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

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

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	-	-
Sample X1	1	H	10418	GeFi	2 µM
Sample X2	2	H	19020	GeFi	1 µM
Sample X3	3	H	6673	GeFi	100 nM
Sample X4	4	H	8559	GeFi	10 nM
Sample X5	5	H	5743	GeFi	100 pM
Sample X6	6	H	9033	GeFi	10 pM
Sample X7	7	H	9090	Ni-NTA	2 µM
Sample X8	8	H	8796	Ni-NTA	1 µM
Sample X9	9	H	5356	Ni-NTA	100 nM
Sample X10	10	H	6684	Ni-NTA	10 nM
Sample X11	11	H	6930	Ni-NTA	100 pM
Sample X12	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.

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

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

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

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

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

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.

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.

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.

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.

	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				20 µL slurry
Strep-bead			x	x	x	30 µL slurry
Hag-D2-Strep	x		x			4 nmol → 4,5 µL
Hag-Strep		x		x		4 nmol → 3,4 µL

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

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

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

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.

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

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

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

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

A colony PCR was carried out.

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.

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).

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.

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°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 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	Blank corrected raw data (485, 520)	Purification	StrepDARPidin concentration [M]
Sample X1	1	F	5782	GeFi	50 µM
Sample X2	2	F	4912	GeFi	25 µM
Sample X3	3	F	3903	GeFi	2,5 µM
Sample X4	4	F	3513	GeFi	250 nM
Sample X5	5	F	3613	GeFi	25 nM
Sample X6	6	F	3211	GeFi	2,5 nM
Sample X7	7	F	7340	Ni-NTA	50 µM
Sample X8	8	F	5329	Ni-NTA	25 µM
Sample X9	9	F	5846	Ni-NTA	2,5 µM
Sample X10	10	F	5718	Ni-NTA	250 nM
Sample X11	11	F	3852	Ni-NTA	25 nM
Sample X12	12	F	6828	Ni-NTA	2,5 nM
Sample X13	5	G	5699	-	-
Sample X14	6	G	9983	-	-
Sample X15	7	G	1760	-	-
Sample X16	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.

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

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

	1	2	3	4	used
StrepDARPidin	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	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.

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

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

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

Negative clones: 1.2, 1.4, 2.3 and 2.9

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

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.

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.

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

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.

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.

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

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

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

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

Result: PstI is contaminated with EcoRI and will be thrown away!

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.

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

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

see above

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

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.

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

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.

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

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

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

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

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

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

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

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

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. (ca 40 ng/µl)	-	-	-	-	1	-	-
piGEM008 + Cu	-	-	-	-	-	1	-
iGEM059 (1:50)	3,3	3,3	3,3	3,3	-	-	-
iGEM073 (1:50)	-	-	-	-	3,3	-	3,125
iGEM055 (1:50)	-	-	-	-	-	3,3	-
iGEM006 (1:50)	3,3	-	-	-	3,3	-	-
iGEM021 (1:50)	-	3,3	-	-	-	-	-
iGEM022 (1:50)	-	-	3,3	-	-	3,3	-
iGEM023 (1:50)	-	-	-	3,3	-	-	3,125
5x HF buffer	5	5	5	5	5	5	5
dNTP mix (10 mM each)	0,3	0,3	0,3	0,3	0,3	0,3	0,3
Phusion DNA polymerase 1:10	1	1	1	1	1	1	1
Water	11,1	11,1	11,1	11,1	11,1	11,1	11,1
Total	25	25	25	25	25	25	25

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

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

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

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

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

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

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

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

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

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

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

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 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 (485, 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.

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.

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.

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.

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

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

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

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

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