Team:Marburg:Project:Notebook:October

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Notebook: October

01.10.2014

18.72 Isolation of flagella from Hag-D2-Strep

Aim: Isolation of filaments for assays with StrepDARPidin

After the purification steps until the ammonium sulfate precipitation a SDS-gel with commassie stain was done and WT3610 as well as Hag-D2_3 from Florian Altegoer were used as a control.

The gel shows that there is a line after the ammonium sulfate precipitation which is running to low to be flagellin. Florian Altegoer made several tries with the same result so that we think of instabile flagellins. He gave us a PY79 strain with a Strep-Tag integrated into the normal Hag-gene for isolation of flagella. They might be more stabile than Hag-D2-Strep filaments.

25.4 Fluorometer assay with A549

Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells

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:

Content Well
Col
Well Row Blank corrected raw data (485, 520) Purification StrepDARPidin concentration [M]
Neg. Control 2 G 14073 - -
Blank B 3 G   - -
Antibody signal 4 G 249059 - -
A549 1 H 10418 GeFi 2 µM
A549 2 H 19020 GeFi 1 µM
A549 3 H 6673 GeFi 100 nM
A549 4 H 8559 GeFi 10 nM
A549 5 H 5743 GeFi 100 pM
A549 6 H 9033 GeFi 10 pM
A549 7 H 9090 Ni-NTA 2 µM
A549 8 H 8796 Ni-NTA 1 µM
A549 9 H 5356 Ni-NTA 100 nM
A549 10 H 6684 Ni-NTA 10 nM
A549 11 H 6930 Ni-NTA 100 pM
A549 12 H 10237 Ni-NTA 10 pM

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.

23.35 Mutagenesis PCR of pSB1C3 Hag-D2-Strep clone 1 and 2

Mutagenesis Primer arrived → PCR with 1C3-Hag-KpnI-Strep clone 1 and 2 (appr. 20 ng/µl) in 30 µl reaction

Plasmid 1
Water 13,2
Phusion Buffer (5x) 6
Phusion 1
dNTPs 1
Primer 81 (1:50) 3,9
Primer 82 (1:50) 3,9

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.

23.36 Gibson assembly and transformation mutated pSB1C3 Hag-D2-Strep clone 1 and 2

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.

Growing cultures for electron microscopy

Aim: cultures in different states of growth for electron microscopy of flagella

The structure of the flagella of the mutant strains Bacillus subtilis 3610 D2-Strep and Bacillus subtilis 3610 D2-cup should be investigated by electron microscopy. Thus cultures of strain WT3619, 3610 D2-Strep and 3610 D2-Cup were inoculated in 5 ml LB medium. The cultures were incubated shaking at 37°C until they reached the exponential phase with an OD600 of 0.7 - 0.8, and until the cells reached the stationary phase. The cells were kept on the same OD by storage on ice until further processing for electron microscopy.

24. Streptavidin-Cup1-1 Fusion

Aim: Fusion of Streptavidin to Cup1-1

24.1 amplification of Streptavidin and cup for Strep-cup

Aim: amplification of Strep-Cup for the fusionprotein Streptavidin-Cup1-1

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.

Mix PCR Single reaction (µl)
piGEM029 1
Primer iGEM078 (1:50) 6,25
Primer iGEM080 (1:50) 6,25
5x HF buffer 10
dNTP mix (10 mM each) 1
Phusion DNA polymerase 1:10 1
Water 24,5
Total Volume 50

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

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

Mix PCR Single reaction (µl)
piGEM029 1
Primer iGEM077 (1:50) 6,25
Primer iGEM079 (1:50) 6,25
5x HF buffer 10
dNTP mix (10 mM each) 1
Phusion DNA polymerase 1:10 1
Water 24,5
Total Volume 50

Step Temperature °C Time
1 98 5 min
2 98 30 sec
3 55 30 sec
4 72 40 sec
5 Go To 2 34x
6 72 5 min
7 4 infinite

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.

13.102 Ligation of piGEM002/007/008/009 with the IPTG inducible promoter

Aim: insertion of the promoter into the nose plasmids

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.

Component Ligation I (µl) Ligation II (µl) Ligation III (µl) Ligation IV (µl)
piGEM-002 (281 ng/µl) 7,9 - - -
piGEM007 (ca 30 ng/µl) - 9,7 - -
piGEM008 (ca 30 ng/µl) - - 9,2 -
piGEM009 (ca 30 ng/µl) -   - 9,5
IPTG inducible promoter (iGEM071/ iGEM072) (54.7 ng/µl) 2,1 0,3 0,3 0,3
ATP (10mM) 1 1 1 1
PNK 0,5 0,5 0,5 0,5
T4 Ligase 1 1 1 1
Ligation Buffer 10x 2 2 2 2
Water 5,5 5,5 5,5 5,5
Total Volume 20 20 20 20

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.

22.17 amplification of KSI with amyE flankII and overhang to the IPTG inducible promoter

Aim: create an overhang in front of the antiholin gene for the modified lac promoter

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.

Mix PCR (µl) Single reaction (µl)
module KSI (amyEI-antiholin-amEII) 1
Primer iGEM037 (1:50) 6,25
Primer iGEM075 (1:50) 6,25
5x HF buffer 10
dNTP mix (10 mM each) 1
Phusion DNA polymerase 1:10 1
Water 24,5
Total Volume 50

Step Temperature °C Time
1 98 5 min
2 98 30 sec
3 55 30 sec
4 72 90 sec
5 Go To 2 34x
6 72 5 min
7 8 infinite

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.

02.10.2014

21.6 new Strep-Bead pulldown

Aim: Checking interactions between Hag-(D2-)Strep and Streptavidin beads

The purified Hag-D2-Strep which was left from crystallization and Florian Altegoer’s 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.

  A280 MW [kDA] Ex.coeff. Conc. [mM]
FliS/ Hag-D2-Strep 28,8 60 23000 0,9 (0,9 nmol/µL)
FliS/ Hag-Strep 206 47,7 17420 1,18 (1,18 nmol/ µL)

  1 2 3 4 5 used
GST-bead x     x x 20 µL slurry
Strep-bead   x x     30 µL slurry
Hag-D2-Strep x   x   x 4 nmol → 4,5 µL
Hag-Strep   x   x   4 nmol → 3,4 µL
  • 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 wash(Strep-wash from Wieland Steinchen)
  • Elution with 40 µL “Strep-Elution” (From Wieland Steinchen)
  • 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)

  Sample
1 Strep-beads without protein
2 Strep-beads + Hag-Strep
3 Strep-beads + Hag-D2-Strep
4 GST-beads + Hag-Strep
5 GST-beads + Hag-D2-Strep
6 Strep-beads + Hag-Strep
7 Strep-beads + Hag-D2-Strep
8 GST-beads + Hag-Strep
9 GST-beads + Hag-D2-Strep

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.

23.37 Addition of the first two biobricks iGEM Team Marburg to the registry

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

Part Number Description
Part:BBa_K1329000 StrepDARPidin
Part:BBa_K1329001 Hag-KpnI

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

23.38 Sequencing results of 1C3-Hag-KpnI-Cup-1

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

23.39 Analyze successful Gibson assembly of Hag-D2-Strep clone 1 and 2 (mutagenesis)

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.

13.103 Screening of the transformands from the ligation of the nose plasmids with the IPTG inducible promoter

Aim: screening for positive clones with the finished plasmids

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.

Mix colony PCR Single reaction
Template part of a colony
Primer iGEM006 (1:10) 1
Primer iGEM071 (1:10) 1
5x HF buffer 4
dNTP mix (10 mM each) 0,5
Phusion DNA polymerase 1:10 1
Water 12,5
Total Volume 20

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

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

Mix colony PCR Single reaction
Template part of a colomy
Primer iGEM006 (1:50) 3,125
Primer iGEM059 (1:50) 3,125
5x HF buffer 4
dNTP mix (10 mM each) 0,3
Phusion DNA polymerase 1:10 1
Water 8,45
Total Volume 20

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

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

13.104 amplification of the strong constitutive promoter for Gram-positives from gDNA of Bacillus cereus

Aim: amplification of the constitutive promoter

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.

Mix PCR Single reaction (µl)
gDNA Bacillus cereus (1:10) 1
Primer iGEM073 (1:50) 6,25
Primer iGEM074 (1:50) 6,25
5x HF buffer 10
dNTP mix (10 mM each) 0,5
Phusion DNA polymerase 1:10 1
Water 25
Total Volume 50

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

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).

24.2 Gibson assembly with Streptavidin and cup

Aim: Fusion of the genes of streptavidin and cup

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.

Component equal amounts volume (µL) equal amounts DNA (µl) with vector (µl)
streptavidin (49.5 ng/µl) 2,5 1,17 0,5
cup (4.9 ng/µl) 2,5 3,83 1,5
pET16b NcoI/SacI - - 3
Gibson mix 15 15 -
Total Volume 20 20 -

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.

25.5 new Fluorometer assay with A549

Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells using a new serial dilution

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:

Content Well
Col
Well Row raw data (485, 520) Purification StrepDARPidin concentration [M]
A549 1 F 5782 GeFi 50 µM
A549 2 F 4912 GeFi 25 µM
A549 3 F 3903 GeFi 2,5 µM
A549 4 F 3513 GeFi 250 nM
A549 5 F 3613 GeFi 25 nM
A549 6 F 3211 GeFi 2,5 nM
A549 7 F 7340 Ni-NTA 50 µM
A549 8 F 5329 Ni-NTA 25 µM
A549 9 F 5846 Ni-NTA 2,5 µM
A549 10 F 5718 Ni-NTA 250 nM
A549 11 F 3852 Ni-NTA 25 nM
A549 12 F 6828 Ni-NTA 2,5 nM
Empty well 5 G 5699 - -
negative control 3T3 fibroblasts 6 G 9983 - -
Blank 7 G 1760 - -
positive control AB 8 G 60182 - -

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.

03.10.2014

21.7 competitive Strep-Bead pulldown

Aim: Checking interactions between Hag-(D2-)Strep and StrepDARPidin

The purified Hag-D2-Strep which was left from crystallization and Florian Altegoer's 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.

  A280 MW [kDA] Ex.coeff. Conc. [M]
FliS/ Hag-D2-Strep 28,8 60 23000 0,9 mM(0,9 nmol/µL)
FliS/ Hag-Strep 206 47,7 17420 1,18 Mm (1,18 nmol/ µL)
StrepDARPidin 6,061 31,8 57410 105,5 µM

  1 2 3 4 5 used
StrepDARPidin     X X X 4 nmol → 40 µl
Hag-D2-Strep   X   X   2 nmol → 2,25 µl
Hag-Strep X   X     2 nmol → 1,7 µl
  • 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

  Sample
1 Hag-Strep
2 Hag D2-Strep
3 Hag Strep + StrepDARPidin
4 Hag-D2-Strep + StrepDARPidin
5 StrepDARPidin
6 Hag-Strep + StrepDARPidin [1:5]
7 Hag-D2-Strep + StrepDARPidin [1:5]
8 Hag-Strep + StrepDARPidin [1:1]
9 Hag-D2-Strep + StrepDARPidin [1:1]

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.

21.8 Western blot with anti-Strep-Tag-AB

Aim: Checking interactions between Hag-D2-Strep & Anti-Strep-Tag-AB

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.

23.40 Plasmid isolation and test restriction of Hag-D2-Strep clone 1 and 2 (mutagenesis)

Aim: Analyze the removal of the PstI restriction site

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.

  1x 18x
Water 14,5 261
CutSmart Buffer (10x) 2 36
PstI 0,5 9
Plasmid 3  

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

Negative clones: 1.2, 1.4, 2.3 and 2.9

BUT: Only one fragment should occur in the gel in case of PstI linearization, why 2??

24.3 amplification of StrepCup from the purified Gibson fragment

Aim: increase the amount of StrepCup to work on

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

Mix PCR Single reaction (µl)
StrepCup (1.5 ng/µl) 3
Primer iGEM077 (1:50) 6,25
Primer iGEM080 (1:50) 6,25
5x HF buffer 10
dNTP mix (10 mM each) 0,5
Phusion DNA polymerase 1:10 1
Water 23
Total Volume 50

Step Temperature °C Time
1 98 5 min
2 98 30 sec
3 55 30 sec
4 72 50 sec
5 Go To 2 34x
6 72 5 min
7 8 infinite

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.

13.105 repeated restriction of the nose plasmids and ligation with the lac promoter and the constitutive promoter

Aim: insert the different promoters into the plasmids for further characterization

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).

Component Ligation I (µl) Ligation II (µl) Ligation III (µl) Ligation IV (µl) Ligation V (µl) Ligation VI (µl) Ligation VII (µl)
piGEM-002 (281 ng/µl) 6,577            
piGEM007 (ca 100 ng/µl)   7,6     8,6    
piGEM008 (ca 100 ng/µl)     7,16     8,3  
piGEM009 (ca 100 ng/µl)       6,76     8
IPTG inducible promoter (iGEM071/ iGEM072) (54.7 ng/µl)         1,4 1,7 2
constitutive promoter (63.2 ng/µl) 3,43 2,4 2,84 3,24      
ATP (10mM) 0,5 0,5 0,5 0,5 0,5 0,5 0,5
PNK 0,5 0,5 0,5 0,5 0,5 0,5 0,5
T4 Ligase 1 1 1 1 1 1 1
Ligation Buffer 10x 2 2 2 2 2 2 2
Water 6 6 6 6 6 6 6
Total Volume 20 20 20 20 20 20 20

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.

04.10.2014

22.17 amplification of amyE-flankI with overhang to the IPTG inducible promoter

Aim: introduce a compatible overhang for fusion PCR/Gibson

In order to fuse the parts of KSI together, analogue overhangs on each part were needed. Thus, amyE flank I was amplified anew with the new primers and the old flank as template.

Mix PCR Single reaction (µl)
amyE flank I 1
Primer iGEM034 (1:50) 6,25
Primer iGEM076 (1:50) 6,25
5x HF buffer 10
dNTP mix (10 mM each) 0,5
Phusion DNA polymerase 1:10 1
Water 25
Total Volume 50

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

The amyE flank with the overhang to the promoter was amplified successfully. The reaction mix was purified with an Omega Gel Extraction kit.

22.18 FusionPCR/Gibson of amyE-flank I, lac promoter and antiholin-amyE-flank II

Aim: Fusion of the single parts to create KS module I

The parts were ready for the assembly. The same FusionPCR/Gibson assembly mixture was used like the last time to build KSI (22.10). The primers were added to the reaction after a first step of incubation at 50°C for 0.5 hours. Then the PCR was carried out as usual.

Mix PCR Single reaction (µl)
amyE flank I (ca 10 ng/µl) 1
IPTG inducible promoter (iGEM071/072; ca 10 ng/µl) 1
antiolin-amyE flank II (10 ng/µl) 1
Primer iGEM034 (1:50) 6,25
Primer iGEM076 (1:50) 6,25
5x HF buffer 10
dNTP mix (10 mM each) 0,7
Phusion DNA polymerase 1:10 1
Water 22,8
Total Volume 50

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


The fusion of the parts with this method did not work. Instead of a distinct band, a smear over the whole lane was visible.

24.4 Restriction of StrepCup and Ligation with pET16b

Aim: insert the StrepCup construct in the overproduction plasmid pET16b

The purified StrepCup fragment was digested with the enzymes NcoI and XhoI, since the Gibson assembly of Strep and Cup with the vector yielded in only one transformand, which was considered as a spontaneous mutation in relation to the control plate, which carried one colony more.

Component Attempt I (µL)
StrepCup 170 ng/µl) 15
NcoI 0,5
XhoI 0,5
Cutsmart Buffer 10x 2
Water 2
Total Volume 20

The reaction was incubated for one hour at 37°C and inactivated at 85°C for 20 minutes afterwards.

The ratio of DNA addition to the ligation was again calculated according to the formula from 13.102.

Component Ligation I (µl)
StrepCup NcoI/XhoI (170.8 ng/µl) 0,5
pET16b NcoI/XhoI (18.1 ng/µl) 16
ATP (10mM) 0,5
PNK 0,5
T4 Ligase 0,5
Ligation Buffer 10x 2
Total Volume 20

The reaction was incubated at room temperature for 1 h and was afterwards completely used to transform cells of E. coli XLIBlue, which were plated out on LB-ampicillin.

13.106 Screening of transformands after ligation of nose plasmids with the lac promoter and the constitutive promoter

Aim: check for correct plasmids

Colony PCRs were performed to screen the clones for the right plasmids. Every plate carried enough colonies to pick multiple clones. Five clones of every plate was picked and this time instead of using the common gfp-reverse primer iGEM006 the primers specific for the degradation tags of the different nose plasmids were used. The ligation with the constitutive promoter should be repeated, since the fragment was not digested with NcoI and SacI before.

Mix PCR 02+const 07+const 08+const 09+const 07+lac 08+lac 09+lac
Template colony colony colony colony colony colony colony
iGEM073 (1:50) 3,125 3,125 3,125 3,125      
iGEM059 (1:50)         3,125 3,125 3,125
iGEM006 (1:50) 3,125            
iGEM021 (1:50)   3,125     3,125    
iGEM022 (1:50)     3,125     3,125  
iGEM023 (1:50)       3,125     3,125
5x HF buffer 4 4 4 4 4 4 4
dNTP mix (10 mM each) 0,3 0,3 0,3 0,3 0,3 0,3 0,3
Phusion DNA polymerase 1:10 1 1 1 1 1 1 1
Water 8,45 8,45 8,45 8,45 8,45 8,45 8,45
Total Volume 20 20 20 20 20 20 20




The ligation with the constitutive promoter had to be repeated, since it was not digested before and could not be ligated correctly. The bands visible on the gel were all of the wrong size, the expected fragment would have a size of 1039 bp. Theoretically, there should not be an amplificate without insertion of the promoter but the primers probably bound randomly. However, some clones with the lac promoter were positive (745 bp), even when the bands were very light. Some clones additionally exhibited other fragments which were probably the result of unspecific primer annealing. The normal plasmids piGEM007, piGEM008 and piGEM009 were used as a positive control for the reverse primers with the ssrA-tags.

25.6a Immunofluorescence microscopy at AG Grosse lab

Aim: Checking the interaction of StrepDARPidin with EpCAM-positive cell lines A549 & Caco-2 via immunofluorescence Antibody staining

We wanted to see if our StrepDARPidin construct binds to EpCAM localized in the cell membrane by staining the cells with an Anti-His-Antibody conjugated with Alexa488 after incubation with our StrepDARPidin which should bind to the N-terminal His-Tag of the StrepDARPidin. DAPI was used to stain the cell core and Phalloidin-Rhodamin to stain the membrane-associated F-actin.

Cover slips fitting into the wells of a 24-well plate were coated in water with gelatin overnight at 4°C. A549 & Caco-2 cells were cultured 2 days before in a small T25 culture flask in DMEM+10% /20% FCS + L-Glutamine until full confluence was reached.

The cells were trypsinized and counted. The cover slips were incubated at 37°C for an hour with 100000 cells per cover slip in medium so that the cells were able to attach to the slips. After an hour the medium was discarded and the slips were washed with PBS several times and the cells observed in the microscope.

 150 µL of a 50 µM StrepDARPidin solution in PBS with 2,5% Glycerin (to keep the protein stable) were used to cover the wells with A549 and Caco-2 cells for half an hour at room temperature. As a negative control we used cells not incubated with the protein.

After discarding the protein solution and wash with PBS the cells were fixed with 3,7% formaldehyde dissolved in PBS for 5 min at room temperature.

After washing PBS+5%BSA was used to block unspecific binding (20 min +wash). The cells were permeabilized with Triton X-100 0,1% in PBS for a minute.

For the staining an Penta-Anti-His-Alexa488 conjugate (QIAGEN) 1:25 (0,2mg/ mL) was used in combination with Phalloidin-Rhodamin (1:200, 300 units stock) and 20 nM DAPI. After washing the slips they were mounted with DePex and dried overnight in the fridge. The microscopy was done with Matthias Plessner (AG Grosse).

05.10.2014

25.6b Immunofluorescence microscopy at AG Grosse lab

Aim: Checking the interaction of StrepDARPidin with EpCAM-positive cell lines A549 & Caco-2 via immunofluorescence Antibody staining

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.

24.5 Screening of transformands from Gibson of StrepCup and pET16b

Aim: check for positive clones

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

Mix colony PCR Single reaction
Template part of a colony
Primer iGEM077 (1:50) 3,125
Primer iGEM080 (1:50) 3,125
5x HF buffer 4
dNTP mix (10 mM each) 0,3
Phusion DNA polymerase 1:10 1
Water 8,45
Total Volume 20

Step Temperature °C Time
1 98 10 min
2 98 30 sec
3 55 30 sec
4 72 50 sec
5 Go To 2 34x
6 72 5 min
7 8 infinite

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.

22.19 FusionPCR of amyE-flank I, lac promoter and antiholin-amyE-flank II

Aim: Fusion of the single parts to create KS module I

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.

Mix PCR Single reaction (µl) Single reaction with overhang primers
amyE flank I (ca 10 ng/µl) 1 1
IPTG inducible promoter (iGEM071/072; ca 10 ng/µl) 1 1
antiholin-amyE flank II (10 ng/µl) 1 1
Primer iGEM034 (1:50) 6,25 6,25
Primer iGEM037 (1:50) 6,25 6,25
Primer iGEM075 (1:50) - 6,25
Primer iGEM076 (1:50) - 6,25
5x HF buffer 10 10
dNTP mix (10 mM each) 0,7 0,7
Phusion DNA polymerase 1:10 1 1
Water 22,8 22,8
Total Volume 50 50

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

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.

23.41 Repeat test restriction

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.

  1x 18x
Water 16,5 148,5 / 144,0
CutSmart Buffer (10x) 2 18
PstI 0,5 4,5
EcoRI 0,5 4,5
Plasmid 1  

PstI restriction should lead to a linearized plasmid, 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 restriction with PstI of AG Graumann and EcoRI of AG Bange.
  • 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 EcoRI and will be thrown away!

06.10.2014

13.107 colony PCR on transformed Bacillus subtilis PY79

Aim: check for positive transformation

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.

Mix colony PCR Single reaction
Colony in 100 µl PBS 5
Primer Flo 56 (1:50) 6,25
Primer Flo 54 (1:50) 6,25
5x HF buffer 10
dNTPs (40 mM) 0,5
Phusion DNA polymerase 1:10 1
Water 21
Total Volume 50

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

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.

23.42 Sequencing of 1C3 Hag-KpnI- D2-Strep
Label number Plasmid Primer (Premix)
AGB0023-564 1C3 Hag-KpnI- D2-Strep 1.1 iGEM 53
AGB0023-565 1C3 Hag-KpnI- D2-Strep 1.1 iGEM 54
AGB0023-566 1C3 Hag-KpnI- D2-Strep 2.1 iGEM 53
AGB0023-567 1C3 Hag-KpnI- D2-Strep 2.1 iGEM 54
23.43 Analysis of the sequencing of 1C3 Hag-KpnI- D2-Strep

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

23.44 Addition of the two new biobricks to the registry (short/long description and sequence)

see above

24.6 Miniprep and restriction of pET16b_StrepCup

Aim: check the plasmid for correct insertion of StrepCup

Clone 6 was picked to inoculate a miniprep in LB-ampicillin. The plasmid was prepped 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.

13.108 Gibson assembly of piGEM008 + Cu promoter and ligation of the nose plasmids with the constitutive promoter

Aim: insert the different promoters into the nose plasmids

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).

Component Gibson purified vector (µL) Gibson unpurified vector (µl) Control  (µL)
Purified piGEM008 (89.6 ng/µL) 4   4
Unpurified piGEM008 (196.7 ng/µl)   3,2  
Cu promotor 1 1  
Gibson mix 15 15 15
Total Volume 20 20 20

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.

Component Ligation I (µl) Ligation II (µl) Ligation III (µl) Ligation IV (µl)
piGEM-002 (201.8 ng/µl) 6,77      
piGEM007 (ca 143.4 ng/µl)   7,4    
piGEM008 (ca 196.7 ng/µl)     6,8  
piGEM009 (ca 218.3 ng/µl)       6,6
constitutive promoter (49.3 ng/µl) 3,23 2,6 3,2 3,4
T4 Ligase 1 1 1 1
Ligation Buffer 10x 2 2 2 2
Water 7 7 7 7
Total Volume 20 20 20 20

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.

22.20 Gibson assembly with separate KSI parts

Aim: Fusion of the single parts to create KS module I

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.

Component Gibson (µL) Gibson with additional primer (µl)
amyE flank I (ca 10 ng/µl) 1,6 1
IPTG inducible promoter (iGEM071/072; ca 10 ng/µl) 1,6 1
antiholin-amyE flank II (10 ng/µl) 1,6 1
Primer iGEM075 (1:50)   0,5
Primer iGEM076 (1:50)   0,5
H2O   1
Gibson Mix 15 15
Total Volume 20 20

Applied onto a gel nothing could be seen.

25.7 new Fluorometer assay with A549 and Caco-2

Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells & Caco-2 cells using a new serial dilution

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:

Content Well Col Well Row raw data (485, 520) Purification StrepDARPidin concentration [M]
Caco-2 1;1 C 344 - 25 µM
Caco-2 2;2 C 538 - 2.5 µM
Caco-2 3;3 C 386 - 250 nM
Caco-2 4;4 C 319 - 25 nM
Caco-2 5;5 C 281 - 2.5 nM
Caco-2 6;6 C 259 - 250 pM
A549 7;7 C 422 - 25 µM
A549 8;8 C 396 - 2,5 µM
A549 9;9 C 273 - 250 nM
A549 10;10 C 247 - 25 nM
A549 11;11 C 288 - 2,5 nM
A549 12;12 C 272 - 250 pM
Blank 5;9 G 197 - -
negative control 3T3 fibroblast 6;10 G 355 - 25 µM
negative control 3T3 fibroblast 7;11 G 305 - 25 µ
positive AB-control 8,12 G 3950 - -

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.

07.10.2014

13.109 Screening for positive transformands from the Gibson and ligation

Aim: check for positive insertion of the promoters

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.

Mix PCR 02+const 07+const 08+const 09+const 08+Cu
Template colony colony colony colony colony
iGEM055 (1:50)         3,125
iGEM073 (1:50) 3,125 3,125 3,125 3,125  
iGEM006 (1:50) 3,125        
iGEM021 (1:50)   3,125      
iGEM022 (1:50)     3,125   3,125
iGEM023 (1:50)       3,125  
5x HF buffer 4 4 4 4 4
dNTP mix (10 mM each) 0,3 0,3 0,3 0,3 0,3
Phusion DNA polymerase 1:10 1 1 1 1 1
Water 8,45 8,45 8,45 8,45 8,45
Total Volume 20 20 20 20 20

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

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.

13.110 Plasmid Maxi Prep of Nose plasmids with Ag and Cu promoter

Aim: Obtain enough plasmid to transform Bacillus subtilis

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 prepped 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.

08.10.2014

23.45 Sending the two new biobricks to the registry

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
Part Number Name
Part:BBa_K1329000 StrepDARPidin
Part:BBa_K1329001 Hag-KpnI
13.111 Bacillus Transformation with isolated plasmids

Aim: Transformation of Bacillus subtilis PY79 with the nose-plasmids

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.

Control PCR with nose-lac plasmids

check the plasmids for correct insertion of the lac promoter

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.


Mix PCR 02 + lac °C 07 + lac 08 + lac 09 + lac
piGEM002/7/8/9 + lac (ca 40 ng/µl) 1,5 1,5 1,5 1,5
iGEM073 (1:50) 3,125 3,125 3,125 3,125
iGEM006 (1:50) 3,125 - - -
iGEM021 (1:50) - 3,125 - -
iGEM022 (1:50) - - 3,125 -
iGEM023 (1:50) - - - 3.125
5x HF buffer 10 10 10 10
dNTP mix (10 mM each) 0,5 0,5 0,5 0,5
Phusion DNA polymerase 1:10 1 1 1 1
Water 23.5 23.5 23.5 23.5
Total 50 50 50 50

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

The PCR did not work. The high bands hat could be seen were probably the plasmid templates.

09.10.2014

24.7 Test expression of StrepCup in E. coli BL21(DE3)

Aim: check the production of StrepCup

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).

13.113 control PCR of nose plasmids with lac promoter / constitutive promoter

Aim: check nose - lac/const plasmids for correct insert

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.


Mix PCR 02+lac with iGEM006 02+lac with iGEM021 02+lac with iGEM022 02+lac with iGEM023 02+const 08+Cu cPCR 09 + const
colony - - - - - - part
piGEM002 (ca 40 ng/µl) 1 1 1 1 - - -
piGEM002/007/008 + const. %#040;ca 40 ng/µl) - - - - 1 - -
piGEM008 + Cu - - - - - 1 -
iGEM059 (1:50) 3,3 3,3 3,3 3,3 - - -
iGEM073 (1:50) - - - - 3,3 - 3,125
iGEM055 (1:50) - - - - - 3,3 -
iGEM006 (1:50) 3,3 - - - 3,3 - -
iGEM021 (1:50) - 3,3 - - - - -
iGEM022 (1:50) - - 3,3 - - 3,3 -
iGEM023 (1:50) - - - 3,3 - - 3,125
5x HF buffer 5 5 5 5 5 5 5
dNTP mix (10 mM each) 0,3 0,3 0,3 0,3 0,3 0,3 0,3
Phusion DNA polymerase 1:10 1 1 1 1 1 1 1
Water 11,1 11,1 11,1 11,1 11,1 11,1 11,1
Total 25 25 25 25 25 25 25

PCR Step Temperature °C Time
1 98 5 min/ colony PCR separate: 10 min
2 98 30 sec
3 55 30 sec
4 72 70 sec
5 Go To 2 34x
6 72 5 min
7 4 infinite


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 ca 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.

13.114 control restriction of nose plasmids + lac and new colony PCR of picked clones

Aim: verify the absence of the lac promoter in the nose plasmids like indicated by the control PCR

The colony PCR for the clones containing the nose plasmids were positive but the control PCR with the prepped 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 restricted 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).

Component piGEM030 (µL) lac plasmids (µL) piGEM008 + const (µL)
piGEM030 5 5 5
nose plasmids + lac promoter - 5 -
piGEM008 + const - - 5
NcoI-HF 0,5 - 0,5
HindIII 1 1 -
BamHI-HF - 0,5 0,5
CutSmart 10x 2 2 2
Water 11,5 11,5 12
Total 20 20 20

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.

Mix colony PCR piGEM002 + lac (µL) piGEM007 + lac (µL) piGEM008 + lac (µL) piGEM009 + lac (µL) Positive control piGEM002 + Ag (µL)
Template part of a colony part of a colony part of a colony part of a colony part of a colony
iGEM073 (1:50) 3,125 3,125 3,125 3,125 -
iGEM057 (1:50) - - - - 3,125
iGEM006 (1:50) 3,125 - - - 3,125
piGEM021 + const - 3,125 - - -
piGEM022 + const - - 3,125 - -
piGEM023 + const - - - 3,125 -
5x HF buffer 5 5 5 5 5
dNTP mix (10 mM each) 0,3 0,3 0,3 0,3 0,3
Phusion DNA polymerase 1:10 1 1 1 1 1
Water 12,45 12,45 12,45 12,45 12,45
Total 20 20 20 20 20

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


The control restriction of the nose plasmids was negative, the plasmid just seemed to be linearized and not cut into two fragments. That means that the HindIII could not cut since the IPTG inducible promoter was not contained in the plasmids. However, also the control piGEM030 did not show the expected bands. It could be possible that HindIII did not work correctly, since it was no HF enzyme and used in the CutSmart buffer of NEB, but according to the NEB Double Digest Finder HindIII was able to cut in CutSmart only with the half of its optimal activity. That was the reason, why the double amount of HindIII was chosen.

Some samples of the new colony PCR showed bands slightly higher than 750 bp, which would resemble the size of the expected fragment of ca 800 bp, but since the controls with the nose plasmids containing the Ag sensitive promoter (all confirmed positive by control PCR on isolated plasmids) were not all positive, it was uncertain, that the visible fragments really were the ones that indicate a positive clone. For the further cloning, the plasmids were isolated and digested directly.

25.9 new Fluorometer assay with A549 and Caco-2

Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells & Caco-2 cells using a new serial dilution

This time Caco-2 (A1-6) and A549 (B1-6) were used to coat row C of a black 96-well plate (100000 cells/ well). 100000 cells of 3T3 WT fibroblasts were used as negative control and as well used to coat C5.

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:

Content Well Col Well Row Blank corrected raw data (85, 520) StrepDARPidin concentration (M)
Caco-2 1 A 1096 25 µM
Caco-2 2 A 380 25 µM
Caco-2 3 A 745 2,5 µM
Caco-2 4 A 565 2,5 µM
Caco-2 5 A 539 250 nM
Caco-2 6 A 635 250 nM
Caco-2 7 A 300 25 nM
Caco-2 8 A 280 25 nM
Caco-2 9 A 1063 2,5 nM
Caco-2 10 A 1024 2,5 nM
Caco-2 11 A 328 250 pM
Caco-2 12 A 488 250 pM
A549 1 B 377 25 µM
A549 2 B 444 25 µM
A549 3 B 365 2,5 µM
A549 4 B 563 2,5 µM
A549 5 B 255 250 nM
A549 6 B 294 250 nM
A549 7 B 376 25 nM
A549 8 B 330 25 nM
A549 9 B 1244 2,5 nM
A549 10 B 531 2,5 nM
A549 11 B 1049 250 pM
A549 12 B 864 250 pM
Blank 1 C 216 -
Empty Well 2 C 767 -
Empty Well 3 C 848 -
Pos. control AB 4 C 4873 -
negative control 3T3 fibroblasts 5 C 512 25 µM

The serial dilution shows that the signal is decreasing for both cell lines. The Caco-2 Cells have a higher signal compared to A549.

10.10.2014

24.8 Test for solubility of StrepCup

Aim: check if StrepCup is soluble or enclosed in inclusion bodies

A second test expression was carried out in 30 ml LB-amp, which was inoculated with E. coli BL21(DE3) and incubated until they reached an OD600 of 0.62. A 1.12 ml preinduction sample was taken and the cells were induced with 30 µl IPTG for 3 hours shaking at 37°C. After this time they reached an OD600 of 1.4, so 500 µl were taken as an induction sample. The taken samples were pelleted by centrifugation, resuspended in 80 µl of water with addition of 20 µl 5x SDS loading dye. The cells were centrifuged at 4000 rpm for 20 minutes and lysed by the microfluidizer. The cell lysate was centrifuged at 20000 rpm for 20 minutes. The supernatant was withdrawn and 80 µl were taken as a sample, which was treated with 20 µl of 5x SDS loading dye. The pellet was resuspended in some buffer A from which 80 µl were taken and 20 µl of 5x SDS loading dye were added. The samples were boiled at 95°C for 10 minutes. Afterwards 10 µl of each sample was loaded onto a 12% SDS polyacrylamide gel.



It could clearly be seen that the StrepCup was in the unsoluble fraction of the lysate.

13.115 Ligation of piGEM008/009 with constitutive promoter

Aim: ligate the restricted vector with the constitutive promoter

Since the control PCR for piGEM008 with the constitutive promoter was negative and not even the colony PCR for piGEM009 with the constitutive promoter was positive, the ligation with these two plasmids was repeated.

Component Ligation I (µl) Ligation II (µl)
piGEM008 (ca 196.7 ng/µl) 5.2 -
piGEM009 (ca 218.3 ng/µl) - 3.05
constitutive promoter (ca 49.3 ng/µl) 2.3 4.45
T4 ligase 0.5 0.5
Ligation buffer 10x 2 2
Water 10 10
Total 20 20

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

Additionally, clones were picked from the old ligation plates to inoculate cultures of LB-ampicillin for plasmid preps.

11.10.2014

25.10 new Fluorometer assay with A549 and Caco-2

Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells & Caco-2 cells using a new serial dilution

This time Caco-2 (F1-6( and A549 (G1-6) were used to coat row C of a black 96-well plate (100000 cells/ well). 100000 cells of 3T3WT fibroblasts were used as negative control and as well used to coat H2 & H3.

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

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


Content Well Col Well Row Blank corrected raw data (485, 520) StrepDARPidin concentration (M)
Caco-2 1 F 382 25 µM
Caco-2 2 F 556 2,5 µM
Caco-2 3 F 550 250 nM
Caco-2 4 A 522 25 nM
Caco-2 5 F 541 2,5 nM
Caco-2 6 F 548 250 pM
A549 1 G 407 25 µM
A549 2 G 491 2,5 µM
A549 3 G 557 250 nM
A549 4 G 359 25 nM
A549 5 G 338 2,5 nM
A549 6 G 632 250 pM
negative control 3T3WT fibroblasts 2 H 409 25 µM
negative control 3T3WT fibroblasts 3 H 468 2,5 µM
Pos. control AB 4 H 3786 -

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

After the previous measurements the results were summarized and a graphic created:



The tendency which could be seen in the previous assays can be reproduced after summarizing the previous measurements as well.

13.116 Making new competent Bacillus subtilis PY79 cells

Aim: making competent cells to transform Bacillus subtilis PY79 with the Nose-plasmids

Since the previous cells were not competent, a new try to make competent Bacillus subtilis PY79 was carried out. The procedure was performed according to the protocol using the SPC- and SPII-media. This time the cells were not centrifuged at 4°C but at room temperature, which should be gentler for the cells, since they do not need to induce cold stress responses that could affect the competence. The cells were tested for transformation efficiency by transforming them with each 25 µl and 50 µl of each nose plasmid with a metallosensitive promoter. After adding the plasmid DNA to 200 µl cells in a test tube, they were incubated shaking a 37°C for 30 minutes. Afterwards they were plated out on LB-chloramphenicol (5 µg/ml) were incubated at 37°C.

13.117 control restriction of nose plasmids with the constitutive promoter

Aim: check the plasmids for the correct insertion of the promoter

The plasmids were isolated from the inoculated cultures (13.115; 2 clones piGEM002 + const, 6 clones piGEM007 + const, 5 clones piGEM008 + const and 4 clones piGEM009 + const) and restricted with the enzymes NcoI-HF and EcoRI-HF. Negative plasmids should yield fragments in size of 476 and 6190 (for piGEM002) or 6235 bp (for piGEM007/008/009). The positive plasmids should result in fragments in the size of 711 and 6190/6235 bp.


Component Volumes (µl)
piGEM002/007/008/009 + const (ca 400 ng/µl) 5
NcoI-HF 0.5
EcoRI-HF 0.5
CutSmart 10x 2
Water 12
Total 20

The reactions were incubated at 37°C for over one hour, 3 µl 6x Purple Loading Dye were added and 7 µl of the restrictions were applied onto a 1% agarose gel.



The undigested piGEM002 was taken as a control. The isolated plasmids from the piGEM002 + const ligation plates did not yield in two fragments but look linearized. The clones 5 and 6 of piGEM007 + const, 4 and 5 of piGEM008 + const were positive. No plasmid of piGEM009 + const was positive.

The plasmids piGEM002 + const and piGEM007 + const were already confirmed as positive by the control PCR (13.113), now piGEM008 + const was positive as well, only piGEM009 + const was missing, so 10 clones from the old and new ligation plates were picked and used to inoculate more cultures LB-amp for plasmid isolation.

12.10.2014

13.118 control restriction of piGEM009 with the constitutive promoter

Aim: check the plasmid for the ccorrect insertion of the promoter

The plasmids were isolated from the cultures (13.117) and restricted again with the enzymes NcoI and EcoRI. The expected fragments were the same like in 13.117.

Component Volumes (µl)
piGEM009 + const (200-400 ng/µl) 5
NcoI-HF 0.5
EcoRI-HF 0.5
CutSmart 10x 2
Water 12
Total 20

The reactions were incubated at 37°C for over one hour, 3 µl 6x Purple Loading Dye were added and 7 µl of the restrictions were applied onto a 1% agarose gel.



The O‘ Gene Ruler 1 kb DNA ladder of Thermo Scientific was used as a marker. All cut fragments were in the size of ca. 500 bp (and the backbone at around 6000 bp), which meant, the plasmids did not contain the constitutive promoter.

13.10.2014

18.74 Isolation of flagella from different Bacillus subtilis strains

Aim: separation of the flagella from the cells

Since the flagella of the Bacillus subtilis mutants Hag-D2-Strep or Hag-Strep were needed for affinity assays together with the StrepDARPidin and the cancer cell lines, the flagella should be isolated. Based on the pictures of the electron microscopy it was observed, that the Hag-D2-Cup strain formed flagella, which are shorter than these of the wildtype, which could mean that they break. However, the mutant flagellin should be expressed and so the Cup on the flagella should be able to fish for ions. In order to confirm that, the flagella of D2-Cup should be isolated as well. 1L LB each were inoculated with Bacillus subtilis 3610wt, Hag-D2-Strep, Hag-D2-Cup and Py79 Hag-Strep in 5 L Erlenmeyer flasks with baffles and incubated shaking at 37°C over night.

24.9 Expression and purification of StrepCup

Aim: expression of Streptavidin-Cup

The Streptavidin-Cup construct sould also be overproduced, purified and tested for ion binding capacity or interaction with StrepTags of the flagella mutants. For this reason 2x 1 L LB-ampicillin was inoculated with E. coli BL21(DE3) pET16b_StrepCup. The medium also contained 50 ml 25 % (w/v; 12.5 g/50ml) lactose as an inducer of the expression. The cultures were incubated shaking at 30°C over night.

14.10.2014

24.9 Expression and purification of StrepCup

Aim: purification of StrepCup

The two 1 L cultures were harvested by centrifugation at 4000 rpm for 20 min. The pellets were resuspended in 10 ml buffer A and centrifuged again in a falcon at 4000 rpm for 15 min. Again the pellet was resuspended in 25 ml buffer A and the cells were broken by a microfluidizer. The lysate was centrifuged with 20000rpm for 20 minutes at 4°C. Samples were taken from the pellet (P1) and the supernatant (S1), which was stored on ice. The pellet was resuspended in IB-wash buffer and centrifuged again at 27000 rpm for 10 minutes. This washing step was repeated. Samples were taken from the pellet (P2) and the supernatant (S2). The pellet was covered in 5 ml 6 M guanidine-hydrochloride and stirred at room temperature until it was completely resuspended, followed by another centrifugation with 20000 rpm at 4°C for 10 min. The pellet (P3) and the supernatant (S3) were stored separately in the refrigerator at 4°C over night after taking samples of both. It was observed that the proteins contained by the supernatant were precipitating instantly as they came in contact with water to dilute the sample and/or SDS loading dye. The precipitate was rich of protein, which resulted in an almost solid, sludgy consistence. After several dilution attempts the sample was diluted 1:1000 in water (protein precipitated by contact with water) the sample was vortexed until the precipitate was gone and 80 µl were taken together with 20 µl of 5x SDS loading dye and bolied at 95°C for 10 min together with the other samples, from which none precipitated as intense as the supernatant of the gua pellet. 10 µl of all samples were transferred onto a 12 % SDS-polyacrylamide gel.



The produced StrepCup was in the unsoluble phase like expected. During the process of isolating StrepCup from the inclusion bodies it could be seen, that StrepCup always remained in the pellet. It was not possible to see the protein in the soluble phase of the guanidinium-hydrochloride treated pellet, since the previous mentioned problems of instant precipitation in contact with water or SDS loading dye.

18.74 Isolation of flagella from different Bacillus subtilis strains

Aim: separation of the flagella from the cells