• piGEM010 = 22ng/µl
• piGEM013 = 46 ng/µl
• piGEM 009 = 13 ng/µl

low concentration: new transformation to obtain more plasmid DNA
Strain: new competent E. coli XL1-blue

The miniprep was performed according to the miniprep protocol.

Concentration 40 ng/µL
low concentration: new transformation to gain more plasmid DNA should be done
Strain: new competent E. coli XL1-blue

Add 5µl plasmid DNA and incubate for 30 min at 37°C, LB plate with 5 ng/µL Cm (prepared freshly)

Starting from 1 mM, eight serial dilutions have been prepared

The resuspended cells from 14.37 were lysed using a french press. The lysate was incubated on ice with 5 µg/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C) to pellet cell debris. The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-1x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-1x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.

We received the synthetized DARPin-gene in a pEC-A2 vector with SpeI restriction sites at the 5' - and 3' -site with an amount of 2,7 µg plasmid per tube.

A stock solution was created by adding 27 µl water to the tube.

Concentration of stock solution = 100 ng/µl

A 1:10 dilution was created by using 1 µl of stock solution adding 9µL of water to an end concentration of 10ng/µl.

Concentration of stock dilution = 10 ng/µl.

1µl of the 1:10 dilution was used for transformation of E. Coli XL1-Blue

3 x 6ml LB-Amp were inoculated with clones from the successful transformed plate (18.2) and incubated at 37°C overnight.

Starting from 1 mM 8 dilutions have been prepared.
In the beginning Minimalmedium S750 has been prepared with 0.5‰ Xylose
In the wells the following mix was added to a total volume of 150 µl:
135µl of MM (90‰) and Silver solution (10‰)
15µl of cells
Finally 70 µl are added to avoid evaporation of the medium

Incubation for appr. 24 hours at 37°C shaking (Viktor AG Waldminghaus)

No positive GFP signal could be detected for the B. subtilis strain with the possible Ag sensitive promoter. The strain was restreaked onto a LB-Cm plate and he did not grow, which proved it to be a wrong strain, which has not been transformed successfully.

Every transformation plate showed a huge growth of bacillus without single colonies. After one day of incubation this seems to be an unrealistic growth, which is why we planned to repeat the transformation after checking the growth of the Bacillus subtilis wildtype 3610 on LB-CM plate with 5 µg/ml chloramphenicol.

Bacillus subtilis Wildtype 3610 was plated on LB-chloramphenicol plate with 5 µg/ml chloramphenicol and incubated overnight at 37°C.

Antibiotics and chemicals:

• Erythromycin 4 mg/ml
• Lincomycin 25mg/ml
• X-Gal 100mg/ml

For MLS selection plates erythromycin with 1 mg/ml were needed. Therefore the 4 mg/ml stock solution was diluted 1:4 by mixing 400 µl eryhtromycin with 1200 µl of 70% ethanol.

3 types of plates were made:

• 1. MLS selectin: 800 mL LB agar + 800 µl eryhtromycin + 800 µl lincomycin
• 2. MLS X-Gal: 800 mL LB agar + 800 µl eryhtromycin + 800 µl lincomycin + 800 µl X-Gal
• 3. X-Gal: 800 mL LB agar + 800 µL X-Gal

Plasmid pEX-A2 + DARPinEc1	Concentration after prep (ng/µl)
Clone 1	398,7
Clone 2	340
Clone 3	309

Making a pre-mix for Eurofins MWG prepairing the sequencing. Primers from Florian Altegoer were used:

• Flo83- Hag11_fw 1:10 with 1µl primer and 9µl water
• Flo86- Hag12_rv 1:10 with 1µl primer and 9µl water

	Pre-mix 1 (µL)	Pre-mix 2 (µL)
piGEM-016 pMAD-cup1-1 40ng/µL	10	10
Flo83- Hag11_fw	2	-
Flo86- Hag12_rv	-	2
Total Volume	12	12

Label	Pre-mix 1	Primer
AGB0023375	Pre-mix 1	Flo83 – Hag11_fw
AGB0023374	Pre-mix 1	Flo86 - Hag12_rv

New plates were made with new chloramphenicol-stocks with 25 mg/ml and 5 mg/ml.

3 aliquots of competent B.s. WT3610 were plated on new LB-chloramphenicol plates and incubated at 37°C.

Restriction	pEX-A2-DARPin digest (µl)	pMAD-Hag-Flank digest (µl)	Spe1-Mastermix
pEX-A2+DARPin (398 µg/µL)	15 = ca. 6µg	-	-
pMAD-Fla (114 µg/µL)	-	10= ca. 1,14 µg	-
SpeI	-	-	1,5
CutSmart-Buffer (10x)	-	-	2
H2O	-	-	16,5
Mastermix	5	10	-
Total	20	20	20

Incubation at 37°C for 2h and heat shock at 81°C for 5 min. After that the pMAD-hag-flank-construct was purified with the OMEGA gel extraction kit according to the protocol.

c(digested piGEM-005)= 4 ng/µl

Result: Bacillus subtilis Wildtype 3610 grew on the chloramphenicol plate. We have already repeated making plates so it might be that the WT has got a resistance while making it competent. A new WT was plated on 2 chloramphenicol plates with 5 µg/µL (old and new one) and 2x LB plates from gly stocks.

Incubation at 37°C overnight.

The PCR with 5' phosphorylated TG_0xGSSG FP und TG_0xGSSG RP should generate the 7491 bp fragment for ligation from the methylated template PheA-Arc1p-C-8x-pET28a from 14.5.

The used Primer concentration was 1 µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.

Content	Volume [µL]
Water	29.5
5x GC Buffer	10
DMSO	2.5
TG_0xGSSG FP	2.5
TG_0xGSSG RP	2.5
PheA-Arc1p-C-8x-pET28a	1
dNTPs	1
Phusion-Polymerase	1
Total Volume	50

Step	Temperature [°C]	Time [min:sec]
1	98	2:00
2	add Polymerase
3	98	0:10
4	67.5	0:30
5	72	4:00
6	go to 3	32x
7	72	10:00
8	4	hold

The amplified fragment was purified on an agarose gel on which it showed the correct size. The extracted linear template was then digested to degrade the original unmutated plasmid using DpnI.

Content	Volume [µL]
10x CutSmart Buffer	3.6
Plasmid	30
DpnI	3
Total Volume	36.6

The reaction mixture was incubated at 37 °C and 300 rpm for 1 h and afterwards heated to 80 °C for 20 min.

In order to create the circular plasmid 100 ng of the linear template were mixed with 1 µL 10x T4-Ligasebuffer, 1 µL T4-Ligase and water to give a total volume of 10 µL. This mixture was incubated for 9 h at 16 °C. The resulting plasmid was dialysed with water to decrease the salt concentration for the following transformation of E. coli Top10 electrocompetent cells with the plasmid.

The flagellin construct was amplified with specifically designed primers (95 and 54 from Florian Altegoer), which allow the amplification of the construct with cup1-1 without flanks (just Hag) including NcoI & BamHI restriction sites.
Template dilution 1:10:
1 µL pET24d-cup1-1 was mixed with 9 µL to an 1:10 dilution of 16,3 ng/µL
Primer dilution 1:10:
The primers were diluted 1:10 by mixing 10 µL of primers with 90 µL millipore water each.

Mix [µl]	reaction with phusion	reaction with Q5 mastermix
pET24d-cup1-1 1:10 dilution	1	1
Primer 54Flo	1	1
Primer 56Flo	1	1
Phusion	1	-
Phusion Buffer 5x	10	-
dNTP Mix	1	-
Water	35	22
Q5 Mastermix	-	25
Total Volume	50	20

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

The PCR was run over night.

Isolated pET24d was cut with NcoI and BamHI in order to create restriction sites for ligating the Flagellin/ Hag-Domain fragments into the vector. The Hag-fragments which were amplified via PCR with primers flo54 and 56 contain NcoI/ BamHI restriction sites for that purpose as well.

Component	Volume [µl]
pET24d (148 ng/µL)	7
CutSmart 10x	2
NcoI	0,5
BamHI	0,5
Water	10
Total Volume	20

Incubation for 1 hour at 37°C and heat shocked at 80°C.

Colonies were picked from plates and 8ml LB with necessary antibiotics each inoculated, followed by incubation at 37°C overnight.

Clones were picked from plates with plasmids:

• pET24d-Fla-Cup1-1 (piGEM-006)
• pMAD-Hag-Flank (piGEM-005)
• pMAD-Hag-Flank -Cup1-1 (piGEM-016)
• pEX-A2+DARPin (piGEM-017)

The sequencing showed the correct insertion of the cup-1-1 gene into the hag-flank construct but contained a stop codon, which was not removed by the used reverse primer. Unfortunately we used primers for amplification of the whole cup1-1 gene and not just for the truncated gene coding for the metallothionein domain only. Therefore new primers had to be designed an the Gibson assembly for integration of the metallothionein domain into pMAD and pET24d has to be repeated.

The with SpeI digested pEX-A2 was purified via electrophoresis with an 1% agarose gel in order to divide pEX-A2 backbone and the DARPin gene (489 bp). The digest attempt was splitted and loaded into two pockets to be sure not to overload the gel. The correct bands were cut out and the DNA fragment was rescued according to the protocol of our OMEGA gel extraction kit.

Expected bands:
DARPin gene: 489 bp
pEX-A2 backbone: 1961bp
The concentration after gel extraction: 9 ng/µl

The DARPin gene with SpeI sites was used as insert for the ligation into linearized pMAD (from 18.5) which contains the designed restriction site. The ligation attempt was done twofold and calculated by the Ligation Calculator from the iGEM-Team Texas in 2011:

Component	Volume [µl]
Insert (DARPin, c= 9 ng/µL)	2
Vector (piGEM-005, c=4ng/µL)	10
Ligase-Buffer 10x	2
T4 Ligase	1
Milipore water	5
Total Volume	20

Incubation was performed overnight at room temperature.

Result: Bacillus subtilis Wildtype 3610 grew on the CM plate.

In order to make out if a selection is possible WT3610 was plated out from an LB plate (13.62)  on 25 ng/ µL CM to check were the threshold of CM for B. subtilis is.

Incubation at 30°C over two days.

Restriction	pEX-A2-DARPin digest (µl)	pMAD-Hag-Flank digest (µl)	SpeI-Mastermix
pEX-A2+DARPin (398 µg/µL)	10 = ca. 4µg	-	-
pMAD-Fla (150 µg/µL)	-	7= ca. 1 µg	-
SpeI	-	-	1
CutSmart-Buffer (10x)	-	-	2,5
Water	-	-	21,5
Mastermix	10	13	-
Total Volume	20	20	25

Incubation was performed for 2 h at 37°C and heat inactivated at 80°C for 5 minutes.

The whole 20µL attempt from the ligations were used to transform E.Coli XL1-Blue, which were plated on LB-Amp plates.  Additionally a negative control was created by transforming E.Coli XL1-Blue with 1 µL SpeI cut piGEM-005 as well.

The SpeI digested pEX-A2 was purified via electrophoresis with an 1% agarose gel like in 18.6. The correct bands were cut out and the DNA fragment was purified according to the protocol of our OMEGA gel extraction kit.

Expected bands:
DARPin gene: 489 bp
pEX-A2 backbone: 1961bp
The concentration after gel extraction: 10 ng/µl

Plate	Amount of colonies
Negative control	6
Ligation attempt 1	7
Ligation attempt 2	0

In order to check the positive insertion of the DARPin gene into pMAD a PCR amplification with primers 54 and 56 for the flagellin should generate an ca. 1500 bp fragment on the gel. Negative clones would just show a 1000 bp band because of the amplification of the Hag-SpeI site construct. The picked clones were transferred on a new LB-amp plate.

Mix	Master-Mix for 7 attempts (µL)	Mix for PCR attempt (µL)
Colony	-	part of a colony
Primer Flo 54	3,5	-
Primer Flo 56	3,5	-
Phusion	7	-
Phusion Buffer 5x	28	-
dNTP Mix	3,5	-
Water	94,5	-
Mastermix	-	20
Total Volume	140	20

Expected bands:
Hag-DARPin-construct: ca. 1500 bp

The gel shows bands at 1000 which means that the clones contain just relegated pMAD vector and are all negative. The ligation had to be repeated.

The cut SpeI cut piGEM-005 was dephosporylated at the 5'-end with antarctic phosphatase to prevent the vector from relegation.

Component	Volume [µl]
Vector (piGEM-005, c=148 ng/µL)	20 (=ca. 2 µg)
Antarctic phos.-Buffer 10x	3
Antarctic phosphatase	1
Milipore Water	6
Total Volume	30

The sample was incubated for 1 h at 37°C at heat inactivated at 70°C for 10 minutes.

Because of the negative colony PCR from 18.13 a new ligation with dephosphorylized piGEM-005 from 18.14 and new extracted DARPin gene from 18.10 was performed in 2 attempts.

Component	Volume [µl]
Insert (DARPin, c= 10 ng/µL)	12,4
Vector (piGEM-005, c= 148 ng/µL)	4,6
Ligase-Buffer 10x	2
T4 Ligase	1
Milipore Water	5
Total Volume	20

Incubation 3h at room temperature

The whole 20 µL reaction of the ligations (18.15) were used to transform E. coli XL1-Blue and plated on LB-Amp plates.  Additionally a negative control was created by transforming E.Coli XL1-Blue with 1µL SpeI digested piGEM-005 as well.

Result: Bacillus subtilis Wildtype 3610 did not grow on the 25 ng/µl CM plate

In order to check at which concentration CM Bacillus cannot survive he was plated out from an LB plate on LB-CM plates with different CM concentrations (5, 10, 15 ng/mL).

Incubation at 30°C over night.

Plate	Amount of colonies
Negative control	1
Ligation attempt 1	2
Ligation attempt 2	>80

In order to check the positive insertion of the DARPin gene into pMAD a PCR amplification with primers 54 and 56 for the flagellin should generate an ca. 1500 bp fragment on the gel. Negative clones would show still just a 1000 bp band because of the amplification of the Hag-Spe1 site construct. The picked clones were transferred on a new LB-amp plate. 12 clones were picked.

Mix	Master-Mix for 14 attempts (µL)	Mix for PCR attempt (µL)
Colony	-	part of a colony
Primer Flo 54	7	-
Primer Flo 56	7	-
Phusion	14	-
Phusion Buffer 5x	56	-
dNTP Mix	7	-
Water	189	-
Mastermix	-	20
Total Volume	280	20

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	1min 45sec
4	72	1 min
5	Go To 2	30x
6	72	4min
7	8	infinite

Expected bands:
Hag-DARPin-construct: ca. 1500 bp

Clone 1, 2, 3, 4, 5, 7, 9, 11 and 12 were the positive clones at 1500 bp.

Six clones of the transformed E. coli Top10 cells from 14.39 were picked and used to inoculate overnight cultures (6 x 5 mL).

Because of the ligation via two SpeI sites, the chance is 50:50 that the clones contain the pMAD-Hag-Flank-DARPin construct in the right orientation (Startcodon on the Hag1-site). For test digests a higher amount of plasmid is needed, which is achieved by inoculation of minipreps.

The clones were used to inoculate 7 mL LB-Amp and incubated at 37°C over night.

Result: Bacillus subtilis Wildtype 3610 grew on the 5, 10 & 15µg/µl LB-CM plate.

We saw that the Bacillus grew on almost every CM concentrations except 25 µg/µL. So we transformed competent WT3610 with Nose plasmid piGEM-003 as a positive control and plated them out on LB-CM with 25 µg/µL and 35 µg/µL.

Additionally, competent wildtype 3610 cells were transformed with piGEM-004 and plated out on LB-CM with 5, 10 and 15 µg/mL CM to check, if these cells can be selected better.

Incubation at 30°C for 2 days.

The concentration of the isolated plasmids was between 100 and 150 ng/µL.

2 x 6 mL of LB were inoculated with Bacillus subtilis WT3610 from an LB plate and incubated at 37°C overnight.

The plasmids were purified from the overnight cultures created in 14.40 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.

The templates (piGEM-005, prepped pasmid from clone 7 & 12 ) were diluted 1:10. piGEM-005 should function as a negative control. Clone 12 was picked in case the second orientation could be used for different experiments. 5 µL PCR product + 1 µL loading dye were analysed on an 1% agarose gel.

Mix	Volume
Template 1:10	1
Primer Flo 54	1
Primer Flo 56	1
Phusion DNA-polymerase	1
Phusion Buffer 5x	10
dNTP Mix	1
Water	35
Total Volume	50

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	1min 45sec
4	72	2 min
5	Go To 2	30x
6	72	4min
7	8	infinite

40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-Arc1p-C-0x-pET28a from two different clones, incubated for 1 h at 37 °C and plated out on LB-Kan⁵⁰ plates that were incubated overnight at 37 °C.

The PCR products from 18.21 were digested with NcoI and BamHI to create restriction sites in order to ligate the fragments into NcoI/BamHI cut pET24d.

Component	Volume [µl]
PCR products	45
Cutsmart 10x	5,2
NcoI	1
BamHI	1
Water	2,8
Total Volume	52

 Incubation for 2 hours at 37°C with heat shock at 80°C.

The NcoI/BamHI digested flagellin PCR products from 18.22 (from clone 7 and piGEM-005) were ligated with NcoI/BamHI digested pET24d.

Component	Pet-Hag attempt (µL)	Pet-Hag-DARPin attempt (µL)
Insert (PCR fragments , c= ca. 400 ng/µL)	2	2
Vector (pet24d, c= 50ng/µL)	5	5
Ligase-Buffer 10x	2	2
T4 Ligase	1	1
Millipore Water	10	10
Total Volume	20	20

Incubation over night at room temperature.

100 µL of overnight culture were added to 10 mL of MNGE-Medium and incubated to an OD₆₀₀ of 1,1-1,3 at 37°C which could take 4-5 hours.

After reaching an OD of 1,1-1,3 2 x 400 µL of the culture were transformed with 1,5 µg piGEM-005 and -018. After one hour incubation at 37°C 100 µL expression mix were added and incubated for one hour as well.

In the end the 500 µL sample was plated out on MLS-X-Gal plates and incubated at 30 °C until colonies could be seen.

The whole 20 µL attempt was used to transform E. coli X1-Blue, plated out on LB-Can plates and incubated at 37°C overnight.

2 x 6 mL of LB-Amp were inoculated with a colony from a piGEM-018 transformation plate and incubated at 37°C overnight.

pMAD-Hag-Flank-DARPin was sent in form of a premix with primers for sequencing. 10 µL with 50-100 ng/µL were mixed with 2 µL of primers for sequencing according to the following scheme:

Premix	Label-Nr.	Construct	Primer
1	391	pMAD-Hag-Flank-DARPin	Flo54 (Hag-F-Nco)
2	392	pMAD-Hag-Flank-DARPin	Flo56 (Hag-R-Bam)

Competent 3610 were transformed with piGEM-002, -003 and -004 by incubating the aliquots with 5 µL plasmid for 30 min at 37°C. After half an hour the whole sample was plated out on LB-Cm (5 ng/µL).

Incubation at 30°C for 2 days.

The miniprep of pMAD-Hag-Flank-DARPin was done according to the protocol of the OMEGA DNA kit.

Because of the unsuccessful ligation from 18.23 new vector (pET24d) was digested with NcoI and BamHI according to the following scheme:

Component	Volume [µl]
pET24d (148 ng/µL)	15
Cutsmart 10x	3
NcoI	0,5
BamHI	0,5
Water	6
Total Volume	30

Incubation at 37°C for 2h.

The DARPin-Hag constructs contain a PstI restriction site which could be used to check if the DARPin gene was inserted into the Hag-construct correctly again. For that purpose PCR products based on piGEM-018 from clone 7 and 12 (right and wrong orientation) as a template were digested with PstI.

PCR products from clone 7: 658 bp and 781 bp fragment.

 PCR products from clone 12: 1000bp and 429 bp fragments.

Component	Volume [µl]
PCR fragment (350 ng/µL)	3
Cutsmart 10x	1
PstI	0,5
Water	5,5
Total Volume	10

Incubation at 37°C for 2h.

piGEM-018 & -005 were used as template for a PCR with the primers 54flo and 56flo. The products could be used for further cloning after NcoI/BamHI digest.

Mix	Volume
Template (piGEM-005 & -018)	1
Primer Flo 54	1
Primer Flo 56	1
Phusion	1
Phusion Buffer 5x	10
dNTP Mix	1
Water	35
Total Volume	50

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	1min 45sec
4	72	2 min
5	Go To 2	30x
6	72	4min
7	8	infinite

The fragments were analysed on an 1% agarose gel.

The cup1-1 domain was amplified via PCR with new primers iGEM-024 and -025. The fragment should be ca. 160 bp.

Mix	Volume
Template (cup1-1 gene)	1
Primer iGEM-024	1
Primer iGEM-025	1
Phusion DNA-polymerase	1
Phusion Buffer 5x	10
dNTP Mix	1
Water	35
Total Volume	50

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

According to the gel analysis the amplification was successful.

5 mL of LB-Kan⁵⁰ medium were inoculated with a clone from 14.42 bearing the PheA-Arc1p-C-0x-pET28a plasmid.

The 6 overnight cultures were used to inoculate 10 mL LB MLS, which were incubated until the culture obtained an OD of 0,1. The cultures were incubated at 30°C for 2h.

Subsequently, the temperature was shifted to 42°C for 6h.

After the heat shock dilutions from 10^-4 to 10^-6 of each culture were plated out on MLS-X-Gal so that 18 plates could be incubated overnight at 42°C.

The cup1-1 domain was amplified via PCR with new primers iGEM-024 and -025. Vector and template were diluted to a concentration under 75 ng/µL. Spe1 cut piGEM-005 was used from the cloning experiments with DARPin.

PCR fragment cup1-1 (464 ng/µL) dilution 1:10

SpeI digested pIGEM-005 (148 ng/µL) dilution 1:2

Component	Volume [µl]
Gibson-Mix	15
Insert (cup1-1 58 ng/µL)	1,3
piGEM-005	1
Water	2,7
Total Volume	20

The mix was incubated 5 min at room temperature and then for 1h at 50°C.

In the end the whole mix was transformed into E.Coli XL1-Blue and plated out on LB-Amp. Incubation was done overnight at 37°C.

Using the preculture from 14.43 60 mL of LB-Kan⁵⁰ medium were inoculated with 600 µL of the preculture and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The culture was cooled down to 18 °C and the protein production was induced with 12 µL of 500 mM IPTG. Over night the culture was incubated at 18 °C and 220 rpm. Afterwards the cell culture was centrifuged (17000 rpm, 20 min, 4 °C), the pellet resuspended in 3 mL buffer A and stored at -20 °C. A glycerolstock of the strain containing the PheA-Arc1p-C-0x-pET28a plasmid was stored at -80 °C.

One blue colony per diluted clone was used to inoculate 4 mL LB. The 6 cultures were incubated at 30°C for 6h and afterwards for 3h at 42°C.

Dilutions from 10^-4 to 10^-4 were plated out on 18 X-Gal plates without MLS selection. The positive clones should not contain the resistance inside the backbone as well as the galactosidase, resulting in normal colored colonies. The plates were incubated at 42°C overnight.

Unfortunately the plate was empty: no colonies. The vector was digested again in order to repeat the assembly.

In order to repeat the Gibson assembly the piGEM-005 was cut with SpeI again.

Component	Volume [µl]
piGEM-005 (108 ng/µL)	10
Cutsmart 10x	2
SpeI	0,5
Water	7,5
Total Volume	20

Incubation 1h at 37°C with following heat shock at 80°C

One white clone was picked from the dilution plates and transferred on a X-Gal Plate as well as on a MLS plate so that 18 clones were tested for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C over night.

As insert cup1-1 PCR product from 15.55 and SpeI digested piGEM-005 from 15.55 were used for a new Gibson assembly.

PCR fragment cup1-1 (464 ng/µL)

SpeI digested pIGEM-005 (53 ng/µL)

Component	Volume [µl]
Gibson-Mix	15
Insert (cup1-1 58 ng/µL)	2,5
piGEM-005	2,5
Total Volume	20

The mix was incubated 5 min at room temperature and then for 1h at 50°C.

In the end the whole mix was used to transform E. coli XL1-Blue and plated out on LB-Amp. Incubation was done overnight at 37°C.

The white colonies grew on the X-Gal Master plates but not on MLS plates so the transformation seemed to be successful. The Backbone with the MLS resistance flipped out of the genome.

No clone was growing on MLS plates so the picked clones seemed to be positive. In order to check the correct integration of the domain constructs, a cPCR with the picked clones on the X-Gal plates was done. The 18 picked B.subtilis clones were cooked in 10 µL PBS for 5 min at 95°C.

Mix	Master-Mix for 18 attempts (µL)	Mix for PCR attempt (µL)
Colony (picked clones 1-9 from Flagellin/-DARPin B.s. plates)	-	Part of a cooked colony
Primer Flo54 Hag-fw	9	-
Primer Flo56 Hag-rv	9	-
Phusion	18	-
Phusion Buffer 5x	72	-
dNTP Mix	9	-
Water	237	-
DMSO	6	-
Mastermix	-	20
Total Volume	100	360

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	1 min 45 sec
4	72	2 min
5	Go To 2	30x
6	72	4min
7	8	infinite

The gel showed no bands. The PCR had to be repeated.

4 x 5 mL of LB-Amp was were inoculated with a picked colony from a transformation plate with XL1-Blue containing the Gibson-assembly product from 15.56.

Incubation at 37°C overnight.

In order to check the correct insertion of the cup1-1 domain into the Hag-Flank-construct in pMAD a cPCR with primers iGEM-025 (Cup1-1 rv) and Flo89 (pMAD-flank1-BamHI fw) was performed. The picked colonies were the same which were used to inoculate minipreps from 15.57. The fragment was supposed to have approx. 1250 bp.

Mix	Master-Mix for 5 attempts (µL)	Mix for PCR attempt (µL)
Colony (picked clones 1-4 for miniprep)	-	Part of a colony
Primer Flo89	2,5	-
Primer piGEM-025	2,5	-
Phusion DNA-polymerase	2,5	-
Phusion Buffer 5x	20	-
dNTP Mix	2,5	-
Water	70	-
Mastermix	-	20
Total Volume	100	20

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

The analysis via 1% agarose gel eletrophoresis showed now bands. The plasmids of all four clones were isolated to use them as template.

This time the isolated plasmid DNA from the picked six clones was used as a template with different primer pairs. First of all the PCR reactions were performed with primers Flo89 and Flo90 in order to receive the whole Hag-Flank-Cup1-1 construct with ca. 2100 bp length. As negative control also piGEM-005 was used as template producing a 2000kb fragment. Because of the small difference, all six reactions were also performed with the primers Flo89 and iGEM-025 (cup1-1 rv) in order to check the insertion of cup1-1 directly with a fragment of 1268 bp.

Mix	Master-Mix 1 for  5 reactions (µL)	Master-Mix 2 for  5 reactions (µL)	Mix 1 for PCR reactions (µL)	Mix 2for PCR reactions (µL)
Plasmid DNA from clone 1-4/ piGEM-005	-	-	1	-
Primer Flo89	2,5	2,5	-	-
Primer Flo90	2,5	-	-	-
Primer piGEM-025	-	2,5	-	-
Phusion DNA-polymerase	2,5	2,5	-	-
Phusion Buffer 5x	20	20	-	-
dNTP-mix	2,5	2,5	-	-
Water	70	70	-	-
Master-Mix 1/ 2	-	-	20	20
Total Volume	100	100	20	20

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	20 sec
5	Go To 2	30x
6	72	4min
7	8	infinite

The gel analysis shows that all 4 clones seem to be positive. An amplification of the cup1-1 fragment with the plasmid DNA as template could proof that.

Because of the unsuccessful cPCR before, the PCR was repeated. The 18 picked B.subtilis clones were cooked in 30 µL PBS for 10 min at 95°C.

Mix	Master-Mix for 18 attempts (µL)	Mix for PCR attempt (µL)
Colony (picked clones 1-9 from Flagellin/-DARPin B.s. plates)	-	Part of a cooked colony
Primer Flo54 Hag-fw	9	-
Primer Flo56 Hag-rv	9	-
Phusion DNA-polymerase	18	-
Phusion Buffer 5x	72	-
dNTP Mix	9	-
Water	237	-
DMSO	6	-
Mastermix	-	20
Total Volume	100	360

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	1 min 45 sec
4	72	2 min
5	Go To 2	30x
6	72	4min
7	8	infinite

All nine clones with piGEM-005 seem to be positive. Clone 1, 3, 7, 8 and 9 of transformed piGEM-018.1 might be positive as well as clone 2-6 from piGEM-018.2. The sequencing of the PCR products should tell us if the insertion was successful.

piGEM-018 & -005 were used as template for a PCR with the primers Flo 54 and Flo 56. The products could be used for further cloning after NcoI/BamHI digest.

Content	Volume [µl]
Template (piGEM-005 & -018)	1
Primer Flo 54	1
Primer Flo 56	1
Phusion	1
Phusion Buffer 5x	10
dNTP Mix	1
Water	35
Total Volume	50

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	30x
6	72	4min
7	8	infinite

More clones from each plate were picked to analyse them via cPCR with the primers flo54 and flo56 (Hag-fw and Hag-rv). The result should be a ca. 1kb fragment for correct pET24d-Hag containing clones and a 1,5 kb fragment for pET24d-Hag-DARPin containing clones.

Content	Volume (µL)	MM for 14 attempts (µL
Colony  (8-15 from pet-Hag,7-14 from pet-Hag-DARPin)	1 colony	-
Primer Flo 54	-	7
Primer Flo 56	-	7
Phusion DNA-polymerase	-	7
Phusion Buffer 5x	-	56
dNTP Mix	-	7
Water	-	196
Mastermix	20	-
Total Volume	20	280

Step	Temperature °C	Time
1	98	10 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

Clone 1 from each pET24d-Hag-SpeI and pET24d-Hag-DARPin clones was positive and were digested with NcoI/BamI to check them again.

Because of the low amount of right clones, new vector (pET24d) was digested with NcoI and BamHI according to the following scheme:

Component	Volume (µL)
pET24d (50 ng/µL)	20
CutSmart 10x	3
NcoI	0,5
BamHI	0,5
Water	6
Total Volume	30

Incubation at 37°C for 2 hours with heat shock in the end at 80°C

Endconcentration: 32 ng/µL

The PCR products from 18.31 were digested with NcoI andBamHI to create restriction sites in order to ligate the fragments into NcoI/BamHI digested pet24d in case of negative clones refering to the cPCR from 18.x

Component	PCR Hag-fragment (µL)	PCR Hag-DARPin fragment (µL)
PCR products	45	45
Cutsmart 10x	5,2	5,2
NcoI	0,5	0,5
BamHI	0,5	0,5
Water	3,8	3,8
Total Volume	52	52

Incubation for 2h at 37°C with heat shock at 80°C.

Endconcentration:
digested Hag-fragment 260 ng/µL
digested Hag-fragment 268 ng/µL

Using the plasmids as template the cup1-1 fragment at 160 bp should be visible on the agarose gel. As positive control PCR products from the cup1-1 amplification used for the Gibson Assembly into piGEM-005 were applied to the gel as well.

Content	Volume (µL)	MM for 5 attempts (µL)
Template (Plasmid from clone 1-4)	0,5	-
Primer iGEM-024	-	2,5
Primer iGEM-025	-	2,5
Phusion DNA-polymerase	-	2,5
Phusion Buffer 5x	-	20
dNTP Mix	-	2,5
Water	-	70
Mastermix	19,5	-
Total Volume	20	100

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	20 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

The bands under the 200 bp mark indicate the positive insertion of cup1-1 into piGEM-005. The plasmid has to be sequenced and is named iGEM-016.

The plasmids are digested with NcoI and BamHI. In case of a correct insertion the pET24d backbone and the insert should be seen on the gel at 5300 bp and 1000/ 1500 bp. As control pET24d-Hag-Flank (piGEM-001) is used to check the mastermix.

Component	Volume (µl)	Mastermix
2x Plasmid from clone 6	5	-
CutSmart 10x	-	5
NcoI	-	0,5
BamHI	-	0,5
Water	-	19
Total Volume	10	25

The used marker was the GeneRuler 1 kb DNA ladder. The lower fragment of the digested pET24d-Hag was too big (2 kb) and pET24d-Hag-DARPin seemed only to be linearized with the exception of two very thin bands below the linear plasmid, which were all of the wrong size.

The NcoI/BamHI digested flagellin PCR products from 18.22 (from clone 7 and piGEM-005) were ligated with NcoI/BamHI digested pET24d.

Component	pET24d-Hag reaction (µL)	pET24d-Hag-DARPin raction (µL)
Insert (PCR fragments) (ca. 260 ng/µL)	2	2
Vector (pET24d, c= 50ng/µL)	5	5
Ligase-Buffer 10x	2	2
T4 Ligase	1	10
Millipore Water	10	10
Total Volume	20	20

The reactions were prepared in double and were incubated at room temperature. One reaction was incubated for 1 hour followed by transformation of E. coli XL1-Blue and the second one over night.

3 clones from ligation plates with pET-Hag and pET-Hag-DARPin were resuspended in 30 µL PBS and cooked at 95 °C for 10 min. After that procedure 1 µL of this suspension was used as PCR template.

Content	Volume (µL)	MM for 7 reactions (µL)
Template ( 1µL cooked suspension)  (8-10 from pET-Hag, 7-9 from pET-Hag-DARPin)	1	-
Primer Flo 54	-	3,5
Primer Flo 56	-	3,5
Phusion DNA-polymerase	-	3,5
Phusion Buffer 5x	-	28
dNTP-mix	-	3,5
Water	-	98
Mastermix	19	-
Total Volume	20	140

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

Run over night

Lysozyme was added to the cell suspension from 14.44 to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 °C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.

60 mL LB-Kan⁵⁰ medium was inoculated from the glycerolstock prepared in 14.44 and incubated at 37 °C and 220 rpm over night.

Gel electrophoresis:

The gel shows negative bands in case of the Hag-DARPin clones. The PCR for Hag 8 and 9 did not work although we cooked the clones in PBS. The transformation efficiency seemed to be very low, so clones from the new ligation attempts should be analysed.

On every plate grew a high amount of colonies. 6 clones of each plate were picked for inoculation of minipreps and for cPCR after cooking with PBS. The clones were transferred to another LB-Can plate.

For checking the insertion of the Hag/-DARPin into pET24d the clones from the transformation plate were transferred to another LB-Can plate  and cooked for 10 min in 30 µL PBS at 95°C. At the same time the colonies were used to inoculate LB-Can for minipreps.

Content	Volume (µL)	MM for 14 attempts (µL)
Template ( 1µL cooked suspension)  (1-6 from pet-Hag, 1-6 from pet-Hag-DARPin)	1	-
Primer Flo 54	-	7
Primer Flo 56	-	7
Phusion DNA-polymerase	-	7
Phusion Buffer 5x	-	198
dNTP-mix	-	7
Water	-	98
Mastermix	19	-
Total Volume	20	280

Step	Temperature °C	Time
1	98	5 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

Run over night

10 x 500 mL LB-Kan⁵⁰ medium were inoculated with 5 mL of the overnight culture prepared in 14.45 and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The flasks were cooled to 18 °C and protein production was induced by adding 100 µL 500 mM IPTG. The cultures were incubated over night at 18 °C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 °C), resuspended in buffer A and stored at -20 °C until further use.

Gel electrophoresis: no bands except for the controls were seen. The PCR has to be repeated with more colony in the PCR tubes or the prepped plasmids

The picked clones from the ligation attempts 18.38 were isolated. In order to check the insertion of the Hag/ Hag-DARPin domain the plasmids were digested with NcoI/BamHI so that the gel should show the insert of 1 or 1,5 bp length and the pET24d backbone at 5300 bp. Because of an unavailable marker as a control the PCR amplified inserts which were used for the ligation will run  with the gel as well. piGEM-001 will also be cut NcoI/BamHI in order to receive a marker band for the pET24d backbone.

Component	Volume (µL)	Mastermix for 8 attempts
Plasmid (ca. 45 ng/µL	5	-
Cutsmart 10x	-	4
NcoI	-	0,5
BamHI	-	0,5
Water	-	35
Mastermix	5	-
Total Volume	10	40

The gel shows that the digested clones were all negative. The ligation had to be performed again.

Purified samples:
clone 3 & 5 from transformed piGEM-005
clone 3 & 9 from transformed piGEM-018

Clone	Concentration (ng/µl)
Hag 3	10
Hag 5	14
Hag-DARPin 3	9
Hag-DARPin 9	9

The concentration of the cPCR samples after purification was too low for sequencing. Therefore the cPCR samples have to be amplified with PCR again.

Content	Volume (µL)	MM for 6 attempts (µL)
Purified cPCR product from 20.6  (3 & 5 from pet-Hag, 3 & 9 from pet-Hag-DARPin)	1	-
Primer Flo 54	-	6
Primer Flo 56	-	6
Phusion DNA-polymerase	-	6
Phusion Buffer 5x	-	6
dNTP-mix	-	60
Water	-	216
Mastermix	19	-
Total Volume	20	300

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

Premix	Label-Nr.	Construct	Primer
1	AGB0023407	Hag-SpeI in B. subtilis genome clone 3	Hag-Nco-fw
2	AGB0023408	Hag-SpeI in B. subtilis genome clone 5	Hag-Nco-fw
3	AGB0023409	Hag-SpeI-DARPin in B. subtilis genome clone3	Hag-Nco-fw
4	AGB0023410	Hag-SpeI-DARPin in B. subtilis genome clone 9	Hag-Nco-fw

The cut pET24d NcoI/BamHI which was used for the previous ligations was not purified and contained an 1 kb insert. For a new clean ligation the reaction was repeated with digested pET24d. The NcoI/BamHI digested flagellin PCR products (from clone 7 and piGEM-005) were ligated with NcoI/BamHI digested pET24d.

The reactions were carried out in double and were incubated 1h at room temperature. Finally they were used to transform E. coli XL1-Blue after inactivation at 65°C for 10 min. The attempts 1.1 and 2.1 were incubated over night at room temperature. 1 µL of digested vector was used for a transformation as a negative control in order to check the amount of religants.

Component	pET-Hag attempt (µL) – 1 & 1.1	pET-Hag-DARPin attempt (µL) 2& 2.1
Insert (PCR fragments) (ca. 260 ng/µL)	3	3
Vector (pET24d, c= 86ng/µL)	5	5
Ligase-Buffer 10x	2	2
T4 Ligase	1	1
Millipore water	9	9
Total Volume	20	20

The ligation between pET24d and the Hag-/Hag-DARPin fragments which were both cut NcoI/BamHI was successful. The ligation plates showed over 20 clones each. However, on the negative control  grew a few clones as well, which were very small and sticking together. In order to analyse the insert of the clones, 5 clones per ligation plate 1 and 2 were used to inoculate minipreps in 5 mL LB-Can. For the Ligation 1 pET-Hag clones 1-5 were picked, as well as clones 6-10 from Plate Ligation 2 pET-Hag-DARPin.

The cultures were inoculated over night at 37°C.

Construct	Clone	Concentration (ng/µL)
pET24d-Hag	1	50
pET24d-Hag	2	87
pET24d-Hag	3	65
pET24d-Hag-DARPin	6	83
pET24d-Hag-DARPin	7	59

Plasmid from isolated clones from each plate were analysed via cPCR with the primers flo54 and flo56 (Hag-fw and Hag-rv). The result should be a ca. 1kb fragment for correct pET24d-Hag containing clones and a 1,5 kb fragment for pET24d-Hag-DARPin containing clones.

Content	Volume (µL)	MM for 6 attempts (µL)
Picked clones from 18.42 (1-3 pET24d-Hag, 6-7 from pET24d-Hag-DARPin)	0,5	-
Primer Flo 54	-	3
Primer Flo 56	-	3
Phusion DNA-polymerase	-	3
Phusion Buffer 5x	-	6
dNTP-mix	-	24
Water	-	81
Mastermix	19,5	-
Total Volume	20	120

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	30x
6	72	4min
7	8	infinite

The samples can be seen on the gel under 18.44. The PCR for the Hag-fragment in pET24d seemed to be positive (PCR.H1 - H3), as well as for the Hag-DARPin fragment (PCR.D7)

The plasmids were digested with NcoI and BamHI. In case of a correct insertion the pet24d backbone and the insert should be seen on the gel at 5300 bp and 1000/ 1500 bp. As control pET24d-Hag-Flank (piGEM-001) is used to check the mastermix.

Component	Volume (µL)	Mastermix(6 attempts)
Plasmid from clone ( pET24d-Hag 1-3, pET24d-Hag-DARPin 6 & 7)	5	-
Cutsmart 10x	-	3
NcoI	-	0,5
BamHI	-	0,5
Water	-	26
Total Volume	10	30

The PCR product of the amplification of cPCR products was not pure considering different bands on the gel next to the right one. In order to prevent sequencing mistakes the PCR attempts were purified via gel extraction of the 1,5 kb band in case of the Hag-DARPin construct.

The purified cPCR product from 20.9 has to be amplified again in order to get a higher concentration for sequencing.

Content	Volume (µL)	MM for 2 attempts (µL)
Purified PCR product from 20.x (Hag-/ Hag-DARPin fragment)	1-2	-
Primer Flo 54	-	1
Primer Flo 56	-	1
Phusion DNA-polymerase	-	1
Phusion Buffer 5x	-	2
dNTP-mix	-	20
Water	-	75
Mastermix	19	-
Total Volume	20	100

Step	Temperature °C	Time
1	98	5 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

Component	Volume (µL)
piGEM-016 (ng/µL)	17
CutSmart 10x	2
SpeI	0,5
Water	0,5
Total Volume	20

Incubation 1 h at 37°C with following heat inactivation at 80°C

As insert cup1-1 PCR product from 15.55 and SpeI digested piGEM-016 from 18.44 were used for a new Gibson assembly.
PCR fragment cup1-1 (464 ng/µL)
SpeI digested pIGEM-005 ( ng/µL)

Component	Volume (µl)
Gibson-mix	15
Insert (cup1-1 464 ng/µL)	2,5
piGEM-019	2,5
Total Volume	20

The mix was incubated 5 min at room temperature and then for 1h at 50°C.

In the end the whole mix was used to transform E. coli XL1-Blue, which were plated out on LB-Amp. Incubation was done over night at 37°C.

In order to screen the clones for the right insertion of cup1-1 four clones were picked from the LB-Can plate and transferred onto a LB-Can plate. Additionally the clones were used for inoculation of minipreps and also cooked in 50 µL dest. Water for 10 min at 95°C. The lysate was used as template.

The PCR with Flo54 and iGEM-025 should generate a 785 bp fragment and the one with iGEM-024 and -025 the 164bp cup domain fragment.

Content	Volume (µL)	MM 1 for 5 attempts (µL)	MM 2 for 5 attempts (µL)
Cooked lysate solution from clones 1-4	3	-
Primer Flo 54	-	2,5	-
Primer iGEM-024	-	-	2,5
Primer iGEM-025	-	-	2,5
dNTP-mix	-	2,5	2,5
Phusion DNA-polymerase	-	5	5
Phusion Buffer 5x	-	20	20
Water	-	67,5	67,5
Mastermix	17	-	-
Total Volume	20	100	100

Step	Temperature °C	Time
1	98	10 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

The PCR showed no bands. Nevertheless, colonies were used to inoculate cultures for minipreps so that the isolated plasmid can be further analysed for the insert.

Construct	Clone	Concentration (ng/µL)
pET24d-Hag-Cup1-1	1	122
pET24d-Hag-Cup1-1	2	85
pET24d-Hag-Cup1-1	3	172
pET24d-Hag-Cup1-1	4	116

The isolated plasmid 1:10 diluted from the picked clones 1-4 were used as PCR template.

The PCR with Flo54 and iGEM-025 should generate a 785 bp fragment and the one with iGEM-024 and -025 the 164bp domain fragment.

Content	Volume (µL)	MM 1 for 5 attempts (µL)	MM 2 for 5 attempts (µL)
Cooked lysate solution from clones 1-4 (1:10)	3	-
Primer Flo 54	-	2,5	-
Primer iGEM-024	-	-	2,5
Primer iGEM-025	-	2,5	2,5
dNTP-mix	-	2,5	2,5
Phusion	-	5	5
Phusion Buffer 5x	-	20	20
Water	-	67,5	67,5
Mastermix	17	-	-
Total Volume	20	100	100

Step	Temperature °C	Time
1	98	3 min
2	98	30 sec
3	55	30 sec
4	72	1 min 45 sec
5	Go To 2	35x
6	72	4min
7	8	infinite

No bands could be observed on the gel. The PCR was repeated with Q5 mastermix to exclude the disfunction of the polymerase.

2 x 6 mL of LB were inoculated with Bacillus subtilis WT3610 from an LB plate and incubated at 37°C over night.

The resuspended cells from 14.46 were lysed using a french press. The lysate was incubated on ice with 5 µg/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C) to pellet cell debris. The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-0x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-0x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.