PBS.

Sequencing results of both plasmids are positive. Send to registry tomorrow.

see above

Clone 6 was picked to inoculate a miniprep in LB-ampicillin. The plasmid was isolated after 8 hours of incubation at 37°C. A control restriction was carried out with the enzymes NcoI, EcoRI and PstI; a negative clone would result in bands of the size of 394 bp, 748 bp and 4569 bp; a positive clone would yield bands in size of 748 bp, 865 bp and 4569 bp. Lane 2 showed the fragments in the expected size.

One plasmid with a metal sensitive promoter was still missing: piGEM008 + Cu promoter. A Gibson assembly with the digested vector and the Copper sensitive promoter was carried out in two parallels: one with purified vector (low concentration) and one with unpurified vector, only heat inactivated after restriction (high concentration).

The reaction was incubated at room temparature for 15 seconds and on 50°C for one hour. The whole reaction mixes were used to transform competent E. coli XLIBlue cells, which were plated out on LB-ampicillin plates.

The ligation with the constitutive promoter was carried out with unpurified vector.

The reactions were incubated for one hour at room temperature and used to transform competent E. coli XLIBlue cells, which were plated out on LB-ampicillin and incubated at 37°C.

Since the first attempts to combine the modules for KSI did not work, a simple Gibson assembly was performed with the separate parts. One reaction also contained the primers iGEM075 and iGEM076 which should introduce the overhangs between the parts.

This time Caco-2 (C1-6) and A549 (C7-12) were used to coat row C of a black 96-well plate (100000 cells/ well). 100000 cells of 3T3 fibroblasts were used as negative control and as well used to coat 2 wells (G10 & G11) which was done overnight.

The same protocol like in 22.5 was used or GeFi-purified StrepDARPidin.

Following concentrations of StrepDARPidin dissolved in PBS (pH 7,4) +2,5 % glycerin were used:

The serial dilution shows that the signal is decreasing for both cell lines. The Caco-2 Cells have a higher signal. The experiment should be repeated.

The plates all carried sufficient colonies to pick up to five clones per transformand. A colony PCR was carried out with specific primers for the constitutive promoter (piGEM073) and the reverse primer for the gfp with or without the degradation tags.

As a positive control E. coli DH5α with the nose plasmids containing the metallosensitive promoter were used. This control only acted as a control for the right vector, since no plasmid was available with the constitutive promoter, which could be used as positive control for the promoter. For every construct positive clones were visible except for piGEM009 + const. The positive fragment for the constitutive promoter should be at ca 1000 bp and the positive construct of piGEM008 + Cu should yield a 923 bp fragment. Both of the fragments could be seen on the gel. Further work was done with the clones 4 of piGEM002 + const, 1 of piGEM007 + const, 5 of piGEM008 + const and 3 of piGEM008 + Cu.

To transform Bacillus a huge amount of plasmid is needed. For this reason 100 ml culture with E. coli for every nose plasmid with a metallosensitive promoter was harvested and the plasmids were isolated according to the protocol in the Qiagen Plasmid Plus Maxi Kit. One exception was made, since no QIAvac plus 24 was available and no vacuum pump. Thus the filtered cell lysate was loaded onto the  columns by centrifugation.

Preparation of both bricks:

• 15 µl plasmid DNA (app. 200 ng/µl) in PCR cup
• Tape with Biobrick number around the PCR cup
• Parafilm wrapped around the whole Eppendorf cup
• 50 ml Falkon with Shipping number, team name and shipping date

Both fragments could be amplified (in a duplicate) and will be used for further cloning processes. The gel for the PheA domain did not run straight, but the approximate expected size can be seen.

The iGEM vector pSB1C3 and the fragments resulting from the previous PCR will be digested with XbaI and PstI for a following ligation and transformation.

Digestion at room temperature over night.

The plasmids isolated with the maxi prep kit were used to transform competent Bacillus subtilis PY79. Ca 15 µg of each plasmid were given to 100 µl of cells in a test tube, which was incubated shaking at 37°C for 30 min before plating the cells out on LB-chloramphenicol plates.

The plasmids isolated with the maxi prep kit were used to transform competent Bacillus subtilis PY79. Ca 15 µg of each plasmid were given to 100 µl of cells in a test tube, which was incubated shaking at 37°C for 30 min before plating the cells out on LB-chloramphenicol plates.

30 ml LB-ampicillin were inoculated with cells of E. coli BL21(DE3) pET16b_StrepCup from plate. This culture was incubated shaking at 37°C until they reached an OD₆₀₀ of 0.58. A preinduction sample was taken (1.2 ml = (0.7 * 0.7) / (0.7 * 0.58)), the cells were pelleted by centrifugation at 14000 rpm for 1 minute, followed by resuspension in 80 µl water and 20 µl 5X SDS loading dye. The culture was induced with 30 µl IPTG for 3 hours. Another sample was taken afterwards, at this time point the culture reached an OD₆₀₀ of 1.2 (0.583 ml = (0.7 * 0.7 / 0.7 * 1.2)). These samples were boiled at 95°C for 10 minutes and then analyzed by SDS-PAGE (12% SDS-polyacrylamide gel).

It could be noticed that there was a difference between the preinduction and the induction sample. According to the Expasy Compute pI/Mw tool StrepCup should be around 18.6 kDa. The protein expressed was slightly bigger, but considering the protein page ruler is just an orientation guide and not 100% accurate the thick band in the induction sample was determined as StrepCup (ca 20 kDa).

Since there could have been a confusion after isolating the plasmids, which could have been the reason for the last negative control PCR on the lac plasmids, every reverse primer for the nose plasmids with the ssrA-tags and without the degradation tag were tested. The following table went for every of the nose plasmids with the lac-promoter, not only for the shown piGEM002 + lac In addition to that, a control PCR was performed with the the three existing nose plasmids with constitutive promoter piGEM002/007 and 008, another control PCR was done for piGEM008 + Cu sensitive promoter, which was the last one missing of the nose-plasmids with the metallosensitive promoters.

Additionally, a colony PCR was made with further 6 clones picked from the ligation plate for piGEM009 + const to check for positive clones.

For the colony PCR several positive clones could be seen that exposed a band in the height of ca 1000 bp, clone 9 was chosen to work with in the further steps. The control PCR on the nose - lac plasmids was negative for all plasmids. Some showed two very thin bands of ca 750 bp and 1000 bp size. The expected fragment should have a size of approx. 818 bp. These findings were striking, since the colony PCR for the clones from which the plasmids were isolated were positive.

The control PCR for the plasmids piGEM002/007 + const and piGEM008 + Cu were positive. The clones containing these plasmids were used to inoculate 100 ml LB+ampicillin for a maxi prep.

The colony PCR for the clones containing the nose plasmids were positive but the control PCR with the isolated plasmids were negative. Since colony PCRs are a less reliable method than the control PCR on the isolated plasmid or a control restriction, the plasmids should be negative. To be sure a control restriction with BamHI and HindIII was carried out with these plasmids. The positive plasmids should result in fragments of 4194 bp and 2620 bp size, the negative plasmid should not be digested by HindIII, because the only restriction site for HindIII is in the IPTG inducible promoter. As a control piGEM030 was cut with HindIII and NcoI (1172 bp, 5246 bp and 241 bp) and piGEM008 + const was cut with BamHI and NcoI (4189 bp + 2757 for a positive plasmid, 4189 bp and 2518 bp for a negative plasmid).

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

Additionally a new colony PCR was done with new picked clones from the ligation plates of the plasmids with the lac promoter.