Purifying the attempts from the 30.04.2014 with gel extraction kit (QIAquick Gel Extraction Kit) according to the protocol.

Fragment	Concentration [ng/µl]
pMAD Digest 1	21
pMAD Digest 2	19
Hag-Flank Construct 1 Digest	16,9
Hag-Flank Construct 2 Digest	17,6
Hag-Flank Construct 3 Digest	11,3

The ligation is carried out with the purified pMAD digest and the hag-flank-construct digest from in order to transform the ligation mix into E.Coli DH5Alpha. For the ligation was the following calculator used: UT Dallas

Mix [µl]	Control 1	Control 2	Construct 1.1	Construct 1.2	Construct 2.1	Construct 2.2	Construct 3.1	Construct 3.2
Plasmid (pMAD digest 1)	1	-	4,5	-	4,7	-	3	-
Plasmid (pMAD digest 2)	-	1	-	5	-	5	-	3,3
Insert (construct)	-	-	5	5	5	4,8	5	5
T Ligase	1	1	1	1	1	1	1	1
T4 Ligase Buffer 10x	2	2	2	2	2	2	2	2
H2O	16	16	7,5	7	7,3	7,2	9	8,7
Total Volume	20	20	20	20	20	20	20	20

Incubation for 1,5 h at room temperature.

To have enough of the construct for further works, a new PCR (as described before) was done overnight.

The yeast was washed from the plate with 2 ml of A. dest. After centrifugation at 11000 rpm for 1 min the supernatant was discarded and 500 µl of Solution 1 of the Omega plasmid prep kit was added. The yeast was vortexed for 5 min after addition of a few glas balls. After that the miniprep was carried out according to the protocol (Omega). (c = 134.5 ng/µl)

E. coli DH5Alpha cells were transformed with 10µl of the prepped plasmid pMa12. They were incubated at room temperature over the weekend.

Purification of the fragments from the 01.05.14. The PCR-products were separated on a 1% agarose-gel.

15 colonies of the transformed E.coli DH5Alpha were picked and incubated in 6 ml LB-Amp at 37°C overnight.

The ligation with pMAD was not successful so it was decided to clone the hag/flank-construct first into pET24d. The ligation was done after using the ligation calculator: UT Dallas

[µl]	Fragment 1	Fragment 2	Fragment 3
pET24d	2,9	3,1	2
Fragment	10	10	10
T4-Ligase-Buffer (10x)	2	2	2
T4 Ligase	1	1	1
H20	4,1	3,9	5
Total	20	20	20

The ligation was incubated at RT for about 2h followed by a transformation of E. coli DH5Alpha. The transformed cells were plated out on LB-Kan and incubated at 37°C over night.

The plasmids were isolated according to the protocol "Miniprep (Plasmid DNA Miniprep Kit II, Omega)".

Clone	Concentration (ng/µl)
1	570,2
2	735,5
3	671,9
4	642,6
5	660,9
6	688
7	982,6
8	637,4
9	635,4
10	1306,6
11	523,9
12	554,4
13	501,3
14	825
15	771,2

The prepped plasmids were restricted with NcoI for 1h 20min and further analyzed on a 1% agarose gel.

The isolated pMa12 of clone 10 was used for a restriction with SacI-HF and NcoI. The restriction leads to fitting ends for the designed oligos which are the promoters we are about to test: PargJ and PykuO. The restriction is incubated at RT over night.

Restriction	Volume [µl]
pMA12 from clone 10	2
NcoI	1
SacI-HF	1
CutSmart Buffer (10x)	2
H2O	14
Total	20

On the plates with the transformands of yesterday were only three colonies. Each 6 ml LB-Kan were inoculated with the colonies and incubated at 27°C for around 9 h. After growth of the bacteria the plasmids were isolated with the Omega Plasmid Prep Kit.

[µl]	Clone 1	Clone 2	Clone 3
Plasmid (500ng)	10	5	5
Water	8	12	12
CutSmart Buffer (10x)	2	2	3
NcoI	0,5	0,5	0,5
BamHI	0,5	0,5	0,5

Restriction for 45 min at 37°C.
Agarose-gel (1%) to compare the cut and uncut plasmid.

Concentration: 3ng/µl

[µl]	Reaction 1	Reaction 2
Oligos	iGEM012/iGEM013 10µl/10µl	iGEM014/iGEM015 10µl/10µl
Water	80	80
Total	100	100

Heat up at 98°C for a few minutes. Let it cool down until room temperature

[µl]	Reaction (Ag)	Reaction (Cu)
Plasmid (3ng/µl)	5,5	5,5
Water	15	15
Buffer (Ligation Buffer 10x)	2,5	2,5
Oligo-Mix	12/13	14/15
T4-DNA-Ligase	1	1

10µl of the ligation reaction will be transformed into chemically competent E.coli DH5Alpha als described before.

See 06.05.2014

Restriction	Volume [µl]
pMA12 from clone 10	2
NcoI	1
SacI-HF	1
CutSmart Buffer (10x)	2
H2O	14
Total	20

No colonies on the plates, repeat ligation and transformation with new digested plasmid.
Concentration plasmid: 17 ng/µl
Ligation: 1.5µl of plasmid used
Tranformation: as usual

1. Sequencing

Label	Description	Primer
AFD001881	piGEM001	T7
AFD001882	piGEM001	T7 Turn
AFD001883	piGEM001	Hag revers (Flo)
AFD001884	piGEM002	TW 260 (Daniel)

Plasmids have to be prepared in a concentration between 50-100 ng/µl in a total volume of 10 to 15µl.
2µl Primers have been added to the premixes, Sample 881 and 882 will be sequenced with primers already present at the company (mwg eurofins).

No colonies on the plates
Repeat annealing of oligos
Step 1:

Amount [µl]
1	Oligo 1
1	Oligo 2
5	Ligase Buffer
1	PNK
42	Water

Incubation at 37°C for 30 min
Step 2:
Heat up water in microwave until boiling, add the eppis bis primer mix
let it cool down to roomtempertur (over lunch)
Step 3:
1:100 dilution
Step 4: Ligation

Amount [µl]
3	Plasmid (17,6ng/µl)
2	Annealed Oligos (1:100)
1	Ligase
2	Ligase Buffer
12	Water

Step 5: Tranformation
Incubation at room temperature over the weekend.
Step 6: Results
Hag Flank construct digestion with speI was positive.

Mix [µl]	Pet24fla1	Pet24fla2	Pet24fla3
Temp. – pet24fla cone 1 (lig1 05.05.14)	0,5	-	-
Temp. – pet24fla cone 2 (lig2 05.05.14)	-	0,5	-
Temp. – pet24fla cone 3 (lig3 05.05.14)	-	-	0,5
Primer iGEM-89flo	1	1	1
Primer iGEM-89flo	1	1	1
Q5-Buffer 5x	10	10	10
Phusion-Polymase	0,5	0,5	0,5
Water	37	37	37
Total Volume	50	50	50

PCR protocol for "PCR Q5 elongation for 2kb 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	5min
7	4	Infinite

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	5min
7	4	Infinite

Result: PCR not successfull – no band at 2,2kb

• 3 aliquots competent E.Coli DH5Alpha were transformed with 1µll of the purified plasmid pet24-fla construct from 05.05.14 from 3 different clones (lig 1-3) each
• After regeneration the cells were plated on LB-canamycin plates and incubated at room temperature until Monday

Aim: Amplification of the fragment for further experiments with pet24-fla from 15.21 as template. The prior PCR was not successful.

Mix [µl]	Pet24fla1	Pet24fla2	Pet24fla3
Temp. – pet24fla cone 1 (lig1 05.05.14)	0,5	-	-
Temp. – pet24fla cone 2 (lig2 05.05.14)	-	0,5	-
Temp. – pet24fla cone 3 (lig3 05.05.14)	-	-	0,5
Primer iGEM-89flo	1	1	1
Primer iGEM-89flo	1	1	1
Q5-Mastermix	25	25	25
Water	22,5	22,5	22,5
Total Volume	50	50	50

Q5 PCR

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

No colonies

[µl]	Mastermix with NcoI - for 4 attempts	Attempt 1-3 Spe1 (20kU/ml)
DNA	-	Ca. 85ng/µl ca. 1µg → 12µl
Spe1	1	-
Master-Mix	-	3
Cutsmart (10x)	6	-
H2O	5	-
Total Volume	12	15
Loading Dye 6x	-	2,5/attempt

Incubation for 60min at 37°C
Heat shock for 2min at 80°C

Colonies were grown on every plate → successful transformation

3x6ml LB + 6µl canamycin each were inoculated with 1 colony of plate 1-3 from 10.05.14. The plates were incubated overnight at 37°C

Mix [µl]	Pet24fla2
Temp. – pet24fla clone 2 (lig2 11.05.14)	0,5
Primer iGEM-89flo	1
Primer iGEM-89flo	1
Q5-Mastermix	25
Water	22,5
Total Volume	50

The PCR was performed according to the "PCRQ5" protocol.

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

The PCR-products were separated on a 1 % agarose gel. The bands of 2.2 kb were cut out and a gel extraction was carried out (c = 100 ng/µl) according to the "Gel Extraction (QIAquick Gel Extraction Kit)" protocol.

Restriction	Volume [µl]
PCR-product hag/flank (100 ng/µl)	5
NcoI-HF	1
BamHI-HF	1
CutSmart-Buffer (10x)	2
H2O	11
Total Volume	20

The restriction is incubated at RT overnight.

For ligation to occur at least one of the DNA ends (insert or vector) should contain a 5´ phosphate. Primers are usually supplied non-phosphorylated; therefore, the oligos or PCR product will not contain a 5´ phosphate. The digestion of DNA with a restriction enzyme will always produce a 5´ phosphate. A DNA fragment can be phosphorylated by incubation with T4 Polynucleotide Kinase in T4-ligase-buffer which already contains the necessary ATP in an appropriate amount (1 mM):

Phosphorylation [µl]	P(argJ)	P(ykuO)
iGEM-012	1	-
iGEM-013	1	-
iGEM-014	-	1
iGEM-015	-	1
T4-ligase buffer	5	5
T4 PNK	1	1
H2O	42	42
Total Volume	50	50

The reaction was done in a PCR cycler with a subsequent heat inactivation of the PNK and annealing reaction (protocol: "Annealing with PNK Inactivation")

5 mL of LB-Kan⁵⁰ medium were inoculated with a clone from 14.24 bearing the PheA-Arc1p-C-2x-pET28a plasmid.

The plasmid pMAD was isolated and restricted with the restriction enzymes BamHI and NcoI to create fitting ends for the ligation with the restricted PCR-fragment.

Restriction	Volume [µl]
pMAD (174.9 ng/µl)	2
NcoI-HF	1
BamHI-HF	1
CutSmart-Buffer (10x)	2
H2O	14
Total Volume	20

The restriction was incubated at 37°C for 1h.

The ligation of pMAD and the restricted PCR-fragment was done according to the previous mentioned ligation calculator.

Ligation	Volume [µl]
pMAD	5
Hag/flank-construct	10
T4 Ligase Buffer (10x)	2
T4 DNA-Ligase	1
H2O	2
Total Volume	20

The ligation was incubated at RT for 1.5 h. After that E. coli DH5Alpha were transformed with the ligation mix and plated out onto LB-Amp plates which were incubated at 37°C overnight.

A ligation of the SacI and NcoI restricted pMa12-construct with the annealed oligos was carried out. The annealed oligos (iGEM-012/013 c = 762.3 ng/µl and iGEM-014/015 c = 825.5 ng/µl) were diluted up to 10-2. 10ng of the insert and 100ng of the vector were used for the ligation.

Ligation [µl]	P(argJ)-pMa12-construct	P(ykuO)-pMa12-construct
iGEM-012/013 (7.6 ng/µl)	1,5	-
iGEM-014/015 (8.3 ng/µl)	-	1,5
T4-ligase-buffer (10x)	2	2
T4-Ligase	1	1
H2O	15,5	15,5
Total Volume	20	20

The ligation was incubated at RT for 2h. The E. coli DH5Alpha were transformed afterwards with the whole ligation mix and plated on LB-Amp, which were incubated at 37°C overnight.

Using the preculture from 14.25 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. A glycerolstock of the strain containing the PheA-Arc1p-C-2x-pET28a plasmid was stored at -80 °C.

The plasmid pMAD from 13.05.14 (ca. 25µl) and restricted with the restriction enzymes BamHI and NcoI to create fitting ends for the ligation with the restricted PCR-fragment. The digest was performed in 2 Eppis with 12,5µl plasmid each.

Restriction [µl]	Mastermix	Volume
pMAD (174.9 ng/µl)	-	12,5
Mastermix	-	7,5
NcoI-HF	1	-
BamHI-HF	1	-
CutSmart-Buffer (10x)	4	-
H2O	9	-
Total Volume	15	20

The restriction was incubated at 37°C for 2h.

The plasmid pet24d with the fla construct from clone 2 was transformed into E.Coli DH5Alpha and plated on LB-Canamycin. Incubation overnight at 37°C.

One clone with plasmid pet24d with the fla construct was used for inoculation of LB-medium. Incubation overnight at 37°C

The plasmid pMA12+Nose-Construct from clone 10 was transformed into E.Coli DH5Alpha and plated on LB-AMP. Incubation overnight at 37°C

The ligation of pMAD and the restricted PCR-fragment was done according to the previous ligation calculator.

Ligation	Volume [µl]
pMAD	5
Hag/flank-construct	20
T4-Ligase-Buffer (10x)	3
T4-DNA-Ligase	1
H2O	1
Total Volume	30

Incubation overnight at room temperature.

Wildtype on LB plate and in LB liquid without an antibiotic.
Bacillus strain 186 on plates containing xylose (Incubation overnight).

Innoculate Bacillus wildtype strain in SPC medium.

Time	OD570
13:30	5,7
14:00	6,2
15:45	6,6
16:30	7
17:00	7,2
	→ new media Incubation for 1,5h

Aliquot and thaw in liquid nitrogen. Store at -80°C.

The cell culture from 14.26 was centrifuged (17000 rpm, 20 min, 4 °C) and the pellet resuspended in 3 mL buffer A. Lysozyme was added 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 14.26 and incubated at 37 °C and 220 rpm over night.

Chemically competent cells from Daniel were transformed with the 20µl ligation-mix, 20µl of the cells were transformed with 1µl pMad uncut (control).
Protocol: see yesterday

PCR Mix	Volume [µl]
Q5-Mastermix	88
Water	110
Primer 89	5,5
Primer 90	5,5
Template	1

Dip in a colony on the trafo plate, streak it on a new plate and dip in the PCR Premix

[µl]	Amount Ag	Amount Cu
Q5-Mastermix	8	8
Water	10	10
TW 260	1	1
Primer 12	1	-
Primer 14	-	1

A PCR ran with 5 picked clones from 16.05.14 and Primers Flo89 and 90 for amplification of inserted construct

	Attempt [µl]
Colony	1 colony
Q5-Mastermix	10
Water	8
Primer Flo89	1
Primer Flo90	1

Innoculate 5 clones in LB liquid and incubation over the weekend at room temperature

10 x 500 mL LB-Kan⁵⁰ medium were inoculated with 5 mL of the overnight culture prepared in 14.27 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.

Restriction: Concentration of plasmid after prep between 20 and 30 ng/µl

	Volume [µl]
Buffer / Water	27
Template	10
NcoI	0,5
BamHI	0,5

	Amount [µl]
Q5-Mastermix	10
Primer 89	1
Primer 90	1
Template	1
Water	7

Bacillus Trafo: rf. Diploma thesis from Dr. Felix Dempwolff
5µl DNA were added to 100µl cells, 30 min incubation at 37°C and plated on LB-Cm.

The resuspended cells from 14.28 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-2x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-2x 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.

The PCR with 5' phosphorylated TG_1xGSSG FP und TG_0xGSSG RP should generate the 7509 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_1xGSSG 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.

	Amount [µl]
Q5-Mix	20
Primer 89	1
Primer 90	1
Template	1
Water	17

No colonies → new trafo
New LB Cm plates for Bacillus (only 5µg/ml instead of 25µg/ml)

	Volume [µl]
Buffer	2,5
Plasmid	20
SpeI	1
Water	-

Concentration of plasmid after clean up only 7 ng/µl

Six clones of the transformed Top10 cells from 14.30 were picked and used to inoculate overnight cultures (6 x 5 mL).

[µl]	Control	With Insert
Gibson Mix	15	15
Plasmid (7ng/µl)	5	5
Insert	-	1
Water	1	-

A part of the aliquot was plated on LB-tet for inoculation of colonies for competent XL1-Blue.

Tranformation from 20.5.14: one colony on the Ag Nose plate
Innoculate shaking culture in the morning together with the wildtype and the gfp strain (186) in LB liquid without antibiotics

Inocculate 96 well plate in the afternoon 15:00

Prepate Cu and Ag solutions to test different concentrations in MTA and see if the Ag nose responds to Ag and also if it is specific for Ag and does not respond to Cu

Ag and Cu in stock solution of 10 mM have been prepared
Added (Concentration): 11 mM, 100µM and 10µM
Cells: Ag Nose mutant, wildtype (AG GM) and gfp (186)

[µl]	piGem 002 clone 7	Nose plasmid 003/004	Amount
Template ≈10µg	15	20	15
SpeI	1	1	1
CutSmart-Buffer	2,5	2,5	2,5
Water	6,5	1,5	6,5

Incubation at room temperature

The plasmids were purified from the overnight cultures created in 14.31 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.

First step: Linearize the following plasmids: piGEM 002 without promotor as well as the plasmids containing the Silver and copper promotor
Second step: PCR to anneal the oligos 20+21, 20+22 and 20+23

	Volume [µl]
Primer A	5
Primer B	5
Phusion	0,25
dNTPs	0,25
Buffer	5
Water	34,5
Cycle	3

Afterwards clean up with kit (all three mixes were carried out in a duplicate, 1 each was frozen away at minus 20)

Third step: Yeast transformation
9 Reactions (3 plasmids and 3 instability tags)

100 ng plasmid plus 1µl of primer mix were filled to 14µll and added to the yeast transformation mix and subsequently transformed into yeast.

To insert the metallothionein gene cup1-1 into the hag/flank-construct the pMAD-fla was restricted with SpeI to linearize it.

Restriction	Volume [µl]
pMAD-fla (330 ng/µl)	4
SpeI	1
CutSmart Buffer (10x)	1
H2O	6
Total Volume	10

The restriction was carried out at 37°C for 1h.
The restriction was cleaned with a DNA Gel Ex Kit (c = 40 ng/µl). 4µl (160 ng) of this restricted plasmid were mixed with 1µl of the cup1-1 fragment (9.6 ng/µl). These 5µl were inserted into the prepared 15µl Gibson-mix. The reaction was incubated at RT for 1 min and then at 50°C for 1h. An extra Gibson-reaction was carried out as a control with just the linearized pMAD without the cup1-1.
E. coli DH5Alpha cells were transformed with the Gibson reactions and plated out on LB-Amp. They were incubated at 37°C over night.

Since a Gibson-Assembly with the same aim was carried out last week, the colonies were checked by a colony PCR with the primers for cup1-1 iGEM-018 and iGEM-019. One colony was picked from the plate from 23rd May and three were taken from the plate from 21st May. The latter was contaminated with Bacillus colonies. For this reason E. coli was picked and streaked out onto a new plate on Friday the 23rd May. The consequence is a plate with just one clone, what is the reason, that only one colony was picked from this plate.

Mix	Volume [µl]
E. coli XLI-Blue pMAD-fla+cup1-1	part of a colony
Primer iGEM-018	0,5
Primer iGEM-019	0,5
Q5-Mastermix	10
Water	9
Total Volume	20

A part of each colony was transferred onto a new LB-Amp plate, which was incubated at 37°C overnight.

40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-Arc1p-C-1x-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 gfp in piGEM002, piGEM002-Ag (and piGEM002-Cu was tagged with different degradation tags by yeast recombination. The plasmids have been prepped form yeast.

Clone	Concentration (ng/µl)
002-21
002-22
002-23
002-Ag-21
002-Ag-22
002-Ag-23
002-Cu-21
002-Cu-22
002-Cu-23

E. coli DH5Alpha cells were transformed with 15µl of each prepped plasmid and plated out on LB-Amp. They were incubated at 37°C over night.

Gly-Stocks - Making of -stocks for long time storage of bacteria strains
For long time freezer storage of bacteria, they have to be treated with glycerin to prevent the crystallization and damaging of the cells. For this reason 2ml of LB with the respective antibiotic were inoculated with the bacteria which were to be stored and were incubated at 37°C overnight.

Strains that are to be stored:

• E. coli XLI-Blue pMAD-fla (piGEM-005)
• E. coli XLI-Blue pMa12-construct (piGEM-002)
• E. coli XLI-Blue pMa12-Cu (piGEM-004)
• E. coli XLI-Blue pMa12-Ag (piGEM-003)
• E. coli BL21(DE3) pLysS

5 mL of LB-Kan⁵⁰ medium were inoculated with a clone from 14.33 bearing the PheA-Arc1p-C-1x-pET28a plasmid.

Since the colony-PCR (26.05.14) was negative and the Gibson-Assembly of linearized pMAD-fla with cup1-1 showed no results the pET24d-fla construct was linearized with SpeI.

Restriction	Volume [µl]
pET24d-fla (98.8 ng/µl)	8
SpeI	1
CutSmart-Buffer (10x)	2
H2O	9
Total Volume	20

The restriction was incubated at 37°C for 1h.

The restriction mix was purified with the Gel Ex Kit. The yield was too low (c = 3.1 ng/µl) to use it for a Gibson-Assembly, so the rest of the pET24d-fla was restricted with SpeI.

Restriction	Volume [µl]
pET24d-fla (98.8 ng/µl)	18,5
SpeI	1
CutSmart-Buffer (10x)	2,5
H2O	3
Total Volume	25

The restriction was incubated at 37°C for 1h.

The restriction mix was purified with the Gel Ex Kit (c = 20.1 ng/µl), which was enough for the Gibson-Assembly. 4µl (80 ng) of this restricted plasmid were mixed with 1 µl of the cup1-1 fragment (9.6 ng/µl). These 5 µl were inserted into the prepared 15µl Gibson-mix. The reaction was incubated at RT for 1 min and then at 50°C for 1 h. An extra Gibson-reaction was carried out as a control with just the linearized pET24d-fla without the cup1-1.

Competent E. coli XLI-Blue from Daniel were transformed with the whole Gibson-reactions. They were plated on LB-Kan an incubated at 37°C overnight.

The transformation with the tag-constructs were nearly in all cases successful except the pMa12-Ag-21. The transformation was repeated, the plate was incubated at 37°C over night.
Four colonies of every plate have been picked, transferred to new LB-Amp plates and were subsequently used for the colony-PCR. The Master Mix was portioned in aliquots of 20µl per PCR-cup and inoculated with the respective colony.

MasterMix for 33 reactions	Volume [µl]
E. coli DH5Alpha with construct	part of a colony
AG TW 260	16
AG TW 316	16
Q5-Mastermix	165
Water	133
Total Volume	330

Step	Temperature °C	Time
1	95	5 min
2	95	20 sec
3	55	20 sec
4	72	20 sec
5	Go To 2	35x
6	72	5min
7	4	Infinite

Gly-Stocks - Making of glycerin-stocks for long time storage of bacteria strains

1 ml of the bacteria which were inoculated yesterday (26.05.2014) were transferred to new, sterile 1.5 ml Eppendorf-cups. 600µl of 50% glycerin were added and the cells were frozen and stored at -80°C in the freezer.

9 ml LB-Tetracycline (1:1000) were inoculated with E. coli XLI-Blue cells as a preculture. They were incubated at 37°C overnight.

93 mL of LB-tet were added to 7 mL of preculture for inculation of main culture. The main culture was incubated at 37°C until an OD of 0,5.

TBF1 recipe	TBF2 recipe
for 50ml       Kaliumacetat 0,15g MnCl2 x 4H2O   1M RbCl2 5 ml 1m CaCl2 0,5 ml 87% Glycerin 8,6 ml A.dest 35,9 ml autoclave or sterile filtration	for 250 ml       1M NaMOPS (pH7) 2,5 ml 1M CaCl2 x 2H2O 8,75 ml 1M RbCl2 2,5 ml 87% Glycerin 43,75 ml A.dest 192,5 ml adjust pH7   store at 4°C in the dark   sterile filtration

• Grow 100ml E.coli culture to OD600=0,5
• Put on ice for 10 min
• Centrifuge 2x 50 ml for 10 min at 3000 rpm and 4°C
• Wash pellets in 10 ml TFB1 (10 min; 3000 rpm; 4°C)
• Resuspend pellets in 2ml TFB2
• Pipett 200µl aliquots in precooled 1,5 ml tubes
• store at -80µC

Using the preculture from 14.34 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. A glycerolstock of the strain containing the PheA-Arc1p-C-1x-pET28a plasmid was stored at -80 °C.

6 clones were picked from transformation plate and a colony PCR was ran with primers iGEM-018 & - 019. The result should be a band at 240 bp in case of a positive clone.

[µl]	Mastermix	Mix for PCR attempts
E. coli XLI-Blue pet24d-fla+cup1-1		part of a colony
Primer iGEM-018	3,5	-
Primer iGEM-019	3,5	-
Q5-Masterix	70	-
Water	63	-
Master-Mix	-	20
Total Volume	140

Step	Temperature °C	Time
1	95	1 min
2	95	20 sec
3	55	20 sec
4	72	20 sec
5	Go To 2	35x
6	72	5min
7	4	Infinite

The Colony-PCR was repeated with pMa12-Ag-21 after successful transformation.
Four colonies have been picked, transferred to new LB-Amp plates and were subsequently used for the colony-PCR.
The Master Mix was portioned in aliquots of 20 µl per PCR-cup and inoculated with the respective colony.

[µl]	Master-Mix for attempts	Mix for PCR attempt
E. coli DH5α with Ag21	-	part of a colony
AG TW 260	2,5	-
AG TW 316	2,5	-
Q5-Mastermix	50	-
Water	45	-
Master-Mix	-	20
Total Volume	100	20

In an additional reaction the colony-PCRs of the negative clones (gel 28.05.14) were repeated with more picked clones per construct. This time 8 colonies of each negative construct were picked, transferred to another LB-Amp-plate and used for the PCR-reaction. Four times eight reactions were carried out, a master mix was prepared for 33 reactions.

[µl]	Master-Mix for 33 reactions	Mix for one PCR reaction
Part of a colony	-	1
AG TW 260	8	0,25
AG TW 316	8	0,25
Q5-Mastermix	330	10
Water	315	9,5
Master-Mix	-	20
Total Volume	660	20

Step	Temperature °C	Time
1	95	5 min
2	95	20 sec
3	55	20 sec
4	72	20 sec
5	Go To 2	35x
6	72	5min
7	4	Infinite

E.Coli XL1-Blue were transformed with 1µl pMAD (189 ng/µl). Another aliquod was transformed with pMAD-fla.
In comparison E.Coli DH5Alpha were transformed the same way.

Every transformation was successful. The higher number of colonies on the pMAD-transformation plate shows a higher transformation efficiency compared with DH5Alpha.

For minipreps 2x6ml of LB-Amp were inoculated with one colony from the pMAD XL1-Blue-Transformation plate. Additionally 3x6ml of LB-Amp were inoculated with one colony from the pMAD-fla XL1-Blue-Transformation plate.
Furthermore 2x 2 mL LB-Amp were inoculated with a clone from pMAD-fla XL1-Blue-Transformation plate for glycerin storage.
Every attempt was incubated at 37°C overnight.

The Colony-PCR was repeated with 002-Cu21 and 002-23.
Four colonies have been picked, transferred to new LB-Amp plates and were subsequently used for the colony-PCR.
The Master Mix was portioned in aliquots of 20 µl per PCR-cup and inoculated with the respective colony.

[µl]	Master-Mix for 18 attempts	Mix for PCR attempt 002-23.1-8	Mix for PCR attempt 002-Cu21.1-8
E. coli DH5α with 002-23	-	part of a colony	-
E. coli DH5α with Cu21	-	-	part of a colony
AG TW 260	9	-	-
AG TW 316	9	-	-
Phusion	3,6	-	-
Q5-Buffer 5x	72	-	-
dNTP-Mix	5	-	-
Water	261,4	-	-
Master-Mix	-	20	20
Total Volume	360	20	20

Step	Temperature °C	Time
1	95	5 min
2	95	20 sec
3	55	20 sec
4	72	20 sec
5	Go To 2	35x
6	72	5min
7	4	Infinite

The cell culture from 14.35 was centrifuged (17000 rpm, 20 min, 4 °C) and the pellet resuspended in 3 mL buffer A. Lysozyme was added 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 14.35 and incubated at 37 °C and 220 rpm over night.

6 mL LB-Amp were inoculated with different colonies cotaining the following plasmid-constructs:

• 002-21
• 002-22
• 002-Cu-23
• 002-Ag-23
• 002-Cu-22
• 002-Ag-22

Incubation Overnight at 37°C

10 x 500 mL LB-Kan⁵⁰ medium were inoculated with 5 mL of the overnight culture prepared in 14.36 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.

Plasmid	Concentration after prep (ng/µL)
pMAD	114 & 108
pMAD-fla (iGEM-005)	114, 128 & 142

Plasmids were frozen away at -20°C

Plasmid	Concentration after prep (ng/µl)
002-21	368
002-22	401
002-Cu-22	425
002-Cu-23	427
002-Ag-22	806
002-Cu-23	402

The positive clone 3 from the colony PCR (28.05.14) was used to inoculate 6 ml LB-Amp for a miniprep in order to receive the plasmid for transforming it into Bacillus subtilis.
Incubation at room temperature over the weekend.

6 mL LB-Amp were inoculated with the positive clone 1 from the Gibson assembly with cup1-1 transformed into E.Coli XL1-Blue from 26.05.14. After miniprep the plasmid can be transformed into Bacillus Subtilis.
Incubation at room temperature over the weekend.

• BS piGEM002 + DT22
• BS piGEM 011 (002 AG + DT22)
• BS piGEM 012 (002 AG + DT23)
• BS piGEM 014 (002 Cu + DT22)
• BS piGEM 002 (Nose)
• BS piGEM 003 (Nose Ag)
• BS piGEM 004 (Nose Cu)

Phusion and Q5 Buffer did not work out so the PCR was repeated with Q5 Mastermix.
PCR performed with 002-Cu21 and 002-23
Result: Positive clones for both Tags have subsequently been inoculated in LB-Amp and incubated at room temperature over the weekend.