Digestion scheme:

[µL]	PSG1164 - uncut	PSG1164 - PciL (10 kU/mL)	PSG1164 - EcoRI-HF
DNA	261,3 ng/µL ca. 1 µg → 3,5 µL	261,3 ng/µL ca. 1 µg → 3,5 µL	261,3 ng/µL ca. 1 µg → 3,5 µL
Enzyme 1	-	PciI 1,0	-
Enzyme 2	-	-	EcoRI-HF 0,5
Buffer	-	10x 3.1 Buffer 1,5	10x Cutsmart 1,5
H2O	11,5	9,0	9,5
Total Volume	15	15	15
Loading Dye (6x)	2,5	2,5	2,5

Incubate samples for 60 min at 37 °C, then induce a heat shock for 2 min at 45 °C. 5 µL marker and 10 µL of the samples are loaded onto the SDS-gel.

Digestion scheme:

[µL]	pMA12 - uncut	pMA12 - HindIII + EcoRI-HF (20 kU/mL)
DNA	479,3 ng/µL ca. 1 µg →2,1 µL	479,3 ng/µL ca. 5 µg → 10,5 µL
Enzyme 1	-	HindIII 0,5
Enzyme 2	-	EcoRI 0,5
Buffer	-	10x -2.0 Buffer 1,5
H2O	12,9	2
Total Volume	15	15
Loading Dye (6x)	2,5	2,5

Dilution of primers:

Stock: dilution of primers with aqua bidest. to 100 µM
Working stock: 1:10 (10 µL primer stock, 90 µL water)

Oligo Name	µL for 100 µM
iGEM-001	292 µL
iGEM-002	236 µL
iGEM-003	397 µL
iGEM-004	291 µL
iGEM-005	364 µL
iGEM-006	273 µL

Dilution of templates:

• Bacillus gDNA: 1:50 → 10 µL gDNA + 490 µL water
• pKH-Stp: 10 ng/µL
• PSG1164: 232 ng/µL → 1:20 → 10 µL plasmid + 190 µL water

Reaction Mix

	1&2 plasmid PSG1146 (856 bp)	3&4 Bacillus gDNA (543 bp)	5&6 plasmid KG-Stp (743 bp)
Fragment	Cat-resistance	amyE-gene	GFP
Template	1,5	1,5	1,5
dNTPs	1	1	1
Primer fwd	1	1	1
Primer rev	1	1	1
Phusion Buffer (5x)	10	10	10
Phusion	1	1	1
H2O	34,5	34,5	34,5
Total Volume	50	50	50

Purification according to the "Purification of PCR Products (QIAquick Gel Extraction Kit) short protocol".

Samples:

• 1' Cat-resistance
• 2' amyE-Gene
• 3' GFP
• 4' (=2+4+6)-Pool, all fragments

[µL]	1''-Control	2''-P+frag	3''-P*+pool	4''-P**(DS)+pool
PEG 3350(50% w/v)	260	260	260	260
LiAc 1 M	36	36	36	36
Salmonsperm(2 mg/mL)	50	50	50	50
H2O	14	10	2	2
Fragm. (GFP, Cat, AmyE)	-	4	-	-
Pool (all fragments)	-	-	12	12
Total Volume	360	360	360	360

Salmonsperm for 10 min on 95 °C, then on ice
*pMA12 - cut with HindIII + EcoRI
**pMA12 - cut by Daniel Schindler

• resuspend yeast pellet in all samples
• induce heat shock at 42 °C for 35 min
• centrifuge for 30 sec - 1 min at 13.000 g
• resuspend pellet in 200 µL sterile water
• plate on selective medium
• incubate at 30 °C for 3 days

Miniprep of Yeast culture 2'', 3'' and 4''

• flush cells from plate with 2 mL water
• centrifuge for 1 min at 11.000 rpm
• pellet cracking: add glass balls
• → miniprep accoding to the miniprep procotol (Omega)

Test PCR with Miniprep 2'', 3'' and 4''

Dilution of elution to 20 ng/µL:

• 2'': 72 ng/µL*X=10*20 ng/µL → 5,3:4,7
• 3'': 38 ng/µL*X=10*20 ng/µL → 3:7
• 4'': 66 ng/µL*X=10*20 ng/µL → 2,7:3,7

	2''' 20 ng	3''' 20 ng	4''' 20 ng
Content	P out of fragments	P out of pool	P (DS) out of pool
Template	1	1	1
dNTPs	1	1	1
Primer001 fwd	1	1	1
Primer006 rev	1	1	1
Buffer (10x)	3	3	3
Taq-Pol	1	1	1
H2O	22,5	22,5	22,5
Total Volume	30	30	30

PCR for 3 h

Amplification of plasmid in E.coli DH5α.

• transformation of 2'', 3'' and 4'' in DH5α
• plating on each ampicillin and canamycin plates

Inoculation of preculture plates: 6 mL LB + 6 µL ampicillin + colony clone

[µL]	Master Mix (7x)	2''' (20 ng) x2	3''' (20 ng) x2	4''' (20 ng) x2
Template	Reaction mix	P out of fragments	P out of pool	P (DS) out of pool
dNTPs	-	1	1	1
Primer001 fwd	3,5	-	-	-
Primer006 rev	3,5	-	-	-
Buffer (10x) Thermo-Pol	17,5	-	-	-
Taq-Pol	0,7	-	-	-
H2O	139,3	-	-	-
Master Mix	-	24	24	24
Total Volume	168	25	25	25

Step	Temperature °C	Time
1	95	2 min
2	95	20 sec
3	58	20 sec
4	68	2,5 min
5	Go To 2	40x
6	68	4 min
7	8	Infinite

Inoculation of 4x6 mL LB with a clone (trafo 13.11.14) from plate 2,3 and 4 (12 falcons). Incubation overnight.

Miniprep according to the miniprep kit protocol (Omega) with overnight preculture.

DNA-concentration: 660 ng/µL

Dilution of Primers

• dilution with aqua bidest to 100 µM
• dilution 1:10 → 10 µL primers (100 µM) with 90 µL aqua bidest

Reaction Mix

[µL]	HagI (1145 bp)	HagII (859 bp)
Content	1	1
Template Flo Pet24-Hag	1	1
dNTPs	1	1
Primer iGEM-007 fwd (Hag-SpeI-F)	1	-
Primer iGEM-008 red (Hag-SpeI)	-	1
Primer 12 rev Flo	1	-
Primer 11 few Flo	-	1
Q5 Reaction Buffer 5x	5	5
Q5-Pol	1	1
H2O	40	40
Total Volume	50	50

Step	Temperature °C	Time
1	95	3 min
2	95	30 sec
3	55	30 sec
4	72	1 min
5	Go To 2	30x
6	72	5 min
7	4	Infinite

Miniprep of transformed E.coli DH5α was performed according to the miniprep kit protocol (Omega) with overnight preculture.

Restriction digest with NcoI and test-plasmids from the miniprep.

[µL]	Mastermix with NcoI - for 14 attempts	Attempt - NcoI (20 kU/mL) - 12 attempts
DNA	-	ca. 350 ng/µL → ca. 1 µg → 2,9 µL
NcoI	3,5 (0,25/attempt)
Master-Mix	-	12,1
Buffer 10x Tango	21
H2O	144,9
Total Volume	169,4
Loading Dye 6x	-	2,5 /attempt

Incubation for 60 min at 37 °C. Then a heat shock was induced for 2 min at 45 °C.

[µL]	Attempt 2.1	Attempt 2.3	Attempt 2.4	Attempt 4.2
DNA	628,8 ng/µL → ca. 1 µg → 1,6 µL	477,2 ng/µL → ca. 1 µg → 2,1 µL	694,9 ng/µL → ca. 1 µg → 1,4 µL	406,8 ng/µL → ca. 1 µg → 2,46 µL
NdeI	0,5	0,5	0,5	0,5
Cutsmart-Buffer 10x	1,5	1,5	1,5	1,5
H2O	11,4	10,9	11,6	10,5
Total Volume	15	15	15	15
Loading Dye 6x	2,5	2,5	2,5	2,5

Dilution of primers

• Hag I: 601,7 ng/µL
• Hag II: 548,2 ng/µL
• Flank I: 226 ng/µL
• Flank II: 221 ng/µL

PCR Hag-Flanks Mix

Mix	Template Mix [µL]	Fla Flank-Hag construct [µL]
Hag I (35 ng/µL)	2	-
Hag II (35 ng/µL)	2	-
Flank I (35 ng/µL)	2	-
Flank II (35 ng/µL)	2	-
Template Mix	-	3
Primer pMAD-Flank I fwd	-	1
Primer pMAD-Flank I rev	-	1
Q5-Mastermix 2x	-	25
H2O	-	20
Total Volume	8	50

Step	Temperature °C	Time
1	95	3 min
2	95	30 sec
3	55	30 sec
4	72	2,5 min
5	Go To 2	35x
6	72	5 min
7	4	Infinite

Results

Expected bands: 2 kb band (whole Flank-Hag fragment)

Gel extraction of 2,2 kb band:
27,6 ng/µL

Attempts with 2 positive clones (2.1 NcoI cut & 2.3 NcoI cut)

Mix for trafo [µL]	p2.1 control	p2.3 control	p2.1 +oligos +amyE	p2.3 +oligos +amyE	p2.1 Anneal+oligo +amyE	p2.3 Anneal+oligos +amyE
p2.1	1	-	1	-	2	-
p2.3	-	1	-	1	-	2
iGEM-009	-	-	1	1	1	1
iGEM-010	-	-	1	1	1	1
iGEM-011	-	-	1	1	1	1
amyE-fragment	-	-	1	1	1	1
H2O	-	-	-	-	14	14
PEG 3350 (50% w/v)	260	260	260	260	260	260
LiAc 1 M	36	36	36	36	36	36
Salmonsperm (2 mg/mL)	50	50	50	50	50	50
H2O	13	13	9	9	-	-
Total Volume	360	360	360	360	-	-

Mix [µL]	2.1 control	2.3 control	3'' - P* + pool	4'' - P** (DS) + pool
PEG 3350 (50% w/v)	260	260	260	260
LiAc 1 M	36	36	36	36
Salmonsperm (2 mg/mL)	50	50	50	50
H2O	14	10	2	2
Fragm. (GFP, Cat, AmyE)	-	4	-	-
Pool (all 3 fragments)	-	-	12	12
Total Volume	360	360	360	360

Salmonsperm for 10 min on 95 °C, then on ice - all attempts on ice!
*pMA12 - cut with HindIII + EcoRI
**pMA12 - cut by Daniel Schindler

• Resuspend yeast pellet in attempts
• Heat shock at 42 °C for 35 min
• Centrifugation for 30 sec - 1 min at 13.000 g
• Resuspend pellet in 200 µL sterile water
• Plate yeast on selective medium
• Incubate at 30 °C till monday

Mix [µL]	Test PCR
Template (Hag-Flank construct)	2
Primer pMAD-Flank I fwd	1
Primer pMAD-Flank II rev	1
Q5-Mastermix 2x	5
H2O	1
Total Volume	10

Step	Temperature °C	Time
1	95	3 min
2	95	30 sec
3	55	30 sec
4	72	2,5 min
5	Go To 2	35x
6	72	5 min
7	4	Infinite

Mix [µL]	Hag-Flank-Fragment digest NcoI/BamHI
DNA	9 ng/µL → 25 µL = 225 ng 27,6 ng/µL → 15 µL = 414 ng → 639 ng in 40 µL
Enzyme NcoI-HF	0,5
Enzyme BamHI-HF	0,5
Cutsmart Buffer (10x)	5
H2O	4
Total Volume	50

From 10.04.2014, performed by Daniel

Plasmids prepared above have been transformed into E.coli, performed by Daniel
Colonies on transformation plates have been inoculated in shaking culture (16 colonies).

Miniprep according to the miniprep kit protocol (Omega).
2.1 and 2.3 different positive colonies from 09.04.2014.

[µL]	Mix
DNA	1
Enzyme NcoI-HF	0,75
Cutsmart Buffer 10x	1,5
H2O	12,25
Total Volume	15

Overnight digestion of two samples with the highest DNA-concentration (2.1 2 and 3.1 A1

Mix [µL]	2.1 2	3.1 A1
DNA	10 µg/8 µL	10 µg/9 µL
Enzyme NcoI-HF	1	1
Cutsmart Buffer 10x	2	2
H2O	9	8
Total Volume	20	20

As template the restriction of the sample 3.1 A1 has been used. The mutagenesis primers have been chosen together with fitting reverse primers to create fragments without the NcoI-restriction sites. These fragments should further be transformed into yeast and assembled into the whole plasmid without the restriction sites for NcoI.

Mix [µL]	Fragment 1	Fragment 2	Fragment 3
Restriction mix 3.1 A1 (2,08 ng/µL)	1	1	1
iGEM-009 (10 mM)	1	-	-
Ag TWp7 (10 mM)	1	-	-
iGEM-010 (10 mM)	-	1	-
iGEM-003 (10 mM)	-	1	-
iGEM-011 (10 mM)	-	-	1
iGEM-006 (10 mM)	-	-	1
Q5-Mastermix 2x	25	25	25
H2O	22	22	22
Total Volume	50	50	50

Every PCR has been carried out in double and was further analyzed on a 1% gel.

The three obtained fragments together with the NcoI-digested plasmid pMA12 have been transformed into S. cerevisiae to get the whole plasmid again. The whole procedure has been done according to the protocol.

Mix for trafo [µL]	Volumes
NcoI-restriced pMA12 (2.1 1)	1
Fragment 1 (146 ng/µL)	1
Fragment 2 (21,7 ng/µL)	9
Fragment 3 (78,6 ng/µL)	3
PEG 3350(50% w/v)	260
LiAc 1 M	36
Salmonsperm (2 mg/mL)	50
H2O	-
Total Volume	360

The transformed yeast cells are incubated at RT over the easter holidays.

Miniprep of Transformed Yeast S.cerevisiae from 21.04.2014 and Trafo of E.coli DH5Alpha.
Miniprep was performed according to the miniprep protocol (Omega).

Plasmid pMa12 with 3 inserts (cat, amyE and gfp) with removed NcoI restriction sites.
Concentration after plasmid prep= 7.5 ng/µL
Transformation of E.coli DH5α with 5 µL DNA (37.5 ng)
Over night incubation on LB-Amp plates.

The transformation of the E.coli cells was not successful. So it has been repeated with the rest of the prepared plasmid.

The concentration of the Hag-Flank-construct was too low (c = 5.8 ng/µL) to use it for the ligation into pMAD. A PCR has been run to increase the amount of the construct.

Mix [µL]	Test PCR
Template (Hag-Flank construct)	2 (12 ng)
Primer 89 from Florian	1
Primer 90 from Florian	1
Q5-Mastermix 2x	25
H2O	21
Total Volume	50

PCR Q5 elongation for 2 kb fragments

Step	Temperature °C	Time
1	95	3 min
2	95	30 sec
3	55	30 sec
4	72	2,5 min
5	Go To 2	35x
6	72	5 min
7	4	Infinite

The PCR-product has been checked on a agarose gel (3 µL + 1 µL 6x Loading-Dye) Expected band: 2000 bp is there, but also 2 additional bands A band with the expected size of ca 2000 bp is visible but there are also two additional DNA-fragments with a lower size. The rest of the PCR-product (47 µL) has been separated on a gel and the expected DNA-fragment has been excised and purified with the Gel Extraction Kit. The purification led to a concentration of 45 ng/µL.

Seven small colonies grew on the plate. The plate was further incubated until the afternoon. Each 6 mL LB-Amp have been inoculated with one of the colonies and incubated over night at 37 °C.

The hag-flank-construct which was amplified yesterday has been digested with NcoI and BamHI to create sticky ends for the following ligation.

Mix [µL]	Hag-Flank-Fragment digest NcoI/BamHI
DNA	1350 ng in 30 µL
Enzyme NcoI-HF	1
Enzyme BamHI-HF	1
Cutsmart-Buffer 10x	5
H2O	13
Total Volume	50

The restriction was carried out at 37°C for 45 min and the digested fragment was purified with the Gel Extraction Kit (c = 24 ng/µL).

(20 ng of vector x kb size of insert/kb size of vector) x molar of ratio of (insert/vector)

0.6 µL of the pMAD-vector (30 ng) and 1 µL of the digested hag-flank-construct (18 ng) were used for the ligation.

Mix [µL]	Control atempt	2x attempt
Plasmid (pMAD)	0,6	0,6
Insert (construct)	-	1
T4 Ligase	1	1
T4 Ligase Buffer (10x)	2	2
H2O	16,4	15,4
Total Volume	20	20

The ligation-mix was incubated at roomtemperature for 1 h followed by a transformation of E. coli DH5α with 10 µL of the ligation-mix. The transformation was incubated on a LB-Amp-plate at 37 °C over night.

[µL]	Attempt 1-7	Mastermix
Plasmid	1,5	-
Enzyme NcoI	-	1
Tango Buffer (10x)	2	16
H2O	15,5	124
Total Volume	20	1

The plates from 15.12 were empty which indicates an unsuccessful ligation. A new ligation was performed after calculation with a Ligation calculator: UT Dallas

Mix [µL]	Control attempt	2x attempt
Plasmid (pMAD)	1	1
Insert (construct)	-	1,9
T4 Ligase	1	1
T4 Ligase Buffer (10x)	2	2
H2O	16	14,4
Total Volume	20	20

The ligation was incubated at roomtemperature for 1.5 h. Afterwards it was stored at -20 °C.

The plasmids were prepared and digested with ask Daniel for restriction used enzymes! Although there is a linear fragment it seems too small. The expected size of the plasmid should be 6666 bp.

The plasmids prepared on Saturday seem to be smaller as expected. For this reason a new attempt to eliminate the restriction sites was carried out. The amplification of fragment 2 (with primers iGEM 003 and iGEM 010 on 15.04.2014) did not happen without any problems such as unspecific smaller bands in addition to the expected one (ca 1000 bp). So this time primer iGEM 003 was replaced by iGEM 002 which would result in a smaller fragment of approximately 600 bp. Later on the fragments 1 and 3 together with the new created one, the digested pMa12 construct and the amyE-fragment (to overlap the region between fragments) would be transformed into S. cerevisiae for recombination.

Mix [µL]	New Fragment 2
Restriction mix 3.1 A1 (2,08 ng/µL)	1
iGEM-010 (10 mM)	1
iGEM-002 (10 mM)	1
Q5-Mastermix 2x	25
H20	22
Total Volume	50

PCR was performed according to the PCR protocol and the PCR-products were analyzed on a 1% agarose gel.

Step	Temperature °C	Time
1	95	3 min
2	95	30 sec
3	58	20 sec
4	72	1 min
5	Go To 2	35x
6	72	5 min
7	4	Infinite

Mix [µL]	Volumes
NcoI-digested pMA12 (2.1 1)	2
Fragment 1 (146 ng/µL)	2
New Fragment 2 (412,9 ng/µL)	2
Fragment 3 (78,6 ng/µL)	3
Fragment amyE (48,3 ng/µL)	5
PEG 3350(50% w/v)	260
LiAc 1 M	36
Salmonsperm(2mg/mL)	50
H2O	-
Total Volume	360

The transformed yeast cells were incubated at 30 °C.

Some new competent E. coli DH5α cells were made because the last batch of cells have not yielded in good results in the last few transformations. The cells were made according to the protocol in the method section below. To test the cells they were plated on normal LB, LB-Amp, LB-Kan and a test-trafo was carried out with pMa12 which provides an ampicillin resistance.

The transformation of E. coli DH5α with the ligation of the pMAD-vector and the hag-flank-construct was not successful. Therefore the PCR for amplification of the construct was repeated by using the hag / flank-parts or the construct itself as template.

Mix [µL]	PCR 1	PCR 2	PCR 3
Template	2 (Template mix Hag I/II + Flanks 15,5)	1 (Flagellin-construct 15,6)	1 (Flagellin-construct digest (15,10)
Primer 89 from Florian	1	1	1
Primer 90 from Florian	1	1	1
Q5-Mastermix 2x	25	25	25
H2O	21	22	22
Total Volume	50

PCR was performed according to the "PCR Q5 elongation for 2kb fragments" protocol and the PCR-products were analyzed on a 1% agarose gel.

Step	Temperature °C	Time
1	95	3 min
2	95	30 sec
3	55	30 sec
4	72	2,5 min
5	Go To 2	35x
6	72	5 min
7	4	Infinite

Since the last ligation attempts were not successful the plasmid pMAD is digested by us instead using the digested pMAD from Florian. The PCR-fragments of the hag-flank-construct were purified (PCR1: c = 62 ng/µL, PCR2: c = 55 ng/µL, PCR3: c = 46 ng/µL) and digested with the enzymes BamHI and NcoI.

[µL]	PCR 1	PCR 2	PCR 3	PCR 4
DNA	all	all	all	5 (300 ng/µL)
BamHI	1	1	1	1
NcoI	1	1	1	1
Cutsmart Buffer	4	4	4	2
H2O	4	4	4	2
Total Volume	40	40	40	20

The restriction was incubated at 37 °C for 1 h and stored at -20 °C.