1) Transformation
Julia Bartels from iGEM 2012 sent us integration vectors for B.subtilis. Sebastian from AG Weber (BIOSS) shared plasmids containing mCherry and GFP.

Vectors:
No.1. pBS0K-Pspac-[RFP]
No.2. pBS1C3-[RFP]
No.3. pBS2E-[RFP]
No.4. pBS3Clux-[RFP]
No.5. pBS4S-[RFP]
No.6. pSBBs4S-Sporovector

7. mCherry
8. GFP

Transformation of the Vectors from Julia Bartels and Sebastian (from AG Weber) into chemically competent E.coli K12 DH5alpha was performed according to protocol. The E.coli were incubated for 1 hour at 37 °C and 250 rpm and spread on LB-Agar plates supplemented with ampicilin. Incubation for o/n at 37 °C.

2) Preparation of competent E.coli
For the preparation for chemically competent E.coli one colony from an agar plate was picked in inoculated in 5 mL LB-medium. Incubation o/n at 37 °C and 250 rpm.

1) Production of competent E.coli
Preparation of chemically competent cells according to the protocol of the manufacturer (Zymoresearch, Z competent Mix&Go kit)
The resulting cell suspension was aliquoted (100 µL) in tubes and stored at -80 °C.

2) Inoculation
From each transformation plate(No. 1-6) 5 colonies were picked and inoculated in 5 mL LB medium supplemented with Ampicilin.
Incubation o/n at 37°C and 250 rpm.
The B.subtilis W168 strain was inoculated in 5 mL of LB medium and incubated o/n at 37 °C and 250 rpm. In parallel, a sample of the W168 strain was spread on a LB agar plate and incubated o/n at 37 °C.

1) MiniPrep
The the plasmids of the inoculated colonies (No.1-6) were prepared using the QiaGen MiniPrep kit according the instructions of the manufacturer and eluted in 20 µL of ultra pure water. The concentration of the DNA was spectroscopically determined by a Nanodrop.

2) Colony PCR Colony PCR for the amplication of the cotZ and cgeA gene was performed using the following oligos:
cotZ: oIG16_1+2 Annealing temperature: 57 °C
cgeA: oIG16_43+34 Annealing temperature: 65 °C

Reaction conditions

Cycler Program

1) Gel electrophoresis
20 µL of the colony PCR samples were supplemented with the appropriate amount of 10X Orange G loading buffer, loaded on a 1 % agarose TAE gel and subjected to electrophoresis for 40 min at 120 V. The DNA was stained with Midori Green. (No bands were observable at all. The amount of Midori Green was not sufficient)

1)Colony PCR
from 09.07.2016 was repeated at a total volume of 50 µL. After electrophoresis the sample were supplemented with the appropriate amount of 10X orange G loading buffer and loaded on an 1 % TAE agarose gel. The gel was subjected to electrophoresis for 40 min at 120 V. The DNA was stained with Midori Green.
Only the amplified cotZ was visible at UV Light.

The cotZ band was excised and the DNA was extracted using the QiaQuick Gel extraction kit according the the instructions of the manufacturer. The DNA was eluted in 20 µL of ultra pure H2O and the concentration was photometrically determined by a NanoDrop.
CotZ gelextraction concentration: 18 ng/µL

1) Colony PCR
Colony-PCR for the amplification of cgeA and cotZ was repeated
Primer:
cgeA: oIG16_034 + 043
cotZ: oIG16_001 + 002

Reaction conditions

Cycler Program

The samples were supplemented with the appropriate amount of 10 X Orange G loading dye and loaded on a 1 % TAE agarose gel. As molecular marker a 2-log marker was used.
No bands were observed at UV light.

1) Gradient colony PCR
To amplify cgeA 7 samples (20 µL each) were amplified by gradient colony PCR applying annealing temperatures from 58 - 68 °C.
An additional 50µl sample was made to amplify CotZ.

The samples were supplemented with 10 X Orange G loading buffer and analyzed by gel electrophoresis. No bands were observable.

1) Repeat of gradient colony PCR
To amplify cgeA 7 samples (20 µL each) were amplified by gradient colony PCR applying annealing temperatures from 58 - 68 °C. The duration of initial denaturation was elongated to 5 min.
The samples were supplemented with 10 X Orange G loading buffer and analyzed by gel electrophoresis. No bands were observable.

1) Lysis of B. subtilis for colony PCR
4 different approaches were tried to lyse the bacteria prior to colony PCR.
1. https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf
2. http://www.scs.illinois.edu/rao/protocol-subtilis-colony.php
3. Mini-Prep lysis buffer.
4. Laemmlie sample buffer 100°C 5 Min.

1 µl of each sample was used for amplification of cotZ using the oligos oIG16_1+2. The extracted cotZ gene from 11.07.16 was amplified as control alongside the lysed samples

Reaction conditions

Cycler Program

2) Testdigestion
PvuII test digestion for verification of the plasmids sent by Julia Bartels.
Reaction conditions:

Incubation at 37 °C for 1 hour. The samples were analyzed by gel electrophoresis on a 1 % TAE agarose gel.

3) Subcloning
Subcloning of the amplified cotZ gene into the linearized pJET1.2 vector was performed using the pJET subcloning kit according to the instructions of the manufacturer. 5 µL of the ligation mixture was used for transformation of chemically competent E.coli DH5alpha (Mix & Go). The transformed bacteria were spread on a LB-agar plate supplemented with amplicilin and incubated o/n at 37 °C. A control ligation was prepared using the DNA fragments supplied by the manufacturer.

1) Colony PCR
for the amplication of the cgeA was performed using the following oligos:
cgeA: oIG16_43+34 Annealing temperature: 65 °C
To lyse the bacteria prior to the colony PCR.
https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf

Reaction conditions

Cycler Program

2) Inoculation
Subcloning and transformation of pJET1.2-cotZ resulted only in a few colonies. Three of them were picked and inoculated in 5 mL LB-medium w/ ampicilin and incubated o/n at 37°C and 250 rpm. The transformation with the control plate yielded >100 colonies.

1) Colony PCR
5 µL of vegetative B.subtilis from a liquid culture were diluted 1:10 in Qiagen EB buffer and incubated at 100°C for 5min to achieve cell lysis
1 µL of the dilution was used for amplification of cgeA by gradient PCR and 8 samples were prepared.

Reaction conditions

Cycler Program

After thermal cycling the samples were supplemented with the appropriate amount of 10X Orange G loading dye and analyed by agarose gel electrophoresis.

2) MiniPrep
The plasmids pJET1.2-cotZ were prepared using the QIAprep Spin Miniprep Kit according the instructions of the manufacturer and eluted in 50µL of EB Buffer. The concentration of the DNA was spectroscopically determined by a nanodrop.

3) Testdigestion
Reaction conditions

Incubation for 1 h at 37 °C. Analysis of digestion by agarose gel electrophoresis.

2-log DNA ladder is bad in the last couple of gels –> discarded.
Not the expected band pattern. Subcloning should be repeated.

3) Colony PCR
5 µL of vegetative B.subtilis from a liquid culture were diluted 1:10 in Qiagen EB buffer and incubated at 100°C for 5min to achieve cell lysis
1 µL of the dilution was used for amplification of cgeA and cotZ by PCR.
oIG16_001+002: CotZ, Tm = 58 °C oIG16_034+043: cgeA, Tm = 63 °C

Cycler Program

The bands were excised from the gel and stored at -20 °C o/n

1) Gelextraction & Subcloning
The DNA was extracted using the QiaGen gel extraction kit according to the protocol of the manufacturer and eluted in 30 µL of ultra pure water. The concentration was determined spectroscopically by a nanodrop.

The extracted DNA was subcloned into a linearized pJET1.2 vector using the CloneJET PCR Cloning Kit according to the protocol of the manufacturer. 5 µL of the ligation mixture were transformed into chemically competent E.coli DH5alpha (Mix&Go) and spread on a LB-agar plate supplemented with ampicilin and incubated at 37 °C o/n.

1) Inoculation
4 colonies per plate were picked and incubated in 5 mL LB-medium (amp) o/n at 37 °C and 250 rpm.

1) MiniPrep
The plasmids pJET1.2-cotZ-I.#1-4;pJET1.2-cotZ-II.#1-4 and pJET1.2-cgeA#1-4 were prepared using the QIAprep Spin Miniprep Kit according the instructions of the manufacturer and eluted in 20µL of ultra pure water (for better results put tubes for 2 minutes on 50°C thermo; centrifuge for 1min at 13000rpm and repeat with 10µL; increases the amount of deluted DNA). The concentration of the DNA was spectroscopically determined by a nanodrop.

2) Test digestion
Due to the concentraion difference of the DNA the samples where divided into two groups

Reaction conditions
DNA concentration < 300ng/µL

DNA concentration > 300ng/µL

Control group < 300ng/µL

Control group > 300ng/µL

Incubation for 1 h at 37 °C. Analysis of digestion by electrophoresis on a 1% TAE agarose gel.

3) Sequencing
Sequencing of pJET1.2-cgeA #2 (IC0225) and pJET1.2-cotZII #2 (IC0224) by GATC biotech.

1) Inoculation
Confirmation of the proper sequences for pJET1.2_cgeA#2 and pJET1.2_cotZII#2. The colonies were re-inoculated in 5 mL LB-medium (w/ amp) and incubated at 37 °C, 250 rpm o/n.

1) Transformation & Inoculation
Nicole provided us with a LB-plate containing E.coli harboring pGEX6P1 and pRP261 (Both plasmids contain glutathion S transferase). Max provided us with a sample of pET303_aGFPnano_TEV_10His (plasmid containing the anti-GFP nanobody). The pET303 plasmid was transformed into chemically competent E.coli DH5alpha and spread on a LB-agar plate supplemented with ampicilin and incubated o/n at 37 °C.

1) Inoculation
Inoculation of the E.coli DH5alpha containing pGEX6P1, pRP261 and pET303_aGFPnano in 5 mL LB medium supplemented with ampicilin. Incubation o/n at 37 °C, 250 rpm.
Glycerol stocks of pJET1.2-cgeA and pJET1.2-cotZ were prepared (15% [v/v] Glycerol).
Inoculation of pBS1C3-[RFP] culture #2 for test-transformation of B.subtilis.

2) Transformation
The pSB1C3-[RFP] vector from the iGEM distribution kit (plate 1, position 23-O) was solubilized with 10 µL of ultra pure water. 1 µL was used for transformation of chemically competent E.coli DH5alpha and spread on a LB-agar plate supplemented with chloramphenicol and incubated o/n at 37 °C.

3) Extension PCR
of cgeA (from pJET1.2_cgeA) and anti GFP-Nanobody (pET303) *Reaction conditions*

1) Gelextraction
Gel Extraction of pET303-aGFPnanobody oIG16_30_fw/oIG16_031_rw PCR product.
37.7ng/µl (18µl) stored at -20 °C

2) MiniPrep
Plasmid preparation of inoculated E.coli DH5alpha transformed with Mini prep of pBS1C3-RFP culture#2 using the QiaQuick MiniPrep kit according to the instructions of the manufacturer. Elution with 30 µL of ultra pure water.
148.6ng/µl (28µl)
placed in freezer -20

Plasmid preparation of inoculated E.coli DH5alpha transformed with pGEX6P1, pRP261, pET303-aGFPnano_TEV_10His using the QiaQuick miniprep kit according to the instructions of the manufacturer. The plasmids were eluted in 30 µL of ultra pure water. The DNA concentration was determined by NanoDrop:

2) Testdigestion
Verification of the plasmid was performed by test digestion with EcoRI and PstI.
Reaction conditions:

The samples were incubated at 37 °C for 1h and analyzed by gel electrophoresis.

1) MiniPrep
Plasmids of pSB1C3 were prepared. DNA concentration was determined by Nanodrop:
Culture #1 127.5ng/µl
Culture #2 88.4ng/µl
Culture #3 116.8ng/µl
Culture #4 126.1ng/µl

2) Testdigestion pSB1C3 was treated with

Digestion mixture:

Agarose Gel of pSB1C3 digestion:

3) Inoculation
Culture #3 was re-inoculated in chloramphenicol-medium.

1) MiniPrep

With culture #3 from the day before a standard mini prep was performed.
cultur #3 169,4ng/µl (28µl)
→ placed in the -20 freezer

2) Colony PCR
Colony PCR of CotG and CotB from B. subtilis 168 genome. PCR for the amplication of the cotG and cgeB gene was performed using the following oligos:
cotG: oIG16_009+010 Annealing temperature: 62 °C
cotB: oIG16_017+018 Annealing temperature: 57 °C
Template DNA preparation: 1 µL of a o/n culture with B.subtilis W168 cells were diluted with EB-Buffer (Qiagen, 1:10) and incubated at 100°C for 5 min for lysis. 1 µL of the lysed cells was used as template for amplificaiton of the genes.

Reaction conditions

Appropriate controls w/o template DNA were included.

The PCR was analyzed by agarose gel electrophoresis.
The bands corresponding to the size of the cotG and cotB were excised and the DNA was extracted using the QiaQuick Gel extraction kit according to the instructions of the manufacturer. The DNA was eluted in 30 µL of ultra pure water.

2) Gel extraction
The concentration of the extracted DNA was determined by a Nanodrop.

3) Subcloning
The amplified genes were subcloned into the pJET1.2 vector according to the protocol of the manufacturer, transformed into chemically competent E.coli DH5alpha, spread on a LB-agar plate supplemented with ampicilin and incubated o/n at 37 °C.

4) Inoculation
The E.coli liquid cultures containing the plasmids pRP261, pGEX6P1 and pET303_aGFPnano_TEV_10His were reinoculated in 5 mL LB medium supplemented with ampicilin and incubated o/n at 37 °C and 250 rpm.

1) Inoculation
4 colonies of plated E.coli DH5alpha with the pJET1.2-cotB and pJET1.2-cotG plasmids were picked and inoculated in 5 mL of LB medium supplemented with ampicilin and incbated o/n at 37 °C and 250 rpm. 2) Sequencing
Sequencing of plasmids sent by Julia Bartels:

1) Mini-prep:

A standard Qiagen Mini-Prep was performed with the following cultures:
pJET1.2-cotB cultures #2/3/4, pJET1.2-cotG cultures #1/2/3/4, pGEX6P1 culture #1 (GST 2), pRP261 culture #1 (GST 1), pET303_aGFPnano_TEV_10His culture #1.

The DNA concentrations were measured with Nanodrop:
pJET1.2-cotB cultures #2=450,9(ng/µl)
pJET1.2-cotB cultures #3=820,3(ng/µl)
pJET1.2-cotB cultures #4=621,2(ng/µl)
pJET1.2-cotG cultures #1=696,5(ng/µl)
pJET1.2-cotG cultures #2=149,6(ng/µl)
pJET1.2-cotG cultures #3=614,8(ng/µl)
pJET1.2-cotG cultures #4=145,2(ng/µl)
pGEX6P1 culture #1=243,1(ng/µl)
pRP261 culture #1=308,2(ng/µl)
pET303_aGFPnano_TEV_10His culture #1=91,1(ng/µl)

µl taken for Test Digestion:
pJET1.2-cotB cultures #2=1(µl)
pJET1.2-cotB cultures #3=1(µl)
pJET1.2-cotB cultures #4=1(µl)
pJET1.2-cotG cultures #1=1(µl)
pJET1.2-cotG cultures #2=4(µl)
pJET1.2-cotG cultures #3=1(µl)
pJET1.2-cotG cultures #4=4(µl)
pGEX6P1 culture #2=(µl)
pRP261 culture #2=(µl)
pET303_aGFPnano_TEV_10His culture #1=6(µl)

2) Test-digestion

Digestion mixture 1:

Digestion mixture 2:

1% Agarose Gel of Test Digestion

1) Mini-prep:

A standard Qiagen Mini-Prep was performed with the following cultures:
pJET1.2-cotB cultures #2/3/4, pJET1.2-cotG cultures #1/2/3/4, pET303_aGFPnano_TEV_10His culture #1.

The DNA concentrations were measured with Nanodrop:
pJET1.2-cotB cultures #2=225,5(ng/µl)
pJET1.2-cotB cultures #3=289,5(ng/µl)
pJET1.2-cotB cultures #4=229,1(ng/µl)
pJET1.2-cotG cultures #1=104,7(ng/µl)
pJET1.2-cotG cultures #2=63,2(ng/µl)
pJET1.2-cotG cultures #3=97,6(ng/µl)
pJET1.2-cotG cultures #4=192,1(ng/µl)
pET303_aGFPnano_TEV_10His culture #1=135,1(ng/µl)

µl taken for Test Digestion:
pJET1.2-cotB cultures #2=2(µl)
pJET1.2-cotB cultures #3=2(µl)
pJET1.2-cotB cultures #4=2(µl)
pJET1.2-cotG cultures #1=5(µl)
pJET1.2-cotG cultures #2=6(µl)
pJET1.2-cotG cultures #3=5(µl)
pJET1.2-cotG cultures #4=3(µl)
pET303_aGFPnano_TEV_10His culture #1=4(µl)

2) Test-digestion

Digestion mixture:

1% Agerose Gel of Test Digestion

After testdigestion pJET1.2-cotB#3 and pJET1.2-cotG#1 were sent to sequencing.

1) Extension PCR
Extension PCR of of cgeA and aGFP-Nanobody to generate overhangs for Gibson cloning.

*Reaction conditions*

*PCR program*
Touchdown PCR

After PCR the samples were analyzed by agarose gel electrophoresis (1 % agarose TAE gel).

2) Gel extraction
The bands corresponding to the appropriate sizes were extracted using the QiaQuick gel extraction kit and the DNA was eluted in 30 µL of ultra pure water. The concentration was photometrically determined by a Nanodrop.

1) Restriction digestion

Linearization of pSB1C3 for Gibson Cloning:

Digestion mixture

2) Agarose Gel of pSB1c3

→ followed by gel extraction for gibson assembly.

1) NEBuilder® HiFi DNA Assembly Cloning:

Assembly of extensionPCR products into pSB1C3.

Reaction conditions
DNA: 1.5 µL
Fragment1: 25 ng
Fragment2: 15 ng
linearized pSB1C3: 25 ng
2X HiFi MasterMix: 5 µL
ultra pure water: 3.5 µL

The reaction was incubated at 50 °C for 1 hour. 2 µL of the reaction mixture was transformed into chemically competent E.coli DH5alpha provided by the NEBuilder HiFi kit according to their protocol, plated on LB-agar plates containing chloramphenicol and incubated o/n at 37 °C.
The control reaction provided by the kit was performed according to the instructions of the manufacturer.

2) Digestion of pBS1C
Digestion mixture:

Agarose gel of pBS1c:

→ followed by a Gel extraction for Transformation of competent B.subtilis W168.

1) Inoculations
Inoculation of 2 colonies from each plate with transformed E.coli containing pSB1C3_cgeA_aHelix_HA-Tag_aGFPnano and pSB1C3_aGFPnano_aHelix_HA-Tag_cgeA. Incubation o/n at 37 °C and 250 rpm.
Remaining E.coli from the transformation (see previous day) were plated on LB-agar plates supplemented with cml and incubated o/n at 37 °C.

2) Digestion of pBS1C (For transformation of B.subtilis)

Digestion mixture:

→ PCR purification was peformed

1) MiniPrep
Plasmid preparation of the inoculated E.coli from the previous day using the QiaQuick Plasmid preparation kit according to the protocol of the manufacturer and eluted in 30 µL of ultra pure water. The DNA concentration was photometrically determined by a NanoDrop:

2) Testdigestion
500 ng of DNA was treated with 5 unit of XbaI and PstI in 1x NEBuffer 2.1 in a total reaction volume of 20 µL. Incubation for 1hour at 37 °C.

2) Inoculation
I Inoculation of E.coli DH5alpha containing an mCherry and GFP plasmid (provided by AG Weber) in 5 mL LB containig (Three colonies per plate were picked)
II E.coli DH5alpha containing plasmids sent from Poland (by Dr. Krystof Hinc) for the construction of fusion proteins for spore surface display arrived:
1)pCotG-N
2)pCotG-C
3)pCotB-N
4)pCotB-C
5)pCgeA-C
6)pCotZ-C
The E.coli were spread on LB-Agar plates containing ampicilin and incubated o/n at 37 °C.
III* 6 colonies per plate of E.coli DH5alpha containing pSB1C3_CgeA_alphaHelix_HA-Tag_aGFPnano or pSB1C3_aGFPnano_alphaHelix_HA-Tag_cgeA were inoculated in 5 mL LB-medium supplemented with chloramphenicol and incubated at 37 °C and 250rpm.

1) MiniPrep pIG16_038 + pIG16_039, GFP + mCherry
Plasmid preparation of inoculated E.coli DH5alpha transformed with plasmids containing GFP and mCherry (from AG Weber) using the QiaQuick MiniPrep kit according to the instructions of the manufacturer. Elution with 30 µL of ultra pure water. The concentration was determined by Nanodrop.

2)Inoculation
Inoculation of the E.coli containing the plasmids sent from Poland. 4 Colonies from each plate were picked and inoculated in 5 mL of LB medium supplemented with Amp.

3)ExtensionPCRs Q5 –> Gel+extraction

Reaction conditions

PCR Program
Touchdown PCRs corresponding to the annealing temperatures.

After amplification the samples were loaded on a 1% Agarose TAE gel.

The bands corresponding to the expected fragment sizes were extracted from the gel, purified and eluted with 30 µL of ultra pure water.

1) Testdigestion
pIG16_038+039 were verified by testdigestion. 500 ng of DNA was treated with XbaI and PstI and analyzed by gel electrophoresis.

2) MiniPrep
The inoculated cultures containing the plasmids sent from poland were preparated using the QiaQuick MiniPrep kit according to the protocol. The DNA was eluted with 30 µL of ultra pure water. The concentration was determined by Nanodrop.

3) GFP purification
Sebastian from AG Weber gave a purified GFP (1.35 mg/mL) and mCherry (2 mg/mL).
Stored at 4 °C, protected from light.

1) ExtensionPCR

After amplification the sample was loaded on a 1% Agarose TAE gel. The band corresponding to the expected size was extracted and purified.

2) 3A assembly

Digestion mixture PCotYZ:
The B.subtilis spore coat promoter PCotYZ was digested from the Sporovector pIG16_017 provided by Julia Bartels from the Mascher Lab at the TU Dresden.

Digestion mixture for pIG16_038+039:

Digestion mixture for pBS1C:

T4 Ligation

Ligation mixture of plG16_38/39 #1:

The reaction was incubated for 30min at RT and transformed into chemically competent mix and go E. coli DH5alpha

3) Gibson assembly
Assembly of extensionPCR products into pSB1C3.

Reaction conditions
DNA: 1.5 µL
Fragment1: 25 ng
Fragment2: 15 ng
linearized pSB1C3: 25 ng
2X HiFi MasterMix: 5 µL
ultra pure water: 3.5 µL
The reaction was incubated at 50 °C for 1 hour. 2µL of the reaction mix were transformed into chemically competent E.coli DH5alpha and spread on agarose LB plates supplemented with chloramphenicol. Incubation o/n at 37 °C.

4) Testdigestion of GFP
For verification 500 ng of the plasmid was treated with XbaI, XhoI, BamHI in 1X NEBuffer 3.1 for 90 min at 37°C. The fragments were analyzed by agarose gel electrophoresis.

5) Test Digestion Plasmids provided by Krystof Hinc
From university Gdansk. A set of vectors for surface display in B.subtilis.

Digestion mixture cgeA-C/cotZ-C:

Digestion mixture cotB-C/N:

Digestion mixture cotG-C:

Digestion mixture cotG-N:

cgeA-C cotB-C/N

cotB-C/N

cot-Z

1) Repeat of 3A Assembly

Ligation mixture of plG_38/39 #1:

2) Inoculation
Inoculation of Culture #1/2 with construct plG16_038 and Culture #1/2 with construct plG16_039.

1)Miniprep

Standard Qiagen Miniprep of Culture #1/2 with construct plG16_38 and Culture #1/2 with construct plG16_39.

2) Digestion

Digestion mixture poG16_38/39:

Digestion mixture pBS1C:

3) Repeat of 3A Assembly

Ligation mixture of plG_38/39 #1:

4) Inoculation
Transformed E.coli containing the plasmids pIG16_040 - 043 were inoculated. 6 colonies per plate were picked and inoculated in 5 mL LB medium supplemented with chloramphenicol. Incubation o/n at 37 °C and 250 rpm.

5) Subcloning of PCotYZ
PCotYZ PCR product was subcloned into pJET2.1 vector according to protocol delivered with the kit.

1) MiniPrep
The inoculated E.coli containing the plasmids pIG16_040 - 043 were preparated using the QiaQuick MiniPrep kit. The DNA was eluted with 30 µL of ultra pure water. Verification of the plasmids was performed by testdigestion. 500 ng of DNA was treated with XbaI and PstI in 1X NEBuffer 3.1 for 90 min at 37 °C.

2) Sequencing
pIG16_038#1 and pIG16_039#1 were sent to sequencing by GATCbiotech.

3) ExtensionPCRs

Reaction conditions

PCR Program
Touchdown PCRs corresponding to the respective annealing temperatures.

3) Inoculation of pJET-PcotYZ colonies

Tree colonies from the the plate 1 (Wlad) and Tree colonies from the the plate 2 (iGEM) were picked and inoculated.

1) Gelextraction of ExtensionPCRs Was performed according to the protocol of the manufacturer. The samples were extracted from the gel from the previous day.

2) Sequence analysis
pIG16_038 + 039 #1
Sequencing showed that the samples were switched at some point. pIG16_039 contained an additional insert of ~9 bp between the alpha helical linker and the HA-tag. New samples have to be prepared for analysis.

3) Reinoculation
New colonies containing pIG16_038#2 039#2 were inoculated in 5 mL of LB medium supplemented with chloramphenicol.

4) Miniprep of pJET-PCotYZ

Standard Qiagen miniprep was performed with 3 cultures from the the plate 1 and 3 cultures from the the plate 2 .

5) Test digestion pJET-PcotYZ

Digestion pJET-PcotYZ:

Culture #1 was sent to sequencing.

1) MiniPrep
Plasmid preparation of pIG16_038#2 and pIG16_039#2 using the QiaQuick MiniPrep kit. The DNA was eluted with 30 µL of ultra pure water.

1) Sequencing The plasmids pIG16_038#2, pIG16_039#2, pIG16_040#2, pIG16_041#3, pIG16_IG16_042#3 and pJET1.2-PCotYZ#1 were sent to sequencing by GATCbiotech.

1) Sequencing Results
pIG16_040 - pIG16_042 could be confirmed by sequencing. Glycerol stock was prepared.
The samples from pIG16_038 and pIG16_039 were switched in previous steps. –> switched back. pIG16_039 was confirmed by sequencing.
pIG16_038 had a 1bp deletion. Further colonies pIG16_038#3 & #4 were sent to sequencing by GATC-Biotech.

2)MiniPrep of pIG16_039-042 and PcotYZ
Standard Quiagen Miniprep was performed.

3) Digestion of pIG16_039-042, PcotYZ

Digestion mixture pIG16_39-42:

Digestion mixture PcotYZ:

4) 3A Assembly

Ligation mixture of plG_38/39 #1:

5) Gibson Cloning
1.33x MasterMix preparation.
5X Iso buffer: 50 µL
T5 Exonuclease (NEB): 1 µL (Diluted 1:10 in ultra pure water)
Taq Ligase (NEB): 25 µL
Phusion Polymerase (NEB): 3.125 µL
Nuclease free water: 108.4 µL
Aliquots: 7.5 µL, stored at -20 °C

Assemblies
All samples were adjusted to 50 ng/µL

0.5 µL of each fragment and 0.5 µL of the digested backbone were mixed with 1µL of ultra pure water and added to the 1.33x Gibson Mix and incubated for 60 min at 50 °C. 5µL of the reaction mix were used to transform chemically competent E.coli DH5alpha. Subsequently, 300 µL of LB medium were added. The Bacteria were incubated at 37 °C and 250 rpm and spread on Agar-LB plates supplemented with chloramphenicol.

1) Extension PCR

Reaction conditions

Cycler Program Touchdown58

The samples analyzed by agarose gel electrophoresis and the bands corresponding to the expected sizes were extracted and purified using the Qiagen gel extraction kit.

2) Inoculation The transformed E.coli from the Gibson assembly (previous day) were inoculated in 5 mL LB-medium supplemented with chloramphenicol and incubated o/n at 37 °C and 250 rpm.

3) Gibson assembly

0.5 µL from each fragment were mixed with 1 µL of ultra pure water, added to 1.33x Gibson Master Mix and incubated at 50 °C for 1 h. 2µL of the reaction mix were transformed into chemically competent E.coli DH5alpha.

1) MiniPrep
Plasmid preparation of pIG16_046 - 055 using the QiaQuick MiniPrep kit. The DNA was eluted in 30 µL of ultra pure water. Verification of the plasmids by Testdigestion using. 500 ng of DNA was treated with 2 unit of XbaI and PstI in 1X NEBuffer 3.1 for 1 hour. . Unexpected band patterns. The DNA was not completly digested.

2) Inoculation
The transformed E.coli with the Gibson assemblies from the previous day were inoculated in 5 mL LB-medium supplemented with chloramphenicol

1) Testdigestion
The digestion of pIG16_046 - 055 from the previous day was repeated. 500 ng of DNA was treated with 4 unit of XbaI and PstI in 1X NEBuffer 2.1 for 90 min at 37 °C.

Positive samples were sent to sequencing:
1) pIG16_047#3
2) pIG16_048#1
3) pIG16_050#5
4) pIG16_051#1
5) pIG16_054#1
6) pIG16_055#2

1) Production of competent DH5-alpha E. coli
E. colis were made competent according to zymo research Mix and go protocol

1) Inoculation
Since pIG16_046, 049, 052 were all negative 5 additional colonies were picked and inoculated in 5 mL of LB medium supplemented with chloramphenicol.
5 colonies of the E.coli transformed with the 3A assembly reaction (pIG16_062 - 065) were pickend and inoculated in 5 mL of LB medium supplemented with ampicilin.
5 colonies of the E.coli transformed with the Gibson assembly reaction (pIG16_057 - 059).

2) Glycerol stocks
Preparation of Glycerol stocks of pIG16_038, 039, 040, 041, 042

1) Plasmid preparation
Preparation of plasmids from transformed E.coli containing the constructs pIG16_46 + 049 + 052+ 058+ 059+ 062+ 063+ 064+ 065

2) Testdigestion
3A assembly: NotI + XhoI
Gibson assembly: XbaI + PstI

Digestions partially incomplete.
Samples sent to sequencing:

3) Sequencing

1) Glycerol stock
Preparation of a 15 % Glycerol stock of pIG16_039 and storage at - 80 °C.

2) Reinoculation
New 6 mL LB cultures were prepared for pIG16_047#3, 050#5, 048#4, 051#1 054#4 and incubated o/n at 37 °C and 250 rpm. For subsequent 3A assembly.

3) Preparation of competent E.coli

The Ecoli strain DH5-alpha was prepered and made competent according to Zymoreserch MIX and GO kit.

1) Extension PCR

Reaction conditions

Thermal Cycling
Touchdown 62

2) Gibson Assembly

The concentration of all fragments was adjusted to 50 ng/µL. 0.5 µL of each fragment were mixed alongside with 0.5 µL of the linearized vector and added to the 1.33x Gibson MasterMix and incubated for 60 min at 50 °C.
5µL of the Gibson mix were transformed into chemically competent E.coli DH5alpha and spread on LB agar plate supplemented with chloramphenicol.

3) Digestion for 3 A Assembly

Digestion of PcotYZ

Digestion of pBS1C and pBS4S

Digestion of pIG16_38 47 48 50 51 54