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Latest revision as of 19:56, 17 October 2014

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 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.

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 pSB1C3-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 DNA-polymerase	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 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.

	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 "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)

	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 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.

	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

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

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
i>EcoRI	0,5	4,5
Plasmid	1

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.

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 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.

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 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.

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

23.46 PCR amplification of the Arc1p-C domain fragment and the PheA domain fragment

Aim: Fragments are needed for the cloning of two new biobricks

	Arc1p (µl)	PheA (µl)
Water	24	24
HF Buffer 5x	10	10
Phusion DNA-polymerase	1	1
Primer for (1:50)	6.5	6.5
Primer rev (1:50)	6.5	6.5
Template (20 ng/µl)	1	1
dNTP mix (10 mM each)	1	1

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.

23.47 Restriction of pSB1C3 and the amplified fragments with XbaI and PstI

Aim: Digested plasmid and DNA fragments are needed

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.

Reaction Mix	pSB1C3 (µl)	Arc1p (µl)	PheA (µl)
PstI	0.5	0.5	0.5
XbaI	0.5	0.5	0.5
Buffer 2.1 10x	2.0	2.0	2.0
Water	14	12	9

Digestion at room temperature over night.

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 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.

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 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).

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.

23.48 Purification of digested plasmid and fragments

Aim: In order to improve the ligation results pure DNA samples are needed

All digested samples have been purified via gel extraction, resulting in the following concentration:

pSB1C3= 19 ng/µl
Arc1p= 25 ng/µl
PheA= 23 ng/µl

23.49 Ligation of digested plasmid and inserts

Aim: Ligate plasmids for further transformation

Ligation Mix	pSB11C3 + Arc1p (µl)	pSB11C3 + PheA (µl)
Water	0	4.3
HF Buffer 10x	2.0	2.0
T4 Ligase	1.0	1.0
Insert	7.6	9
Vector	9.4	3.7

The ligation was performed at 16°C for 3h.

23.50 Transformation of competent E. coli XL-1-Blue with ligated plasmid

Aim: Ligate plasmids for further transformation

After the ligation the reaction was inactivated by incubating the reaction mix at 85%deg;C for 10 minutes. Afterwards chemically competent E. coli XL-1-blue were transformed with the whole reaction mix. Standard E. coli transformation protocol was used.

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 digested 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.

23.51 Inoculation of presumably positive clones from the ligation plates

Aim: Grow cultures for the isolation of plasmid psB1C3 with the two new Biobricks

Two clones for every biobrick plasmid were inoculated in liquid LB-CM (10:30) for plasmid isolation.

23.52a Plasmid isolation and test digest

Aim: Analyze the plasmids for the insertion of the fragments into the plasmid

Plasmid isolation of the pSB1C3 plasmid from E. coli was performed with the Quiagen Mini-prep kit. Plasmid concentration was determined with a Nanodrop spectrophotometer (100-120 ng/µl).

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

Component	Restriction mix (µl)
Water	12.0
CutSmart 10x	2.0
XbaI	0.5
PstI	0.5
Plasmid (600 ng/µl)	5.0

Gel analysis of the digested plasmids reveals that all clones are negative, new ligation will be performed with a higher amount of insert.

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 digested 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.

23.53 New Ligation and transformation

Aim: Analyze the plasmids for the insertion of the fragments into the plasmid

In order to calculate the needed amount of insert and vector the iGEM ligation calculator was used. For this reaction a higher amount of insert was used (1:5)

Ligation Mix	pSB11C3 + Arc1p (µl)	pSB11C3 + PheA (µl)
Water	0.6	5.5
HF Buffer 10x	2.0	2.0
T4 Ligase	1.0	1.0
Insert	9.0	9.0
Vector	7.4	2.5

The ligation was performed at 16°C for 3 hours.

After the ligation the reaction was inactivated by incubating the reaction mix at 85°C for 10 minutes. Afterwards chemically competent E. coli XL-1-blue were transformed with the whole reaction mix. Standard E. coli transformation protocol was used.

12.10.2014

13.118 control restriction of piGEM009 with the constitutive promoter

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

The plasmids were isolated from the cultures (13.117) and digested 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.

23.51b Inoculation of presumably positive clones from the ligation plates

Aim: Grow cultures for the isolation of plasmid psB1C3 with the two new Biobricks

Two clones for every biobrick plasmid were inoculated in liquid LB-CM for plasmid isolation.

23.52b Plasmid isolation and test restriction

Aim: Analyse the plasmids for the insertion of the fragments into the plasmid

Plasmid isolation of the pSB1C3 plasmid from E.coli was performed with the Quiagen Mini-prep kit. Plasmid concentration was determined with a Nanodrop spectrophotometer (90-135 ng/µl).

Component	Restriction mix (µl)
Water	12.0
CutSmart 10x	2.0
XbaI	0.5
PstI	0.5
Plasmid (600 ng/µl)	5.0

Both plasmids have been cloned successfully as visible in the two agarose gels. The expected inserts could be cut out of the iGEM plasmid pSB1C3.

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.

23.53 Sequencing of BBa-K1329004 and BBa-K1329005

Aim: check for the correct sequence of the biobricks

Sequencing samples have been prepared in a concentration of 50 - 100 ng/µl and primers were added. Plasmids were then sequenced by eurofins mwg.

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.

18.74a Isolation of flagella from different Bacillus subtilis strains

Aim: separation of the flagella from the cells

Strains: Wildtype 3610, PY-Strep, Hag-D2-Strep, Hag-D2-Cup

The different cultures were harvested by centrifugation at 4000 rpm for 20 minutes. The pellets were resuspended in 30 ml 0.1 M TRIS/HCl buffer pH 8.0 containing 0.5 % Brij-58. 330 µl of a 10 mg/ml lysozyme solution were added and the lysis was performed in a roller at 4°C until the samples were mostly clear.

20 µl of RNase free DNase was added and lysis continued for another 30 min. The bacterial debris was removed by centrifugation at 10.000 rpm for 10 min, followed by an ultracentrifugation step of the supernatant at 100.000 rpm for 90 min (4°C).

For the analysis of flagella via SDS-PAGE 1 ml with OD₆₀₀ 0.7 was prepared for every sample by centrifugation and subsequent heating to 95°C (10 min). Cell pellets were centrifuged and resuspended in 40 µL water and 10 µL SDS loading dye (5x).

Calculated size of flagellin is about 37 kDa, running slightly higher than the fourth band of the unstained protein marker (first line). Flagellin is present in the wildtype sample before and after cell lysis. Other proteins can be seen for PY-Strep before (higher than Hag) and after (lower than Hag) cell lysis. The two Hag constructs with the inserted D2 domain run a bit higher than wildtype flagellin and are present after cell lysis in a small amount.

Flagellin is present in the wildtype supernatant before and after cell lysis. Before lysis no flagellin can be seen in the supernatant of the other three strains. Differing proteins bands can be seen for these three strains in the supernatant after cell lysis.

A high amount of flagellin is present after cell lysis of wildtype B. subtilis. A thin band can be seen for Hag-D2-Cup before cell lysis and a thicker band for Hag-D2-Strep that does not fit the size of flagellin. The supernatant of the ultra-centrifugation step was transferred to a new falcon and the pellet was resuspended in 3 ml standard saline citrate buffer (pH 7.3) and both fractions were stored at 4°C overnight.

15.10.2014

18.74b Isolation of flagella from different Bacillus subtilis strains II

20% ammonium sulphate were added to the pellet resulting from the ultracentrifugation resuspended in standard saline citrate buffer (pH 7.3). The obtained precipitant was analysed in an SDS PAGE gel.

A high amount of flagellin is present in the ultracentrifugation pellet of the wildtype cells after lysis. A mixture of proteins can be seen for Hag-D2-Strep after cell lysis. It can be assumed that flagellin hag-D2-Strep is partially unstable. Anyway, the analysis of presumably isolated flagella was continued.

16.10.2014

23.54 Sequencing results

Both plasmid have been sequenced and the correct inserts are in the iGEM backbone pSB1C3.

23.55 Send Biobricks to 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_K1329004	synthetic catalyst part 1: tRNA scaffold
Part:BBa_K1329005	Synthetic catalyst part 2

18.75 Investigation the interaction between the isolated flagella and StrepDARPidin

Aim: A possible interaction of the flagella with StrepDARPidin should be visualized by electron microscopy

In order to visualize the interaction of the StrepDARPidin, the protein was incubated with the precipitated flagella and subsequently stained for electron microscopy.

Samples prepared:

1 = Hag-D2-Strep (1.2 mg/ml)
2 = Wildtype (2 mg/ml)
3 = PY-Strep (0.4 mg/ml)
4 = Hag-D2-Strep + StrepDARPidin (1:10)
5 = Wildtype+ StrepDARPidin (1:10)
6 = PY-Strep+ StrepDARPidin (1:10)
7 = StrepDARPidin (7 mg/ml)

Number	Flagellin	StrepDARPidin	Buffer (Saline citrate)
1	50 µl	-	50 µl
2	200 µl	-	-
3	80 µl	-	20 µl
4	3.5 µl	10 µl	36.5 µl
5	17.5 µl	10 µl	50 µl
6	6 µl	10 µl	34 µl
7	-	10 µl	40 µl

Incubation on ice for 19 minutes before samples were stained for electron microscopy.

@@ Line 20: / Line 20: @@
 	</div>
 	<div class="exp-content">
-	<p>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. </p>
+	<p>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. </p>
      <img src="https://static.igem.org/mediawiki/2014/2/2c/MR_2014-10-01_18.72.jpg" width="30%" />
-     <p>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.</p>
+     <p>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 <i>B. subtilis</i> 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.</p>
 	</div>
 </fieldset>
-<fieldset class="exp22">
+<fieldset class="exp25">
-     <legend><a name="exp22.4c">22.4 Fluorometer assay with A549</a></legend>
+     <legend><a name="exp25.4c">25.4 Fluorometer assay with A549</a></legend>
 	<div class="aim">
 	<p>Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells</p>
@@ Line 70: / Line 70: @@
    </tr>
    <tr>
-     <th scope="row">Sample X1</th>
+     <th scope="row">A549</th>
      <td>1</td>
      <td>H</td>
@@ Line 78: / Line 78: @@
    </tr>
    <tr>
-     <th scope="row">Sample X2</th>
+     <th scope="row">A549</th>
      <td>2</td>
      <td>H</td>
@@ Line 86: / Line 86: @@
    </tr>
    <tr>
-     <th scope="row">Sample X3</th>
+     <th scope="row">A549</th>
      <td>3</td>
      <td>H</td>
@@ Line 94: / Line 94: @@
    </tr>
    <tr>
-     <th scope="row">Sample X4</th>
+     <th scope="row">A549</th>
      <td>4</td>
      <td>H</td>
@@ Line 102: / Line 102: @@
    </tr>
    <tr>
-     <th scope="row">Sample X5</th>
+     <th scope="row">A549</th>
      <td>5</td>
      <td>H</td>
@@ Line 110: / Line 110: @@
    </tr>
    <tr>
-     <th scope="row">Sample X6</th>
+     <th scope="row">A549</th>
      <td>6</td>
      <td>H</td>
@@ Line 118: / Line 118: @@
    </tr>
    <tr>
-     <th scope="row">Sample X7</th>
+     <th scope="row">A549</th>
      <td>7</td>
      <td>H</td>
@@ Line 126: / Line 126: @@
    </tr>
    <tr>
-     <th scope="row">Sample X8</th>
+     <th scope="row">A549</th>
      <td>8</td>
      <td>H</td>
@@ Line 134: / Line 134: @@
    </tr>
    <tr>
-     <th scope="row">Sample X9</th>
+     <th scope="row">A549</th>
      <td>9</td>
      <td>H</td>
@@ Line 142: / Line 142: @@
    </tr>
    <tr>
-     <th scope="row">Sample X10</th>
+     <th scope="row">A549</th>
      <td>10</td>
      <td>H</td>
@@ Line 150: / Line 150: @@
    </tr>
    <tr>
-     <th scope="row">Sample X11</th>
+     <th scope="row">A549</th>
      <td>11</td>
      <td>H</td>
@@ Line 158: / Line 158: @@
    </tr>
    <tr>
-     <th scope="row">Sample X12</th>
+     <th scope="row">A549</th>
      <td>12</td>
      <td>H</td>
@@ Line 178: / Line 178: @@
      <legend><a name="exp23.35">23.35 Mutagenesis PCR of pSB1C3 Hag-D2-Strep clone 1 and 2</a></legend>
 	<div class="exp-content">
-	<p>Mutagenesis Primer arrived &rarr; PCR with 1C3-Hag-KpnI-Strep clone 1 and 2 (appr. 20 ng/µl) in 30 µl reaction</p>
+	<p>Mutagenesis Primer arrived &rarr; PCR with pSB1C3-Hag-<i>Kpn</i>I-Strep clone 1 and 2 (appr. 20 ng/&micro;l) in 30 &micro;l reaction</p>
      <table width="100%" border="1">
    <tr>
@@ Line 193: / Line 193: @@
    </tr>
    <tr>
-     <th scope="row">Phusion</th>
+     <th scope="row">Phusion DNA-polymerase</th>
      <td>1</td>
    </tr>
@@ Line 224: / Line 224: @@
 	</div>
 	<div class="exp-content">
-	<p>The  structure of the flagella of the mutant strains <em>Bacillus subtilis</em> 3610 D2-Strep and <em>Bacillus subtilis</em> 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&deg;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.</p>
+	<p>The  structure of the flagella of the mutant strains <em>Bacillus subtilis</em> 3610 D2-Strep and <em>Bacillus subtilis</em> 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&deg;C until  they reached the exponential phase with an OD<sub>600</sub> 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.</p>
 </div>
 </fieldset>
@@ Line 612: / Line 612: @@
 	</div>
 	<div class="exp-content">
-	<p>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 received from Wieland Steinchen. We wanted to see if the Strep-Tag in the flagellin monomers can interact with the Streptavidin beads.</p>
+	<p>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.</p>
      <table width="100%" border="1">
    <tr>
@@ Line 649: / Line 649: @@
    <tr>
      <th scope="row">GST-bead</th>
-    <td>x</td>
      <td>x</td>
      <td>&nbsp;</td>
      <td>&nbsp;</td>
-     <td>&nbsp;</td>
+     <td>x</td>
+    <td>x</td>
      <td>20 &micro;L slurry</td>
    </tr>
    <tr>
      <th scope="row">Strep-bead</th>
-    <td>&nbsp;</td>
      <td>&nbsp;</td>
      <td>x</td>
      <td>x</td>
-     <td>x</td>
+     <td>&nbsp;</td>
+    <td>&nbsp;</td>
      <td>30 &micro;L slurry</td>
    </tr>
@@ Line 671: / Line 671: @@
      <td>x</td>
      <td>&nbsp;</td>
-     <td>&nbsp;</td>
+     <td>x</td>
      <td>4 nmol &rarr; 4,5 &micro;L</td>
    </tr>
@@ Line 686: / Line 686: @@
 	<ul class="list">
      <li>Pd-column + bead-slurry/ 1 min. 4000 rpm, RT</li>
-     <li>Add 400 µL GeFi(old) - wash/ 1 min. 4000 rpm, RT</li>
+     <li>Add 400 &micro;L GeFi(old) - wash/ 1 min. 4000 rpm, RT</li>
-     <li>Discard Flow through, add 400 µL GeFi(old) + Protein &rarr; 20 min on Turning wheel at RT</li>
+     <li>Discard Flow through, add 400 &micro;L GeFi(old) + Protein &rarr; 20 min on Turning wheel at RT</li>
      <li>1 min at 4000 rpm, RT</li>
-     <li>2 x 400 &micro;L wash(Strep-wash from Wieland Steinchen)</li>
+     <li>2 x 400 &micro;L "Strep-wash" </li>
-     <li>Elution with 40 &micro;L “Strep-Elution” (From Wieland Steinchen)</li>
+     <li>Elution with 40 &micro;L 'Strep-Elution'</li>
      <li>5 min. incubation at RT</li>
      <li>1 min. 4000 rpm at RT, Elution in 10 &micro;L Loading Buffer in 1,5 mL Eppi</li>
@@ Line 774: / Line 774: @@
 	<div class="exp-content">
 	<p>Inoculate 16 clones from the Gibson assembly (1.10.) in Lb-Cm for plasmid preparation and test restriction.</p>
-	<p>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.</p>
+	<p>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 <i>Pst</i>I in order to analyze the removal of the <i>Pst</i>I  restriction site in the insert.</p>
 </div>
 </fieldset>
@@ Line 1,052: / Line 1,052: @@
 	</div>
 	<div class="exp-content">
-	<p>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.</p>
+	<p>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 <i>Nco</i>I and <i>Sac</i>I the reaction was prepared with the vector as well.</p>
      <table width="100%" border="1">
    <tr>
@@ Line 1,096: / Line 1,096: @@
 	</div>
 </fieldset>
-<fieldset class="exp22">
+<fieldset class="exp25">
-     <legend><a name="exp22.5">22.5 new Fluorometer assay with A549</a></legend>
+     <legend><a name="exp25.5">25.5 new Fluorometer assay with A549</a></legend>
 	<div class="aim">
 	<p>Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells using a new serial dilution</p>
 	</div>
 	<div class="exp-content">
-	<p>In order to check the interaction of StrepDARPidin with the EpCAM on  A549 Lung Carcinoma cells we planned an ELISA-Like assay. 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&deg;C. </p>
+	<p>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&deg;C. 100000 cells of 3T3 fibroblasts were used as negative control and as well used to coat well G6.</p>
 	<p>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). </p>
 	<p>In wells H1-6 gel filtration purified StrepDARPidin with 87 &micro;M was  tested, in well H 7-12 Ni-NTA purified StrepDARPidin with 280 &micro;M.</p>
@@ Line 1,112: / Line 1,112: @@
 Col</th>
      <th scope="col">Well Row</th>
-     <th scope="col">Blank  corrected raw data (485, 520)</th>
+     <th scope="col">raw data (485, 520)</th>
      <th scope="col">Purification</th>
      <th scope="col">StrepDARPidin  concentration [M]</th>
    </tr>
    <tr>
-     <th scope="row">Sample  X1</th>
+     <th scope="row">A549</th>
      <td>1</td>
      <td>F</td>
@@ Line 1,125: / Line 1,125: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X2</th>
+     <th scope="row">A549</th>
      <td>2</td>
      <td>F</td>
@@ Line 1,133: / Line 1,133: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X3</th>
+     <th scope="row">A549</th>
      <td>3</td>
      <td>F</td>
@@ Line 1,141: / Line 1,141: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X4</th>
+     <th scope="row">A549</th>
      <td>4</td>
      <td>F</td>
@@ Line 1,149: / Line 1,149: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X5</th>
+     <th scope="row">A549</th>
      <td>5</td>
      <td>F</td>
@@ Line 1,157: / Line 1,157: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X6</th>
+     <th scope="row">A549</th>
      <td>6</td>
      <td>F</td>
@@ Line 1,165: / Line 1,165: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X7</th>
+     <th scope="row">A549</th>
      <td>7</td>
      <td>F</td>
@@ Line 1,173: / Line 1,173: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X8</th>
+     <th scope="row">A549</th>
      <td>8</td>
      <td>F</td>
@@ Line 1,181: / Line 1,181: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X9</th>
+     <th scope="row">A549</th>
      <td>9</td>
      <td>F</td>
@@ Line 1,189: / Line 1,189: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X10</th>
+     <th scope="row">A549</th>
      <td>10</td>
      <td>F</td>
@@ Line 1,197: / Line 1,197: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X11</th>
+     <th scope="row">A549</th>
      <td>11</td>
      <td>F</td>
@@ Line 1,205: / Line 1,205: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X12</th>
+     <th scope="row">A549</th>
      <td>12</td>
      <td>F</td>
@@ Line 1,213: / Line 1,213: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X13</th>
+     <th scope="row">Empty well</th>
      <td>5</td>
      <td>G</td>
@@ Line 1,221: / Line 1,221: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X14</th>
+     <th scope="row">negative control 3T3 fibroblasts</th>
      <td>6</td>
      <td>G</td>
@@ Line 1,229: / Line 1,229: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X15</th>
+     <th scope="row">Blank</th>
      <td>7</td>
      <td>G</td>
@@ Line 1,237: / Line 1,237: @@
    </tr>
    <tr>
-     <th scope="row">Sample  X16</th>
+     <th scope="row">positive control AB</th>
      <td>8</td>
      <td>G</td>
@@ Line 1,252: / Line 1,252: @@
        <p>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&rsquo;s position so that it was not possible to remove the whole preincubated  material which might be left when measuring the control.</p>
        <p>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. </p>
+	  <p>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.</p>
 </div>
 </fieldset>
@@ Line 1,268: / Line 1,269: @@
 	</div>
 	<div class="exp-content">
-	<p>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.</p>
+	<p>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.</p>
      <table width="100%" border="1">
    <tr>
@@ Line 1,307: / Line 1,308: @@
      <td><strong>3</strong></td>
      <td><strong>4</strong></td>
+	<td><strong>5</strong></td>
      <td><strong>used</strong></td>
    </tr>
    <tr>
      <td><strong>StrepDARPidin</strong></td>
-    <td>X</td>
      <td>&nbsp;</td>
-    <td>X</td>
      <td>&nbsp;</td>
+    <td>X</td>
+    <td>X</td>
+	<td>X</td>
      <td>4 nmol &rarr; 40 &micro;l</td>
    </tr>
@@ Line 1,323: / Line 1,326: @@
      <td>&nbsp;</td>
      <td>X</td>
+	<td>&nbsp;</td>
      <td> 2 nmol &rarr; 2,25 &micro;l</td>
    </tr>
    <tr>
      <td><strong>Hag-Strep</strong></td>
-     <td>&nbsp;</td>
+     <td>X</td>
      <td>&nbsp;</td>
      <td>X</td>
-     <td>X</td>
+     <td>&nbsp;</td>
+	<td>&nbsp;</td>
      <td>2 nmol &rarr; 1,7 &micro;l</td>
    </tr>
@@ Line 1,341: / Line 1,346: @@
        <li>Pellet resuspended in 10 &micro;L LB</li>
      </ul>
+	<img src="https://static.igem.org/mediawiki/2014/b/bd/SDS_gel_competitive_Strep_beat_pulldown.jpg" width="30%" />
+	<br />
      <table width="100%" border="1">
    <tr>
@@ Line 1,348: / Line 1,355: @@
    <tr>
      <td><strong>1</strong></td>
-     <td>Strep-beads without protein</td>
+     <td>Hag-Strep</td>
    </tr>
    <tr>
      <td><strong>2</strong></td>
-     <td>Strep-beads + Hag-Strep</td>
+     <td>Hag D2-Strep</td>
    </tr>
    <tr>
      <td><strong>3</strong></td>
-     <td>Strep-beads + Hag-D2-Strep</td>
+     <td>Hag Strep + StrepDARPidin</td>
    </tr>
    <tr>
      <td><strong>4</strong></td>
-     <td>GST-beads + Hag-Strep</td>
+     <td>Hag-D2-Strep + StrepDARPidin</td>
    </tr>
    <tr>
      <td><strong>5</strong></td>
-     <td>GST-beads + Hag-D2-Strep</td>
+     <td>StrepDARPidin</td>
    </tr>
    <tr>
      <td><strong>6</strong></td>
-     <td>Strep-beads + Hag-Strep</td>
+     <td>Hag-Strep + StrepDARPidin [1:5]</td>
    </tr>
    <tr>
      <td><strong>7</strong></td>
-     <td>Strep-beads + Hag-D2-Strep</td>
+     <td>Hag-D2-Strep + StrepDARPidin [1:5]</td>
    </tr>
    <tr>
      <td><strong>8</strong></td>
-     <td>GST-beads + Hag-Strep</td>
+     <td>Hag-Strep + StrepDARPidin [1:1]</td>
    </tr>
    <tr>
      <td><strong>9</strong></td>
-     <td>GST-beads + Hag-D2-Strep</td>
+     <td>Hag-D2-Strep + StrepDARPidin [1:1]</td>
    </tr>
 </table>
-	<p>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. </p>
+	<p>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.</p>
 </div>
+</fieldset>
+<fieldset class="exp21">
+	<legend><a name="exp21.8">21.8 Western blot with anti-Strep-Tag-AB</a></legend>
+	<div class="aim">
+	<p>Aim: Checking interactions between Hag-D2-Strep & Anti-Strep-Tag-AB</p>
+	</div>
+	<div class="exp-content">
+	<p>To 2ml of culture of Bacillus subtilis (OD=1.01) 120&micro;l H20 and 80 &micro;l were added for lysation. 10 &micro;l 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&deg;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&deg;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.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/3/36/Western_Blot_AntiStrep.png" width="30%" />
+	<br />
+	<p>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.</p>
+	</div>
 </fieldset>
 <fieldset class="exp23">
      <legend><a name="exp23.40">23.40 Plasmid isolation and test restriction of Hag-D2-Strep clone 1 and 2 (mutagenesis)</a></legend>
 	<div class="aim">
-	<p>Aim: Analyze the removal of the PstI restriction site </p>
+	<p>Aim: Analyze the removal of the <i>Pst</i>I restriction site </p>
 	</div>
 	<div class="exp-content">
-	<p>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.</p>
+	<p>Plasmid preparation of the inoculated E. coli cultures was performed after over night incubation. 500-600 ng of all plasmids have been digested with <i>Pst</i>I for 2h (37&deg;C). In case the <i>Pst</i>I 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.</p>
      <table width="100%" border="1">
    <tr>
@@ Line 1,410: / Line 1,430: @@
    </tr>
    <tr>
-     <th scope="row">PstI</th>
+     <th scope="row"><i>Pst</i>I</th>
      <td>0,5</td>
      <td>9</td>
@@ Line 1,423: / Line 1,443: @@
      <img src="https://static.igem.org/mediawiki/2014/6/62/MR_2014-10-03_23.40.png" width="40%" />
      <p>Negative clones: 1.2, 1.4, 2.3 and 2.9</p>
-     <p>BUT: Only one fragment should  occur in the gel in case of PstI linearization, why 2??</p>
+     <p>Only one fragment should  occur in the gel in case of <i>Pst</i>I linearization.</p>
 </div>
 </fieldset>
@@ Line 1,525: / Line 1,545: @@
 	</div>
 	<div class="exp-content">
-	<p>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).</p>
+	<p>Since the last ligation yielded in barely any colonies on the plates, the vectors were not further purified after restriction with <i>Nco</i>I and <i>Sac</i>I, 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).</p>
      <table width="100%" border="1">
    <tr>
@@ Line 1,766: / Line 1,786: @@
 	</div>
 	<div class="exp-content">
-	<p>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.</p>
+	<p>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&deg;C for 0.5 hours. Then the PCR was carried out as usual.</p>
       <table width="100%" border="1">
    <tr>
@@ Line 1,862: / Line 1,882: @@
 </div>
 </fieldset>
-<fieldset class="exp22">
+<fieldset class="exp24">
-     <legend><a name="exp22.4">24.4 Restriction of StrepCup and Ligation with pET16b</a></legend>
+     <legend><a name="exp24.4">24.4 Restriction of StrepCup and Ligation with pET16b</a></legend>
 	<div class="aim">
 	<p>Aim: insert the StrepCup construct in the overproduction plasmid pET16b</p>
 	</div>
 	<div class="exp-content">
-	<p>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 tansformand, which was considered as a spontaneous mutation in relation to the control plate, which carried one colony more.</p>
+	<p>The purified StrepCup fragment was digested with the enzymes <i>Nco</i>I and <i>Xho</i>I, 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.</p>
      <table width="100%" border="1">
    <tr>
@@ Line 1,879: / Line 1,899: @@
    </tr>
    <tr>
-     <th scope="row">NcoI</th>
+     <th scope="row"><i>Nco</i>I</th>
      <td>0,5</td>
    </tr>
@@ Line 1,907: / Line 1,927: @@
      </tr>
    <tr>
-     <th scope="row">StrepCup NcoI/XhoI  (170.8 ng/&micro;l)</th>
+     <th scope="row">StrepCup <i>Nco</i>I/<i>Xho</i>I  (170.8 ng/&micro;l)</th>
      <td>0,5</td>
      </tr>
    <tr>
-     <th scope="row">pET16b NcoI/XhoI  (18.1 ng/&micro;l)</th>
+     <th scope="row">pET16b <i>Nco</i>I/<i>Xho</i>I  (18.1 ng/&micro;l)</th>
      <td>16</td>
      </tr>
@@ Line 1,944: / Line 1,964: @@
 	</div>
 	<div class="exp-content">
-	<p>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.</p>
+	<p>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 <i>Nco</i>I and <i>Sac</i>I before.</p>
      <table width="100%" border="1">
    <tr>
@@ Line 2,084: / Line 2,104: @@
 </div>
 </fieldset>
-<fieldset class="exp22">
+<fieldset class="exp25">
-     <legend><a name="exp22.6a">22.6 Immunofluorescence microscopy  at AG Grosse lab</a></legend>
+     <legend><a name="exp25.6a">25.6a Immunofluorescence microscopy  at AG Grosse lab</a></legend>
 	<div class="aim">
 	<p>Aim: Checking the interaction of StrepDARPidin with EpCAM-positive cell lines A549 & Caco-2 via immunofluorescence Antibody staining</p>
@@ Line 2,106: / Line 2,126: @@
 	<a name="05.10.2014">05.10.2014</a>
 </h2>
-<fieldset class="exp22">
+<fieldset class="exp25">
-     <legend><a name="exp22.6b">22.6 Immunofluorescence microscopy  at AG Grosse lab</a></legend>
+     <legend><a name="exp25.6b">25.6b Immunofluorescence microscopy  at AG Grosse lab</a></legend>
 	<div class="aim">
 	<p>Aim: Checking the interaction of StrepDARPidin with EpCAM-positive cell lines A549 & Caco-2 via immunofluorescence Antibody staining</p>
@@ Line 2,113: / Line 2,133: @@
 	<div class="exp-content">
 	<p>The dried samples were analysed under a Laser scanning microscope Zeiss LSM Series.</p>
+	<img src="https://static.igem.org/mediawiki/2014/3/39/MR_fuorescence_microscopy_anti-His_Caco2_2014_10_05.jpg" width="50%" />
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/8/89/MR_fuorescence_microscopy_anti-His_A549_2014_10_05.jpg" width="50%" />
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/8/8e/MR_fuorescence_microscopy_anti-His_NIH3T3_2014_10_05.jpg" width="50%" />
+	<br />
+	<p>The dried samples were analysed under a Laser scanning microscope Zeiss LSM Series.</p>
+	<p>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.</p>
+	<p>We were able to prove that the StrepDARPidin is binding specifically to EpCAM-positive cells.</p>
 	</div>
 </fieldset>
@@ Line 2,330: / Line 2,359: @@
      <legend><a name="exp23.41">23.41 Repeat test restriction</a></legend>
 	<div class="exp-content">
-	<p>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.</p>
+	<p>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 <i>Pst</i>I restriction site 8 clones will be digested with <i>Pst</i>I and <i>Pst</i>I/<i>Eco</i>RI.</p>
      <table width="100%" border="1">
    <tr>
@@ Line 2,348: / Line 2,377: @@
    </tr>
    <tr>
-     <th scope="row">PstI</th>
+     <th scope="row"><i>Pst</i>I</th>
      <td>0,5</td>
      <td>4,5</td>
    </tr>
    <tr>
-     <th scope="row">EcoRI</th>
+     <th scope="row">i>Eco</i>RI</th>
      <td>0,5</td>
      <td>4,5</td>
@@ Line 2,365: / Line 2,394: @@
 	<br />
 	<img src="https://static.igem.org/mediawiki/2014/9/9e/MR_2014-10-05_23.41.png" width="50%" />
-     <p>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.</p>
+     <p><i>Pst</i>I restriction should lead to a linearized plasmid, i>Eco</i>RI/<i>Pst</i>I shows the expected size of 1500 and 2000 bp. Additionally some undigested plasmid is left in the <i>Pst</i>I restriction.</p>
      <ul>
-       <li>Contamination of plasmid? New restriction with  PstI of AG Graumann and EcoRI of AG Bange.</li>
+       <li>Contamination of plasmid? New digestion with new batch of <i>Pst</i>I and <i>Eco</i>RI.</li>
-       <li>PstI restriction lead to a linearized plasmid  and PstI/EcoRI double digest lead to two fragments of a size of 1400 and 2000  bp</li>
+       <li><i>Pst</i>I restriction lead to a linearized plasmid  and <i>Pst</i>I/<i>Eco</i>RI double digest lead to two fragments of a size of 1400 and 2000  bp</li>
        <li>Due to limited time clones 1.1 and 2.1 will be  sent for sequencin</li>
      </ul>
      <img src="https://static.igem.org/mediawiki/2014/b/b8/MR_2014-10-05_23.41_2.png" width="50%" />
-     <p>Result: PstI is contaminated with EcoRI and will be thrown away!</p>
+     <p>Result: <i>Pst</i>I is contaminated with i>Eco</i>RI.</p>
 	</div>
 </fieldset>
@@ Line 2,526: / Line 2,555: @@
 	</div>
 	<div class="exp-content">
-	<p>Clone 6 was picked to inoculate a miniprep in LB-ampicillin. The plasmid was prepped after 8 hours of incubation at 37&deg;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.</p>
+	<p>Clone 6 was picked to inoculate a miniprep in LB-ampicillin. The plasmid was isolated after 8 hours of incubation at 37&deg;C. A control restriction was carried out with the enzymes <i>Nco</i>I, <i>Eco</i>RI and <i>Pst</i>I; 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.</p>
 	</div>
 	<img src="https://static.igem.org/mediawiki/2014/5/56/PET16b_StrepCup_restriction.png" width="20%" />
@@ Line 2,708: / Line 2,737: @@
 	</div>
 <p>Applied onto a gel nothing could be seen.</p>
+</fieldset>
+<fieldset class="exp25"
+	<legend><a name="exp25.7">25.7 new Fluorometer assay with A549 and Caco-2</a></legend>
+	<div class="aim">
+	<p>Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells & Caco-2 cells using a new serial dilution</p>
+	</div>
+	<div class="exp-content">
+	<p>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.</p>
+	<p>The same protocol like in 22.5 was used or GeFi-purified StrepDARPidin.</p>
+	<p>Following concentrations of StrepDARPidin dissolved in PBS (pH 7,4) +2,5 % glycerin were used:</p>
+	<table width="100%" border="1">
+  <tr>
+    <th scope="col">Content</th>
+    <th scope="col">Well Col</th>
+    <th scope="col">Well Row</th>
+    <th scope="col">raw data (485, 520)</th>
+    <th scope="col">Purification</th>
+    <th scope="col">StrepDARPidin  concentration [M]</th>
+  </tr>
+  <tr>
+    <th scope="row">Caco-2</th>
+    <td>1;1</td>
+    <td>C</td>
+    <td>344</td>
+    <td>-</td>
+    <td>25 &micro;M</td>
+  </tr>
+  <tr>
+    <th scope="row">Caco-2</th>
+    <td>2;2</td>
+    <td>C</td>
+    <td>538</td>
+    <td>-</td>
+    <td>2.5 &micro;M</td>
+  </tr>
+  <tr>
+    <th scope="row">Caco-2</th>
+    <td>3;3</td>
+    <td>C</td>
+    <td>386</td>
+    <td>-</td>
+    <td>250 nM</td>
+  </tr>
+  <tr>
+    <th scope="row">Caco-2</th>
+    <td>4;4</td>
+    <td>C</td>
+    <td>319</td>
+    <td>-</td>
+    <td>25 nM</td>
+  </tr>
+    <tr>
+    <th scope="row">Caco-2</th>
+    <td>5;5</td>
+    <td>C</td>
+    <td>281</td>
+    <td>-</td>
+    <td>2.5 nM</td>
+  </tr>
+    <tr>
+    <th scope="row">Caco-2</th>
+    <td>6;6</td>
+    <td>C</td>
+    <td>259</td>
+    <td>-</td>
+    <td>250 pM</td>
+  </tr>
+    <tr>
+    <th scope="row">A549</th>
+    <td>7;7</td>
+    <td>C</td>
+    <td>422</td>
+    <td>-</td>
+    <td>25 &micro;M</td>
+  </tr>
+    <tr>
+    <th scope="row">A549</th>
+    <td>8;8</td>
+    <td>C</td>
+    <td>396</td>
+    <td>-</td>
+    <td>2,5 &micro;M</td>
+  </tr>
+    <tr>
+    <th scope="row">A549</th>
+    <td>9;9</td>
+    <td>C</td>
+    <td>273</td>
+    <td>-</td>
+    <td>250 nM</td>
+  </tr>
+    <tr>
+    <th scope="row">A549</th>
+    <td>10;10</td>
+    <td>C</td>
+    <td>247</td>
+    <td>-</td>
+    <td>25 nM</td>
+  </tr>
+    </tr>
+    <tr>
+    <th scope="row">A549</th>
+    <td>11;11</td>
+    <td>C</td>
+    <td>288</td>
+    <td>-</td>
+    <td>2,5 nM</td>
+  </tr>
+    </tr>
+    <tr>
+    <th scope="row">A549</th>
+    <td>12;12</td>
+    <td>C</td>
+    <td>272</td>
+    <td>-</td>
+    <td>250 pM</td>
+  </tr>
+    </tr>
+    <tr>
+    <th scope="row">Blank</th>
+    <td>5;9</td>
+    <td>G</td>
+    <td>197</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+  <tr>
+    <th scope="row">negative control 3T3 fibroblast</th>
+    <td>6;10</td>
+    <td>G</td>
+    <td>355</td>
+    <td>-</td>
+    <td>25 &micro;M</td>
+  </tr>
+  <tr>
+    <th scope="row">negative control 3T3 fibroblast</th>
+    <td>7;11</td>
+    <td>G</td>
+    <td>305</td>
+    <td>-</td>
+    <td>25 &micro;</td>
+  </tr>
+  <tr>
+    <th scope="row">positive AB-control</th>
+    <td>8,12</td>
+    <td>G</td>
+    <td>3950</td>
+    <td>-</td>
+    <td>-</td>
+  </tr>
+</table>
+<br />
+<p>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.</p>
+</div>
 </fieldset>
 </div>
@@ Line 2,884: / Line 3,067: @@
 	</div>
 	<div class="exp-content">
-	<p>To  transform Bacillus a huge amount of plasmid is needed. For this reason 100 ml  culture with <em>E. coli</em> 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&nbsp; columns by centrifugation. </p>
+	<p>To  transform Bacillus a huge amount of plasmid is needed. For this reason 100 ml  culture with <em>E. coli</em> 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&nbsp; columns by centrifugation. </p>
 </div>
 </fieldset>
@@ Line 2,920: / Line 3,103: @@
 </table>
 	</div>
+</fieldset>
+<fieldset class="exp23">
+	<legend><a name="exp23.46">23.46 PCR amplification of the Arc1p-C domain fragment and the PheA domain fragment</a></legend>
+	<div class="aim">
+		<p>Aim: Fragments are needed for the cloning of two new biobricks</p>
+	</div>
+	<div class="exp-content">
+	<table width="100%" border="1">
+          <tr>
+            <th scope="col">&nbsp;</th>
+            <th scope="col">Arc1p (&micro;l)</th>
+            <th scope="col">PheA (&micro;l)</th>
+          </tr>
+          <tr>
+            <th scope="row">Water</th>
+            <td>24</td>
+			<td>24</td>
+          </tr>
+          <tr>
+            <th scope="row">HF Buffer 5x</th>
+            <td>10</td>
+			<td>10</td>
+          </tr>
+          <tr>
+            <th scope="row">Phusion DNA-polymerase</th>
+            <td>1</td>
+			<td>1</td>
+          </tr>
+          <tr>
+            <th scope="row">Primer for (1:50)</th>
+            <td>6.5</td>
+            <td>6.5</td>
+          </tr>
+          <tr>
+            <th scope="row">Primer rev (1:50)</th>
+            <td>6.5</td>
+            <td>6.5</td>
+          </tr>
+		  <tr>
+            <th scope="row">Template (20 ng/&micro;l)</th>
+            <td>1</td>
+            <td>1</td>
+          </tr>
+		  <tr>
+            <th scope="row">dNTP mix &#040;10 mM each&#041;</th>
+            <td>1</td>
+            <td>1</td>
+		  <tr>
+      </table>
+	  <br />
+	<img src="https://static.igem.org/mediawiki/2014/2/27/MR_20141008_amplification_Arc1p_bb_cloning.png" width="30%" />
+	<br />
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/0/0a/MR_20141008_amplification_PheA_bb_cloning.png" width="30%" />
+	<br />
+	<p>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.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+	<legend><a name="exp23.47">23.47 Restriction of pSB1C3 and the amplified fragments with <i>Xba</i>I and <i>Pst</i>I</a></legend>
+	<div class="aim">
+	<p>Aim: Digested plasmid and DNA fragments are needed</p>
+	</div>
+	<div class="exp-content">
+	<p>The iGEM vector pSB1C3 and the fragments resulting from the previous PCR will be digested with <i>Xba</i>I and <i>Pst</i>I for a following ligation and transformation.</p>
+	<br />
+	<table width="100%" border="1">
+          <tr>
+            <th scope="col">Reaction Mix</th>
+            <th scope="col">pSB1C3 (&micro;l)</th>
+			<th scope="col">Arc1p (&micro;l)</th>
+            <th scope="col">PheA (&micro;l)</th>
+          </tr>
+          <tr>
+            <th scope="row"><i>Pst</i>I</th>
+            <td>0.5</td>
+			<td>0.5</td>
+			<td>0.5</td>
+          </tr>
+          <tr>
+            <th scope="row"><i>Xba</i>I</th>
+            <td>0.5</td>
+			<td>0.5</td>
+			<td>0.5</td>
+          </tr>
+          <tr>
+            <th scope="row">Buffer 2.1 10x</th>
+            <td>2.0</td>
+			<td>2.0</td>
+			<td>2.0</td>
+          </tr>
+          <tr>
+            <th scope="row">Water</th>
+            <td>14</td>
+            <td>12</td>
+			<td>9<td>
+          </tr>
+      </table>
+	  <br />
+		<p>Digestion at room temperature over night.</p>
 </fieldset>
 <fieldset class="exp13">
@@ Line 3,084: / Line 3,367: @@
 	</div>
 	<div class="exp-content">
-	<p>30 ml LB-ampicillin were inoculated with cells of <i>E. coli</i> BL21&#040;DE3&#041; pET16b_StrepCup from plate. This culture was incubated shaking at 37&deg;C until they reached an OD600 of 0.58. A preinduction sample was taken &#040;1.2 ml = &#040;0.7 * 0.7&#041; / &#040;0.7 * 0.58&#041;&#041;, the cells were pelleted by centrifugation at 14000 rpm for 1 minute, followed by resuspension in 80 &micro;l water and 20 &micro;l 5X SDS loading dye. The culture was induced with 30 &micro;l IPTG for 3 hours. Another sample was taken afterwards, at this time point the culture reached an OD600 of 1.2 &#040;0.583 ml = &#040;0.7 * 0.7 / 0.7 * 1.2&#041;&#041;. These samples were boiled at 95&deg;C for 10 minutes and then analyzed by SDS-PAGE (12% SDS-polyacrylamide gel).
+	<p>30 ml LB-ampicillin were inoculated with cells of <i>E. coli</i> BL21&#040;DE3&#041; pET16b_StrepCup from plate. This culture was incubated shaking at 37&deg;C until they reached an OD<sub>600</sub> of 0.58. A preinduction sample was taken &#040;1.2 ml = &#040;0.7 * 0.7&#041; / &#040;0.7 * 0.58&#041;&#041;, the cells were pelleted by centrifugation at 14000 rpm for 1 minute, followed by resuspension in 80 &micro;l water and 20 &micro;l 5X SDS loading dye. The culture was induced with 30 &micro;l IPTG for 3 hours. Another sample was taken afterwards, at this time point the culture reached an OD<sub>600</sub> of 1.2 &#040;0.583 ml = &#040;0.7 * 0.7 / 0.7 * 1.2&#041;&#041;. These samples were boiled at 95&deg;C for 10 minutes and then analyzed by SDS-PAGE (12% SDS-polyacrylamide gel).
 	<br />
 	<img src="https://static.igem.org/mediawiki/2014/f/f7/SDS.png" width="30%" />
@@ Line 3,095: / Line 3,378: @@
 	<div class="aim">
 	<p>Aim: check nose - lac/const plasmids for correct insert
-        </div>
+	</div>
 	<div class="exp-content">
 	<p>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.</p>
@@ Line 3,132: / Line 3,415: @@
            </tr>
            <tr>
-             <th scope="row">piGEM002/007/008 + const. &#040;ca 40 ng/&micro;l&#041;</th>
+             <th scope="row">piGEM002/007/008 + const. %#040;ca 40 ng/&micro;l&#041;</th>
 			<td>-</td>
              <td>-</td>
@@ Line 3,315: / Line 3,598: @@
        </table>
 	  <br />
-	  <img src="https://static.igem.org/mediawiki/2014/4/42/CPCR_lac_plasmids_09.10.2014.png" width="40%" />
+	  <img src="https://static.igem.org/mediawiki/2014/4/42/CPCR_lac_plasmids_09.10.2014.png" width="30%" />
 	  <br />
-	  <p>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.</p>
+	  <p>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.</p>
 	  <p>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.</p>
 	 </div>
@@ Line 3,327: / Line 3,610: @@
 	</div>
 	<div class="exp-content">
-	<p>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 &#040;1172 bp, 5246 bp and 241 bp&#041; and piGEM008 + const was cut with BamHI and NcoI &#040;4189 bp + 2757 for a positive plasmid, 4189 bp and 2518 bp for a negative plasmid&#041;.
+	<p>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 <i>Bam</i>HI and <i>Hind</i>III 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 <i>Hind</I>III, because the only restriction site for <i>Hind</I>III is in the IPTG inducible promoter. As a control piGEM030 was cut with <i>Hind</I>III and <i>Nco</i>I &#040;1172 bp, 5246 bp and 241 bp&#041; and piGEM008 + const was cut with <i>Bam</i>HI and <i>Nco</i>I &#040;4189 bp + 2757 for a positive plasmid, 4189 bp and 2518 bp for a negative plasmid&#041;.
 	<br />
 	<table width="100%" border="1">
@@ Line 3,542: / Line 3,825: @@
        </table>
 	  <br />
-	  <img src="https://static.igem.org/mediawiki/2014/9/95/Kontroll_verdau_und_Colony_PCR_lac_plasmide_09.10.2014.png" width="50%" />
+	  <img src="https://static.igem.org/mediawiki/2014/9/95/Kontroll_verdau_und_Colony_PCR_lac_plasmide_09.10.2014.png" width="40%" />
 	  <br />
 	  <p>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 <i>Hind</i>III 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 <i>Hind</i>III 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 <i>Hind</i>III was able to cut in CutSmart only with the half of its optimal activity. That was the reason, why the double amount of <I>Hind</i>III was chosen.</p>
@@ Line 3,548: / Line 3,831: @@
 	</div>
 </fieldset>
-<fieldset class="exp22">
+<fieldset class="exp25">
-     <legend><a name="exp22.9">22.9 new Fluorometer assay with A549 and Caco-2</a></legend>
+     <legend><a name="exp25.9">25.9 new Fluorometer assay with A549 and Caco-2</a></legend>
 	<div class="aim">
 	<p>Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells & Caco-2 cells using a new serial dilution</p>
 	</div>
 	<div class="exp-content">
-	<p>This time Caco-2 &#040;A1-6&#041; and A549 &#040;B1-6&#041; were used to coat row C of a black 96-well plate &#040;100000 cells/ well&#041; 100000 cells of 3T3 fibroblasts were used as negative control and as well used to coat C5.</p>
+	<p>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.</p>
 	<p>The same protocol like in 22.5 was used or GeFi-purified StrepDARPidin.</p>
 	<p>Following concentrations of StrepDARPidin dissolved in PBS pH 7,4 +2,5 % glycerin were used:</p>
@@ Line 3,772: / Line 4,055: @@
 	  <p>The serial dilution shows that the signal is decreasing for both cell lines. The Caco-2 Cells have a higher signal compared to A549.</p>
 	  </div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.48">23.48 Purification of digested plasmid and fragments</a></legend>
+	<div class="aim">
+	<p>Aim: In order to improve the ligation results pure DNA samples are needed</p>
+	</div>
+	<div class="exp-content">
+	<p>All digested samples have been purified via gel extraction, resulting in the following concentration:</p>
+	<ul class="list">
+    <li>pSB1C3= 19 ng/&micro;l</li>
+    <li>Arc1p= 25 ng/&micro;l</li>
+    <li>PheA= 23 ng/&micro;l</li>
+	</ul>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.49">23.49 Ligation of digested plasmid and inserts</a></legend>
+	<div class="aim">
+	<p>Aim: Ligate plasmids for further transformation</p>
+	</div>
+	<div class="exp-content">
+	<br />
+	<table width="100%" border="1">
+          <tr>
+            <th scope="col">Ligation Mix</th>
+            <th scope="col">pSB11C3 + Arc1p (&micro;l)</th>
+            <th scope="col">pSB11C3 + PheA (&micro;l)</th>
+          </tr>
+          <tr>
+            <th scope="row">Water</th>
+			<td>0</td>
+			<td>4.3</td>
+		  </tr>
+		  <tr>
+            <th scope="row">HF Buffer 10x</th>
+			<td>2.0</td>
+			<td>2.0</td>
+		  </tr>
+		  <tr>
+            <th scope="row">T4 Ligase</th>
+			<td>1.0</td>
+			<td>1.0</td>
+		  </tr>
+		  <tr>
+            <th scope="row">Insert</th>
+			<td>7.6</td>
+			<td>9</td>
+		  </tr>
+		  <tr>
+		    <th scope="row">Vector</th>
+			<td>9.4</td>
+			<td>3.7</td>
+		  </tr>
+	</table>
+	<br />
+	<p>The ligation was performed at 16&deg;C for 3h.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.50">23.50 Transformation of competent <i>E. coli</i> XL-1-Blue with ligated plasmid</a></legend>
+	<div class="aim">
+	<p>Aim: Ligate plasmids for further transformation</p>
+	</div>
+	<div class="exp-content">
+	<p>After the ligation the reaction was inactivated by incubating the reaction mix at 85%deg;C for 10 minutes. Afterwards chemically competent <i>E. coli</i> XL-1-blue were transformed with the whole reaction mix. Standard <i>E. coli</i> transformation protocol was used.</p>
+	</div>
 </fieldset>
 </div>
+<!-- 10.10.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="10.10.2014">10.10.2014</a>
+</h2>
+<fieldset class="exp24">
+    <legend><a name="exp24.8">24.8 Test for solubility of StrepCup</a></legend>
+	<div class="aim">
+	<p>Aim: check if StrepCup is soluble or enclosed in inclusion bodies</p>
+	</div>
+	<div class="exp-content">
+	<p>A second test expression was carried out in 30 ml LB-amp, which was inoculated with <i>E. coli</i> BL21&#040;DE3&#041; and incubated until they reached an OD<sub>600</sub> of 0.62. A 1.12 ml preinduction sample was taken and the cells were induced with 30 &micro;l IPTG for 3 hours shaking at 37&deg;C. After this time they reached an OD<sub>600</sub> of 1.4, so 500 &micro;l were taken as an induction sample. The taken samples were pelleted by centrifugation, resuspended in 80 &micro;l of water with addition of 20 &micro;l 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 &micro;l were taken as a sample, which was treated with 20 &micro;l of 5x SDS loading dye. The pellet was resuspended in some buffer A from which 80 &micro;l were taken and 20 &micro;l of 5x SDS loading dye were added. The samples were boiled at 95&deg;C for 10 minutes. Afterwards 10 &micro;l of each sample was loaded onto a 12% SDS polyacrylamide gel.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/6/63/SDS_PAGE_StrepCup_soluble_unsoluble_10.10.2014.png" width="30%" />
+	<br />
+	<p>It could clearly be seen that the StrepCup was in the unsoluble fraction of the lysate.</p>
+	</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.115">13.115 Ligation of piGEM008/009 with constitutive promoter</a></legend>
+	<div class="aim">
+	<p>Aim: ligate the digested vector with the constitutive promoter</p>
+	</div>
+	<div class="exp-content">
+	<p>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.</p>
+	<table width="100%" border="1">
+          <tr>
+            <th scope="col">Component</th>
+            <th scope="col">Ligation I &#040;&micro;l&#041;</th>
+            <th scope="col">Ligation II &#040;&micro;l&#041;</th>
+          </tr>
+          <tr>
+            <td>piGEM008 &#040;ca 196.7 ng/&micro;l&#041;</td>
+			<td>5.2</td>
+			<td>-</td>
+		  </tr>
+		  <tr>
+            <td>piGEM009 &#040;ca 218.3 ng/&micro;l&#041;</td>
+			<td>-</td>
+			<td>3.05</td>
+		  </tr>
+		  <tr>
+            <td>constitutive promoter &#040;ca 49.3 ng/&micro;l&#041;</td>
+			<td>2.3</td>
+			<td>4.45</td>
+		  </tr>
+		  <tr>
+            <td>T4 ligase</td>
+			<td>0.5</td>
+			<td>0.5</td>
+		  </tr>
+		  <tr>
+            <td>Ligation buffer 10x</td>
+			<td>2</td>
+			<td>2</td>
+		  </tr>
+		  <tr>
+            <td>Water</td>
+			<td>10</td>
+			<td>10</td>
+		  </tr>
+		  <tr>
+            <td>Total</td>
+			<td>20</td>
+			<td>20</td>
+		  </tr>
+	</table>
+	<br />
+	<p>The reaction was incubated at room temperature for over one hour and afterwards used to transform competent <i>E. coli</i> XLIBlue, which were plated out on LB-ampicillin and incubated at 37&deg;C over night.</p>
+	<p>Additionally, clones were picked from the old ligation plates to inoculate cultures of LB-ampicillin for plasmid preps.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.51">23.51 Inoculation of presumably positive clones from the ligation plates</a></legend>
+	<div class="aim">
+	<p>Aim: Grow cultures for the isolation of plasmid psB1C3 with the two new Biobricks</p>
+	</div>
+	<div class="exp-content">
+	<p>Two clones for every biobrick plasmid were inoculated in liquid LB-CM (10:30) for plasmid isolation.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.52a">23.52a Plasmid isolation and test digest</a></legend>
+	<div class="aim">
+	<p>Aim: Analyze the plasmids for the insertion of the fragments into the plasmid</p>
+	</div>
+	<div class="exp-content">
+	<p>Plasmid isolation of the pSB1C3 plasmid from <i>E. coli</i> was performed with the Quiagen Mini-prep kit. Plasmid concentration was determined with a Nanodrop spectrophotometer (100-120 ng/&micro;l).</p>
+	<p>Restriction: 4 &micro;l plasmid in a total 10 &micro;l restriction mix</p>
+	<br />
+	<table width="100%" border="1">
+          <tr>
+            <th scope="col">Component</th>
+            <th scope="col">Restriction mix (&micro;l)</th>
+          </tr>
+          <tr>
+            <th scope="col">Water</th>
+			<td>12.0</td>
+		  </tr>
+		  <tr>
+            <th scope="col">CutSmart 10x</th>
+			<td>2.0</td>
+		  </tr>
+		  <tr>
+            <th scope="col"><i>Xba</i>I</th>
+			<td>0.5</td>
+		  </tr>
+		  <tr>
+            <th scope="col"><i>Pst</i>I</th>
+			<td>0.5</td>
+		  </tr>
+		  <tr>
+            <th scope="col">Plasmid (600 ng/&micro;l)</th>
+			<td>5.0</td>
+		  </tr>
+	</table>
+	<br />
+	<p>Gel analysis of the digested plasmids reveals that all clones are negative, new ligation will be performed with a higher amount of insert.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 11.10.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="11.10.2014">11.10.2014</a>
+</h2>
+<fieldset class="exp25">
+    <legend><a name="exp25.10">25.10 new Fluorometer assay with A549 and Caco-2</a></legend>
+	<div class="aim">
+	<p>Aim: Checking interaction of StrepDARPidin with EpCAM on A549 Lung carcinoma cells & Caco-2 cells using a new serial dilution</p>
+	</div>
+	<div class="exp-content">
+	<p>This time Caco-2 &#040;F1-6&#040; and A549 &#040;G1-6&#041; were used to coat row C of a black 96-well plate &#040;100000 cells/ well&#041;. 100000 cells of 3T3WT fibroblasts were used as negative control and as well used to coat H2 & H3.</p>
+	<p>The same protocol like in 22.5 was used for GeFi-purified StrepDARPidin.</p>
+	<p>Following concentrations of StrepDARPidin dissolved in PBS pH 7,4 +2,5 % glycerin were used:</p>
+	<br />
+	<table width="100%" border="1">
+          <tr>
+            <th scope="col">Content</th>
+            <th scope="col">Well Col</th>
+            <th scope="col">Well Row</th>
+			<th scope="col">Blank corrected raw data &#040;485, 520&#041;</th>
+			<th scope="col">StrepDARPidin concentration &#040;M&#041;</th>
+          </tr>
+          <tr>
+            <td>Caco-2</td>
+			<td>1</td>
+			<td>F</td>
+			<td>382</td>
+			<td>25 &micro;M</td>
+		  </tr>
+		  <tr>
+            <td>Caco-2</td>
+			<td>2</td>
+			<td>F</td>
+			<td>556</td>
+			<td>2,5 &micro;M</td>
+		  </tr>
+		  <tr>
+            <td>Caco-2</td>
+			<td>3</td>
+			<td>F</td>
+			<td>550</td>
+			<td>250 nM</td>
+		  </tr>
+		  <tr>
+            <td>Caco-2</td>
+			<td>4</td>
+			<td>A</td>
+			<td>522</td>
+			<td>25 nM</td>
+		  </tr>
+		  <tr>
+            <td>Caco-2</td>
+			<td>5</td>
+			<td>F</td>
+			<td>541</td>
+			<td>2,5 nM</td>
+		  </tr>
+		  <tr>
+            <td>Caco-2</td>
+			<td>6</td>
+			<td>F</td>
+			<td>548</td>
+			<td>250 pM</td>
+		  </tr>
+		  <tr>
+            <td>A549</td>
+			<td>1</td>
+			<td>G</td>
+			<td>407</td>
+			<td>25 &micro;M</td>
+		  </tr>
+		  <tr>
+            <td>A549</td>
+			<td>2</td>
+			<td>G</td>
+			<td>491</td>
+			<td>2,5 &micro;M</td>
+		  </tr>
+		  <tr>
+            <td>A549</td>
+			<td>3</td>
+			<td>G</td>
+			<td>557</td>
+			<td>250 nM</td>
+		  </tr>
+		  <tr>
+            <td>A549</td>
+			<td>4</td>
+			<td>G</td>
+			<td>359</td>
+			<td>25 nM</td>
+		  </tr>
+		  <tr>
+            <td>A549</td>
+			<td>5</td>
+			<td>G</td>
+			<td>338</td>
+			<td>2,5 nM</td>
+		  </tr>
+		  <tr>
+            <td>A549</td>
+			<td>6</td>
+			<td>G</td>
+			<td>632</td>
+			<td>250 pM</td>
+		  </tr>
+		  <tr>
+            <td>negative control 3T3WT fibroblasts</td>
+			<td>2</td>
+			<td>H</td>
+			<td>409</td>
+			<td>25 &micro;M</td>
+		  </tr>
+		  <tr>
+            <td>negative control 3T3WT fibroblasts</td>
+			<td>3</td>
+			<td>H</td>
+			<td>468</td>
+			<td>2,5 &micro;M</td>
+		  </tr>
+		  <tr>
+            <td>Pos. control AB</td>
+			<td>4</td>
+			<td>H</td>
+			<td>3786</td>
+			<td>-</td>
+		  </tr>
+      </table>
+	  <br />
+	  <p>The serial dilution shows that the signal is decreasing for both cell lines. The Caco-2 Cells have a higher signal.</p>
+	  <p>After the previous measurements the results were summarized and a graphic created:</p>
+	  <br />
+	  <img src="https://static.igem.org/mediawiki/2014/d/da/Fluorescence_assay.jpg" width="30%" />
+	  <br />
+	  <p>The tendency which could be seen in the previous assays can be reproduced after summarizing the previous measurements as well.</p>
+	  </div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.116">13.116 Making new competent <i>Bacillus subtilis</i> PY79 cells</a></legend>
+	<div class="aim">
+	<p>Aim: making competent cells to transform Bacillus subtilis PY79 with the Nose-plasmids</p>
+	</div>
+	<div class="exp-content">
+	<p>Since the previous cells were not competent, a new try to make competent <i>Bacillus subtilis</i> 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&deg;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 &micro;l and 50 &micro;l of each nose plasmid with a metallosensitive promoter. After adding the plasmid DNA to 200 &micro;l cells in a test tube, they were incubated shaking a 37&deg;C for 30 minutes. Afterwards they were plated out on LB-chloramphenicol &#040;5 &micro;g/ml&#041; were incubated at 37&deg;C.</p>
+	</div>
+</fieldset>
+<fieldset class="exp13">
+    <legend><a name="exp13.117">13.117 control restriction of nose plasmids with the constitutive promoter</a></legend>
+	<div class="aim">
+	<p>Aim: check the plasmids for the correct insertion of the promoter</p>
+	</div>
+	<div class="exp-content">
+	<p>The plasmids were isolated from the inoculated cultures &#040;13.115; 2 clones piGEM002 + const, 6 clones piGEM007 + const, 5 clones piGEM008 + const and 4 clones piGEM009 + const&#041; and digested with the enzymes <i>Nco</i>I-HF and <i>Eco</i>RI-HF. Negative plasmids should yield fragments in size of 476 and 6190 &#040;for piGEM002&#041; or 6235 bp &#040;for piGEM007/008/009&#041;. The positive plasmids should result in fragments in the size of 711 and 6190/6235 bp.</p>
+	<br />
+	<table width="100%" border="1">
+		<tr>
+			<th scope="col">Component</th>
+			<th scope="col">Volumes &#040;&micro;l&#041;</th>
+		</tr>
+		<tr>
+			<td>piGEM002/007/008/009 + const &#040;ca 400 ng/&micro;l&#041;</td>
+			<td>5</td>
+		</tr>
+		<tr>
+			<td><i>Nco</i>I-HF</td>
+			<td>0.5</td>
+		</tr>
+		<tr>
+			<td><i>Eco</i>RI-HF</td>
+			<td>0.5</td>
+		</tr>
+		<tr>
+			<td>CutSmart 10x</td>
+			<td>2</td>
+		</tr>
+		<tr>
+			<td>Water</td>
+			<td>12</td>
+		</tr>
+		<tr>
+			<td>Total</td>
+			<td>20</td>
+		</tr>
+	</table>
+	<br />
+	<p>The reactions were incubated at 37&deg;C for over one hour, 3 &micro;l 6x Purple Loading Dye were added and 7 &micro;l of the restrictions were applied onto a 1% agarose gel.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/e/e7/Control_restriction_nose_%2B_const_11.10.2014.png" width="50%" />
+	<br />
+	<p>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.</p>
+	<p>The plasmids piGEM002 + const and piGEM007 + const were already confirmed as positive by the control PCR &#040;13.113&#041;, 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.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.53">23.53 New Ligation and transformation</a></legend>
+	<div class="aim">
+	<p>Aim: Analyze the plasmids for the insertion of the fragments into the plasmid</p>
+	</div>
+	<div class="exp-content">
+	<p>In order to calculate the needed amount of insert and vector the iGEM ligation calculator was used. For this reaction a higher amount of insert was used (1:5)</p>
+	<br />
+		<table width="100%" border="1">
+          <tr>
+            <th scope="col">Ligation Mix</th>
+            <th scope="col">pSB11C3 + Arc1p (&micro;l)</th>
+            <th scope="col">pSB11C3 + PheA (&micro;l)</th>
+          </tr>
+          <tr>
+            <th scope="row">Water</th>
+			<td>0.6</td>
+			<td>5.5</td>
+		  </tr>
+		  <tr>
+            <th scope="row">HF Buffer 10x</th>
+			<td>2.0</td>
+			<td>2.0</td>
+		  </tr>
+		  <tr>
+            <th scope="row">T4 Ligase</th>
+			<td>1.0</td>
+			<td>1.0</td>
+		  </tr>
+		  <tr>
+            <th scope="row">Insert</th>
+			<td>9.0</td>
+			<td>9.0</td>
+		  </tr>
+		  <tr>
+		    <th scope="row">Vector</th>
+			<td>7.4</td>
+			<td>2.5</td>
+		  </tr>
+	</table>
+	<br />
+	<p>The ligation was performed at 16&deg;C for 3 hours.
+	<br />
+	<p>After the ligation the reaction was inactivated by incubating the reaction mix at 85&deg;C for 10 minutes. Afterwards chemically competent <i>E. coli</i> XL-1-blue were transformed with the whole reaction mix. Standard <i>E. coli</i> transformation protocol was used.
+	</div>
+</fieldset>
+</div>
+<!-- 12.10.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="12.10.2014">12.10.2014</a>
+</h2>
+<fieldset class="exp13">
+    <legend><a name="exp13.118">13.118 control restriction of piGEM009 with the constitutive promoter</a></legend>
+	<div class="aim">
+	<p>Aim: check the plasmid for the correct insertion of the promoter</p>
+	</div>
+	<div class="exp-content">
+	<p>The plasmids were isolated from the cultures &#040;13.117&#041; and digested again with the enzymes <i>Nco</i>I and <i>Eco</i>RI. The expected fragments were the same like in 13.117.</p>
+	<table width="100%" border="1">
+		<tr>
+			<th scope="col">Component</th>
+			<th scope="col">Volumes &#040;&micro;l&#041;</th>
+		</tr>
+		<tr>
+			<td>piGEM009 + const &#040;200-400 ng/&micro;l&#041;</td>
+			<td>5</td>
+		</tr>
+		<tr>
+			<td><i>Nco</i>I-HF</td>
+			<td>0.5</td>
+		</tr>
+		<tr>
+			<td><i>Eco</i>RI-HF</td>
+			<td>0.5</td>
+		</tr>
+		<tr>
+			<td>CutSmart 10x</td>
+			<td>2</td>
+		</tr>
+		<tr>
+			<td>Water</td>
+			<td>12</td>
+		</tr>
+		<tr>
+			<td>Total</td>
+			<td>20</td>
+		</tr>
+	</table>
+	<br />
+	<p>The reactions were incubated at 37&deg;C for over one hour, 3 &micro;l 6x Purple Loading Dye were added and 7 &micro;l of the restrictions were applied onto a 1% agarose gel.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/c/c6/Restriktion_piGEM009_const_12.10.2014.png" width="40%" />
+	<br />
+	<p>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 &#040;and the backbone at around 6000 bp&#041;, which meant, the plasmids did not contain the constitutive promoter.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.51b">23.51b Inoculation of presumably positive clones from the ligation plates</a></legend>
+	<div class="aim">
+	<p>Aim: Grow cultures for the isolation of plasmid psB1C3 with the two new Biobricks</p>
+	</div>
+	<div class="exp-content">
+	<p>Two clones for every biobrick plasmid were inoculated in liquid LB-CM for plasmid isolation.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.52b">23.52b Plasmid isolation and test restriction</a></legend>
+	<div class="aim">
+	<p>Aim: Analyse the plasmids for the insertion of the fragments into the plasmid</p>
+	</div>
+	<div class="exp-content">
+	<p>Plasmid isolation of the pSB1C3 plasmid from <i>E.coli</i> was performed with the Quiagen Mini-prep kit. Plasmid concentration was determined with a Nanodrop spectrophotometer (90-135 ng/&micro;l).</p>
+	<br />
+		<table width="100%" border="1">
+          <tr>
+            <th scope="col">Component</th>
+            <th scope="col">Restriction mix (&micro;l)</th>
+          </tr>
+          <tr>
+            <th scope="col">Water</th>
+			<td>12.0</td>
+		  </tr>
+		  <tr>
+            <th scope="col">CutSmart 10x</th>
+			<td>2.0</td>
+		  </tr>
+		  <tr>
+            <th scope="col"><i>Xba</i>I</th>
+			<td>0.5</td>
+		  </tr>
+		  <tr>
+            <th scope="col"><i>Pst</i>I</th>
+			<td>0.5</td>
+		  </tr>
+		  <tr>
+            <th scope="col">Plasmid (600 ng/&micro;l)</th>
+			<td>5.0</td>
+		  </tr>
+	</table>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/3/32/MR_20141012_test_digest_pSB1C3_Arc1p_PstI_XbaI.png" width="30%" />
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/c/c8/MR_20141012_test_digest_pSB1C3_PheA_PstI_XbaI.png" width="30%" />
+	<br />
+	<p>Both plasmids have been cloned successfully as visible in the two agarose gels. The expected inserts could be cut out of the iGEM plasmid pSB1C3.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 13.10.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="13.10.2014">13.10.2014</a>
+</h2>
+<fieldset class="exp18">
+    <legend><a name="exp18.74">18.74 Isolation of flagella from different <i>Bacillus subtilis</i> strains</a></legend>
+	<div class="aim">
+	<p>Aim: separation of the flagella from the cells</p>
+	</div>
+	<div class="exp-content">
+	<p>Since the flagella of the <i>Bacillus subtilis</i> 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 <i>Bacillus subtilis</i> 3610wt, Hag-D2-Strep, Hag-D2-Cup and Py79 Hag-Strep in 5 L Erlenmeyer flasks with baffles and incubated shaking at 37&deg;C over night.</p>
+	</div>
+</fieldset>
+<fieldset class="exp24">
+    <legend><a name="exp24.9">24.9 Expression and purification of StrepCup</a></legend>
+	<div class="aim">
+	<p>Aim: expression of Streptavidin-Cup</p>
+	</div>
+	<div class="exp-content">
+	<p>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 <i>E. coli</i> BL21&#040;DE3&#041; pET16b_StrepCup. The medium also contained 50 ml 25 % &#040;w/v; 12.5 g/50ml&#041; lactose as an inducer of the expression. The cultures were incubated shaking at 30&deg;C over night.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.53">23.53 Sequencing of BBa-K1329004 and BBa-K1329005</a></legend>
+	<div class="aim">
+	<p>Aim: check for the correct sequence of the biobricks</p>
+	</div>
+	<div class="exp-content">
+	<p>Sequencing samples have been prepared in a concentration of 50 - 100 ng/&micro;l and primers were added. Plasmids were then sequenced by eurofins mwg.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 14.10.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="14.10.2014">14.10.2014</a>
+</h2>
+<fieldset class="exp24">
+    <legend><a name="exp24.9">24.9 Expression and purification of StrepCup</a></legend>
+	<div class="aim">
+	<p>Aim: purification of StrepCup</p>
+	</div>
+	<div class="exp-content">
+	<p>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&deg;C. Samples were taken from the pellet &#040;P1&#041; and the supernatant &#040;S1&#041;, 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 &#040;P2&#041; and the supernatant &#040;S2&#041;. 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&deg;C for 10 min. The pellet &#040;P3&#041; and the supernatant &#040;S3&#041; were stored separately in the refrigerator at 4&deg;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 &#040;protein precipitated by contact with water&#041; the sample was vortexed until the precipitate was gone and 80 &micro;l were taken together with 20 &micro;l of 5x SDS loading dye and bolied at 95&deg;C for 10 min together with the other samples, from which none precipitated as intense as the supernatant of the gua pellet. 10 &micro;l of all samples were transferred onto a 12 % SDS-polyacrylamide gel.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/3/34/SDS_expression_StrepCup_isolation_from_IB_14.10.2014.jpg" width="30%" />
+	<br />
+	<p>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.</p>
+	</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.74a">18.74a Isolation of flagella from different <i>Bacillus subtilis</i> strains</a></legend>
+	<div class="aim">
+	<p>Aim: separation of the flagella from the cells</p>
+	</div>
+	<div class="exp-content">
+	<p>Strains: Wildtype 3610, PY-Strep, Hag-D2-Strep, Hag-D2-Cup</p>
+	<p>The different cultures were harvested by centrifugation at 4000 rpm for 20 minutes. The pellets were resuspended in 30 ml 0.1 M TRIS/HCl buffer pH 8.0 containing 0.5 % Brij-58. 330 &micro;l of a 10 mg/ml lysozyme solution were added and the lysis was performed in a roller at 4&deg;C until the samples were mostly clear.</p>
+	<p>20 &micro;l of RNase free DNase was added and lysis continued for another 30 min. The bacterial debris was removed by centrifugation at 10.000 rpm for 10 min, followed by an ultracentrifugation step of the supernatant at 100.000 rpm for 90 min (4&deg;C).</p>
+	<p>For the analysis of flagella via SDS-PAGE 1 ml with OD<sub>600</sub> 0.7 was prepared for every sample by centrifugation and subsequent heating to 95&deg;C (10 min). Cell pellets were centrifuged and resuspended in 40  &micro;L water and 10  &micro;L SDS loading dye (5x).</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/1/18/MR_20141014_flagella_isolation_cells_before_after_lysis.png" width="30%" />
+	<br />
+	<p>Calculated size of flagellin is about 37 kDa, running slightly higher than the fourth band of the unstained protein marker (first line). Flagellin is present in the wildtype sample before and after cell lysis. Other proteins can be seen for PY-Strep before (higher than Hag) and after (lower than Hag) cell lysis. The two Hag constructs with the inserted D2 domain run a bit higher than wildtype flagellin and are present after cell lysis in a small amount.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/9/92/MR_20141014_flagella_isolation_supernatant_before_after_lysis.png" width="30%" />
+	<br />
+	<p>Flagellin is present in the wildtype supernatant before and after cell lysis. Before lysis no flagellin can be seen in the supernatant of the other three strains. Differing proteins bands can be seen for these three strains in the supernatant after cell lysis.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/3/3f/MR_20141014_flagella_isolation_pellets_after_ultracentrifugation.png" width="30%" />
+	<br />
+	<p>A high amount of flagellin is present after cell lysis of wildtype <i>B. subtilis</i>. A thin band can be seen for Hag-D2-Cup before cell lysis and a thicker band for Hag-D2-Strep that does not fit the size of flagellin. The supernatant of the ultra-centrifugation step was transferred to a new falcon and the pellet was resuspended in 3 ml standard saline citrate buffer (pH 7.3) and both fractions were stored at 4&deg;C overnight.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 15.10.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="15.10.2014">15.10.2014</a>
+</h2>
+<fieldset class="exp18">
+    <legend><a name="exp18.74b">18.74b Isolation of flagella from different <i>Bacillus subtilis</i> strains II</a></legend>
+	<div class="exp-content">
+	<p>20% ammonium sulphate were added to the pellet resulting from the ultracentrifugation resuspended in standard saline citrate buffer (pH 7.3). The obtained precipitant was analysed in an SDS PAGE gel.</p>
+	<br />
+	<img src="https://static.igem.org/mediawiki/2014/8/8c/MR_20141014_flagella_isolation_ammoniumsulphate_precipitation.png" width="30%" />
+	<br />
+	<p>A high amount of flagellin is present in the ultracentrifugation pellet of the wildtype cells after lysis. A mixture of proteins can be seen for Hag-D2-Strep after cell lysis. It can be assumed that flagellin hag-D2-Strep is partially unstable. Anyway, the analysis of presumably isolated flagella was continued.</p>
+	</div>
+</fieldset>
+</div>
+<!-- 16.10.14 -->
+<div class="notebooky-entry">
+<h2 class="title">
+	<a name="16.10.2014">16.10.2014</a>
+</h2>
+<fieldset class="exp24">
+    <legend><a name="exp23.54">23.54 Sequencing results</a></legend>
+	<div class="exp-content">
+	<p>Both plasmid have been sequenced and the correct inserts are in the iGEM backbone pSB1C3.</p>
+	</div>
+</fieldset>
+<fieldset class="exp23">
+    <legend><a name="exp23.55">23.55 Send Biobricks to registry</a></legend>
+	<div class="exp-content">
+	<p>Preparation of both bricks:</p>
+	<ul class="list">
+    <li>15 &micro;l plasmid DNA (app. 200 ng/&micro;l) in PCR cup</li>
+    <li>Tape with Biobrick number around the PCR cup</li>
+    <li>Parafilm wrapped around the whole Eppendorf cup</li>
+    <li>50 ml Falkon with Shipping number, team name and shipping date</li>
+	</ul>
+	<br />
+	<table width="100%" border="1">
+		<tr>
+            <th scope="col">Part number</th>
+            <th scope="col">Name</th>
+        </tr>
+        <tr>
+            <th scope="col">Part:BBa_K1329004</th>
+			<td>synthetic catalyst part 1: tRNA scaffold</td>
+		  </tr>
+		  <tr>
+            <th scope="col">Part:BBa_K1329005</th>
+			<td>Synthetic catalyst part 2</td>
+		  </tr>
+	</table>
+	</div>
+</fieldset>
+<fieldset class="exp18">
+    <legend><a name="exp18.75">18.75 Investigation the interaction between the isolated flagella and StrepDARPidin</a></legend>
+	<div class="aim">
+	<p>Aim: A possible interaction of the flagella with StrepDARPidin should be visualized by electron microscopy</p>
+	</div>
+	<div class="exp-content">
+	<p>In order to visualize the interaction of the StrepDARPidin, the protein was incubated with the precipitated flagella and subsequently stained for electron microscopy.</p>
+	<p>Samples prepared:</p>
+	<ul class="list">
+    <li>1 = Hag-D2-Strep (1.2 mg/ml)</li>
+	<li>2 = Wildtype (2 mg/ml)</li>
+	<li>3 = PY-Strep (0.4 mg/ml)</li>
+	<li>4 = Hag-D2-Strep + StrepDARPidin (1:10)</li>
+	<li>5 = Wildtype+ StrepDARPidin (1:10)</li>
+	<li>6 = PY-Strep+ StrepDARPidin (1:10)</li>
+	<li>7 = StrepDARPidin (7 mg/ml)</li>
+	</ul>
+	<br />
+	<table width="100%" border="1">
+		<tr>
+			<th scope="col">Number</th>
+			<th scope="col">Flagellin</th>
+			<th scope="col">StrepDARPidin</th>
+			<th scope="col">Buffer (Saline citrate)</th>
+		</tr>
+		<tr>
+			<td>1</td>
+			<td>50 &micro;l</td>
+			<td>-</td>
+			<td>50 &micro;l</td>
+		</tr>
+		<tr>
+			<td>2</td>
+			<td>200 &micro;l</td>
+			<td>-</td>
+			<td>-</td>
+		</tr>
+		<tr>
+			<td>3</td>
+			<td>80 &micro;l</td>
+			<td>-</td>
+			<td>20 &micro;l</td>
+		</tr>
+		<tr>
+			<td>4</td>
+			<td>3.5 &micro;l</td>
+			<td>10 &micro;l</td>
+			<td>36.5 &micro;l</td>
+		</tr>
+		<tr>
+			<td>5</td>
+			<td>17.5 &micro;l</td>
+			<td>10 &micro;l</td>
+			<td>50 &micro;l</td>
+		</tr>
+		<tr>
+			<td>6</td>
+			<td>6 &micro;l</td>
+			<td>10 &micro;l</td>
+			<td>34 &micro;l</td>
+		</tr>
+		<tr>
+			<td>7</td>
+			<td>-</td>
+			<td>10 &micro;l</td>
+			<td>40 &micro;l</td>
+		</tr>
+	</table>
+	<br />
+	<p>Incubation on ice for 19 minutes before samples were stained for electron microscopy.</p>
+	</div>
+</fieldset>
 </div>

Team:Marburg:Project:Notebook:October

From 2014.igem.org

Latest revision as of 19:56, 17 October 2014

Notebook: October