Lab Book Cloning

To keep an overview of the cloned constructs every plasmid was assigned to an ID: pIG16_000.
All used oligos were assigned to an ID as well: oIG16_000.
The complete list of the resulting bacterial strains and oligos can be found in the attached Excel files. The spore coat proteins cotZ, cotG, cotB and cgeA were amplified from the genome of B. subtilis 168. The anti-GFP nanobody and the GST were amplified from plasmids provided by Dr. Nicole Gensch and Dr. Maximilian Ulbrich. For the cloning strategy see Project - Approach (LINK ZU PROJECT _ APPROACH)

07.07.16

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.

08.07.16

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.

09.07.16

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

component	concentration	volume [µL]
1 B.subtilis colony	-	-
Primer fw	10 µM	1.0
Primer rv	10 µM	1.0
dNTPs	40 µM	0.4
HF Buffer	5X	4.0
Phusion Pol	2U/µL	0.2
ddH2O	-	ad20 µL

Cycler Program

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	1 min	1
Denaturation	98	10 s	30
Annealing	58 or 65	10 s	30
Elongation	72	30 s	30
Final elongation	72	5 min	1
Storage	8	-

10.07.16

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)

11.07.16

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

12.07.16

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

component	concentration	volume [µL]
1 B.subtilis colony W168	-	-
Primer fw	10 µM	2.5
Primer rv	10 µM	2.5
dNTPs	40 µM	0.4
HF Buffer	5X	10
Phusion Pol	2U/µL	0.5
ddH2O	-	ad50 µL

Cycler Program

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	1 min
Denaturation	98	10 s	30X
Annealing	58 or 65	10 s	30X
Elongation	72	30 s	30X
Final elongation	72	5 min
Storage	8	-

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.

13.07.16

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.

14.07.16

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.

15.07.16

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

component	concentration	volume [µL]
lysed B.subtilis W168	-	1 µL
Primer fw	10 µM	2.5
Primer rv	10 µM	2.5
dNTPs	40 µM	0.4
HF Buffer	5X	10
Phusion Pol	2U/µL	0.5
ddH2O	-	ad50 µL

Cycler Program

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	5 min
Denaturation	98	10 s	30X
Annealing	65	10 s	30X
Elongation	72	30 s	30X
Final elongation	72	5 min
Storage	8	-

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

component	concentration	volume
MiniPrep DNA	200-600 ng/µL	2 µL
CutSmart Buffer (NEB)	10X	2 µL
PvuII-HF	20 U/ µL	0.1 µL
ddH2O	-	ad20 µL

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.

16.07.16

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

component	concentration	volume [µL]
lysed B.subtilis W168	-	1 µL
Primer fw	10 µM	2.5
Primer rv	10 µM	2.5
dNTPs	40 µM	0.25
HF Buffer	5X	10
Phusion Pol	2U/µL	0.5
ddH2O	-	ad50 µL

Cycler Program

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	5 min
Denaturation	98	10 s	30X
Annealing	65	10 s	30X
Elongation	72	30 s	30X
Final elongation	72	5 min
Storage	8	-

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.

17.07.16

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

component	concentration	volume [µL]
lysed B.subtilis W168	-	1 µL
Primer fw	10 µM	1
Primer rv	10 µM	1
dNTPs	40 µM	0.1
HF Buffer	5X	5
Phusion Pol	2U/µL	0.2
ddH2O	-	ad20 µL

Cycler Program

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	5 min
Denaturation	98	10 s	30X
Annealing	gradient 58-68	20 s	30X
Elongation	72	30 s	30X
Final elongation	72	5 min
Storage	10	-

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.

DNA	concentration[ng/µL]
pJet_cotZ_#1	120
pJet_cotZ_#2	89
pJet_cotZ_#3	68

3) Testdigestion
Reaction conditions

component	concentration	volume
DNA	68 - 120 ng/µL	7 µL
XbaI	20U/µL	0.2
XhoI	20U/µL	0.2
CutSmart	10X	2 µL
dH2O	-	11 µL

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

component	concentration	volume [µL]
lysed B.subtilis W168	-	1 µL
Primer fw	10 µM	2.5
Primer rv	10 µM	2.5
dNTPs	40 µM	0.25
HF Buffer	5X	10
Phusion Pol	2U/µL	0.5
ddH2O	-	ad50 µL

Cycler Program

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	5 min
Denaturation	98	10 s	30X
Annealing	58 or 63	10 s	30X
Elongation	72	30 s	30X
Final elongation	72	5 min
Storage	8	-

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

18.07.16

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.

gene	concentration
cgeA	6 ng/µL
cotZ#1	8 ng/µL
cotZ#2	11 ng/µL

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.

19.07.16

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

20.07.16

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.

DNA	concentration[ng/µL]
pJet1.2_cgeA_#1	127
pJet1.2_cgeA_#2	214 –> Seq
pJet1.2_cgeA_#3	279
pJet1.2_cgeA_#4	141
pJet1.2_cotZ-I_#1	260
pJet1.2_cotZ-I_#2	101
pJet1.2_cotZ-I_#3	409
pJet1.2_cotZ-I_#4	435
pJet1.2_cotZ-II_#1	475
pJet1.2_cotZ-II_#2	350 –> Seq
pJet1.2_cotZ-II_#3	353
pJet1.2_cotZ-II_#4	342

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

Reaction conditions
DNA concentration < 300ng/µL

component	concentration	volume
DNA	101 - 279 ng/µL	5 µL
XbaI	20U/µL	0.2
XhoI	20U/µL	0.2
CutSmart	10X	2 µL
dH2O	-	14.1 µL

DNA concentration > 300ng/µL

component	concentration	volume
DNA	342-475 ng/µL	1.5 µL
XbaI	20U/µL	0.2
XhoI	20U/µL	0.2
CutSmart	10X	2 µL
dH2O	-	12.6 µL

Control group < 300ng/µL

component	concentration	volume
DNA	101 - 279 ng/µL	5 µL
dH2O	-	5 µL

Control group > 300ng/µL

component	concentration	volume
DNA	342-475 ng/µL	1.5 µL
dH2O	-	8,5 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.

21.07.16

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.

22.07.16

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.

23.07.16

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*

component	concentration	volume
DNA	~10 ng/µL	1 µL
Primer fw	10 µM	2.5
Primer rv	10 µM	2.5
Q5 High-Fidelity Master Mix (NEB)	2x	25 µL
ultra pure H2O	-	ad 50 µL

Touchdown-PCR Template: pJET1.2_cgeA
Primer:olG16_44fw + olG16_45rw

Cycler Program GFP-Nanobody

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	5 min
Denaturation	98	10 s	10X
Annealing	72* (-1°C per cycle)	10 s	10X
Elongation	72	30 s	10X
Denaturation	98	10 s	20X
Annealing	63	10 s	20X
Elongation	72	30 s	20X
Final elongation	72	5 min
Storage	8	-

Template: pET303_aGFPnano
Primer:olG30_fw/olG31_rw

Cycler Program CgeA

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	5 min
Denaturation	98	10 s	10X
Annealing	72* (-1°C per cycle)	10 s	10X
Elongation	72	30 s	10X
Denaturation	98	10 s	20X
Annealing	62	10 s	20X
Elongation	72	30 s	20X
Final elongation	72	5 min
Storage	8	-

After amplification the samples were analyzed by agarose gel electrophoresis.

Gel GFP-Nanobody/CgeA

24.07.16

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:

Sample	concentration[ng/µL]
pRP261 #1	248.9
pRP261 #2	175.0
pRP261 #3	235.9
pRP261 #4	221.0
pGEX-6P1 #1	198.7
pGEX-6P1 #2	177.3
pGEX-6P1 #3	149.1
pGEX-6P1 #4	131.0
pET303-aGFPnano_TEV_10His #1	167.4
pET303-aGFPnano_TEV_10His #2	151.1
pET303-aGFPnano_TEV_10His #3	161.7
pET303-aGFPnano_TEV_10His #4	165.0

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

Component	concentration	volume [µL]
DNA	131-248ng/µL	3
PstI	20u/µL	0.1
EcoRI	20u/µL	0.1
NEBuffer 2.1	10X	2
ultra pure water	-	ad 20 µL

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

25.07.16

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:

Component	concentration	volume [µL]
DNA	500ng/µL	5-6
PstI	20u/µL	0.1
EcoRI-HF	20u/µL	0.1
NEBuffer 2.1	10X	2
ultra pure water	-	ad 20 µL

Agarose Gel of pSB1C3 digestion:

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

26.07.16

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

component	concentration	volume [µL]
Q5 HiFi MasterMix	2x	25
Primer fw	10 µM	2.5
Primer rv	10 µM	2.5
lysed cells	-	1
ultra pure water	-	ad 50 µL

Appropriate controls w/o template DNA were included.

The PCR was analyzed by agarose gel electrophoresis.
colony_pcr_cotb_cotg 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.

sample	concentration
CotB	50.2 ng/µL
CotG	42.1 ng/µL

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.

27.07.16

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:

Plasmid	colony#	Primer:oIG16_
pBS1C-[RFP]	#2	025
		026
		028
		029
		032
pBS2E-[RFP]	#5	025
pBS4S-[RFP]	#5	025
		26
		27
SporoVector-[RFP]	#1	025
		026
		027

28.07.16

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:

Component	concentration	volume [µL]
DNA	500ng/µL	5-6
XbaI	20u/µL	0.1
XhoI	20u/µL	0.1
NEBuffer 2.1	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture 2:

Component	concentration	volume [µL]
DNA	500ng/µL	5-6
PstI	20u/µL	0.1
EcoRI-HF	20u/µL	0.1
NEBuffer 2.1	10X	2
ultra pure water	-	ad 20 µL

1% Agarose Gel of Test Digestion

29.07.16

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:

Component	concentration	volume [µL]
DNA	500ng/µL	5-6
PstI	20u/µL	0.1
EcoRI-HF	20u/µL	0.1
NEBuffer Smart-Cut	10X	2
ultra pure water	-	ad 20 µL

1% Agerose Gel of Test Digestion

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

30.07.16

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

Template	Primer	Annealing Temp. [°C]
pJET1.2_cgeA #2	oIG16_044 + 045	62
	oIG16_050 + 035	62
pET303_aGFPnano_TEV_10His	oIG16_030 + 031	67
	oIG16_051 + 042	67

*Reaction conditions*

Component	concentration	Volume[µL]
Primer fw	10 µM	2.5
Primer rv	10 µM	2.5
Q5 HiFi MasterMix	2x	25
ultra pure H2O	-	19

*PCR program*
Touchdown PCR

Step	Temperature [°C]	Duration	Repeats
Initial denaturation	98	5 min
Denaturation	98	10 s	10X
Annealing	Annealing Temp + 10°C (-1°C per cycle)	10 s	10X
Elongation	72	30 s	10X
Denaturation	98	10 s	20X
Annealing	Annealing temp.	10 s	20X
Elongation	72	30 s	20X
Final elongation	72	5 min
Storage	8	-

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.

Sample	Concentration [ng/µL]
cgeA_oIG16_044+045	85.2
cgeA_oIG16_50+35	62.4
aGFPnano_oIG16_31+30	93.7
aGFPnano_oIG16_51+42	104

31.07.16

1) Restriction digestion

Linearization of pSB1C3 for Gibson Cloning:

Digestion mixture

Component	concentration	volume [µL]
DNA	100ng/µL	5-6
XbaI	20u/µL	0.5
XhoI	20u/µL	0.5
NEBuffer 2.1	10X	10
ultra pure water	-	ad 50 µL

2) Agarose Gel of pSB1c3

→ followed by gel extraction for gibson assembly.

01.08.16

1) NEBuilder® HiFi DNA Assembly Cloning:

Assembly of extensionPCR products into pSB1C3.

No.	amplified fragment1	amplified fragment 2	digested Backbone	Resulting Plasmid
1	cgeA oIG16_50+35	aGFPnano_oIG16_51+42	XbaI - pSB1C3 - SpeI	pSB1C3_cgeA_aHelix_HA_aGFPnano pIG16_038
2	aGFPnano oIG16_30+47	cgeA oIG16_46+45	EcoRI - pSB1C3 - SpeI	pSB1C3_aGFPnano_HA_aHelix_cgeA pIG16_039

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:

Component	concentration	volume [µL]
DNA	148ng/µL	16
XhoI	20u/µL	0.5
NEBuffer CutSmart	10X	10
ultra pure water	-	ad 50 µL

Agarose gel of pBS1c:

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

02.08.16

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:

Component	concentration	volume [µL]
DNA	700ng/µL	10
XhoI	20u/µL	1.5
NEBuffer CutSmart	10X	10
ultra pure water	-	ad 50 µL

→ PCR purification was peformed

03.08.16

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:

sample	concentration [ng/µL]
pSB1C3_CgeA_alphaHelix_HA-Tag_aGFPnano #1	69.1
pSB1C3_CgeA_alphaHelix_HA-Tag_aGFPnano #2	126.7
pSB1C3_aGFPnano_alphaHelix_HA-Tag_cgeA #1	120.4
pSB1C3_aGFPnano_alphaHelix_HA-Tag_cgeA #2	182.1

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.

04.08.16

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.

No	Sample ID	Nucleic Acid Conc.
1	h2o	0,9	ng/µl
2	pIG16_038 #1	106,9ng/µl
3	pIG16_038 #2	92,4ng/µl
4	pIG16_038 #3	77,3ng/µl
5	pIG16_038 #4	64,4ng/µl
6	pIG16_038 #5	63,2ng/µl
7	pIG16_038 #6	55,4ng/µl
8	pIG16_039 #1	105,2ng/µl
9	pIG16_039 #2	101,3ng/µl
10	pIG16_039 #3	73,2ng/µl
11	pIG16_039 #4	85,2ng/µl
12	pIG16_039 #5	80,4ng/µl
13	pIG16_039 #6	86,7ng/µl
14	pIG16_024 GFP #1	93,2ng/µl
15	pIG16_024 GFP #2	76,3ng/µl
16	pIG16_024 GFP #3	100,9ng/µl
17	pIG16_025mCherry#3	143,2ng/µl
18	pIG16_025mCherry#2	175,8ng/µl
19	pIG16_025mCherry#3	132,8ng/µl

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

No.	Template	Primer: oIG16_#	Annealing Temp[°C]	Resulting construct
1	pET303_aGFPnano	030 + 047	67	Nano_HA_G4S
2	pET303_aGFPnano	053 + 042	67	G4S_HA_Nano
3	pJET_cotG	015 + 016	62	HA_aHelix_CotG
4	pJET_cotG	011 + 013	62	CotG_aHelix_HA
5	pSBBs4S_Sporovector	048 + 049	55	Bb-Prefix_PCotYZ_Bb-Suffix
6	pJET_cgeA	046 + 045	62	HA_G4S_CgeA
7	pJET_cgeA	050 + 052	62	CgeA_G4S_HA
8	dH2O	046 + 045	62	negative control
9	dH2O	015 + 016	62	negative control
10	dH2O	030 + 047	67	negative control

Reaction conditions

Component	Volume µL
2XHiFi MasterMix	25
Primer1	2.5
Primer2	2.5
template DNA	0.1
ultra pure water	19.9

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.

05.08.16

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.

06.08.16

1) ExtensionPCR

template	Primer oIG16_	Annealing Temp. [°C]	Resulting construct
pJet1.2_cotG	011 + 013	62	CotG_aHelix_HA

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.

Component	concentration	volume [µL]
DNA	500ng/µL	13.5
EcoRI	20u/µL	0.1
SpeI	20u/µL	0.1
NEBuffer Cut Smart	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture for pIG16_038+039:

Component	concentration	volume [µL]
DNA	500ng/µL	variable
XbaI	20u/µL	0.1
PstI	20u/µL	0.1
NEBuffer 3.1	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture for pBS1C:

Component	concentration	volume [µL]
DNA	500ng/µL	variable
EcoRI	20u/µL	0.2
PstI	20u/µL	0.2
NEBuffer 2.1	10X	2
ultra pure water	-	ad 20 µL

T4 Ligation

Ligation mixture of plG16_38/39 #1:

Component	concentration	volume [µL]
pBS1C	2000ng/µL	1
plG_38	105ng/µL	4
plG_39	104ng/µL	4
PcotYZ	24ng/µL	0.2
T-4 Ligase		1µl
NEBuffer T-4 Ligase	10X	2
ultra pure water	-	ad 20 µL

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.

No.	amplified fragment1	amplified fragment 2	digested Backbone	Resulting Plasmid
1	cgeA oIG16_050+052	aGFPnano_oIG16_53+42	XbaI - pSB1C3 - SpeI	pSB1C3_cgeA_G4S_HA_aGFPnano pIG16_040
2	aGFPnano oIG16_30+47	cotG oIG16_46+45	EcoRI - pSB1C3 - SpeI	pSB1C3_aGFPnano_HA_G4S_cgeA pIG16_041
3	aGFPnano oIG16_030+031	cotG oIG16_015+016	EcoRI - pSB1C3 - SpeI	pSB1C3_aGFPnano_HA_aHelix_cotG pIG16_042
4	cotG oIG16_011+013	aGFPnano oIG16_051+042	EcoRI - pSB1C3 - SpeI	pSB1C3_cotG_aHelix_HA_aGFPnano pIG16_043

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:

Component	concentration	volume [µL]
DNA	500ng/µL	cgeA: #1/2/4=5 #3=8 cotZ: #1/2/3/4=7
XhoI	20u/µL	0.1
PstI	20u/µL	0.1
NEBuffer 3.1	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture cotB-C/N:

Component	concentration	volume [µL]
DNA	500ng/µL	cotB-C: #1/2=5 #3/4=6 cotB-N #1=5 #2=6 #3/4=7
PstI	20u/µL	0.1
NEBuffer 3.1	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture cotG-C:

Component	concentration	volume [µL]
DNA	500ng/µL	#1/2/3/4=2
BamHI	20u/µL	0.1
NEBuffer 3.1	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture cotG-N:

Component	concentration	volume [µL]
DNA	500ng/µL	#1/2/3=2 #4=6
XbhI	20u/µL	0.1
XhoI	20u/µL	0.1
NEBuffer CutSmart	10X	2
ultra pure water	-	ad 20 µL

cgeA-C cotB-C/N

cotB-C/N

cot-Z

07.08.16

1) Repeat of 3A Assembly

Ligation mixture of plG_38/39 #1:

Component	concentration	volume [µL]
pBS1C	2000ng/µL	1
plG16_038	105ng/µL	7
plG16_039	104ng/µL	7
PcotYZ	24ng/µL	0.4
T4 Ligase		1µl
T4 Ligase reaction buffer	10X	2
ultra pure water	-	ad 20 µL

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

08.08.16

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:

Component	concentration	volume [µL]
DNA	500ng/µL
XbaI	20u/µL	0.1
PstI	20u/µL	0.1
NEBuffer 3.1	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture pBS1C:

Component	concentration	volume [µL]
DNA	500ng/µL
EcoRI	20u/µL	0.2
PstI	20u/µL	0.2
NEBuffer 2.1	10X	2
ultra pure water	-	ad 20 µL

3) Repeat of 3A Assembly

Ligation mixture of plG_38/39 #1:

Component	concentration	volume [µL]
pBS1C	2000ng/µL	0.5
plG_38	500ng/µL	1.5
plG_39	500ng/µL	1.5
PcotYZ	24ng/µL	0.5
T-4 Ligase		1µl
NEBuffer T-4 Ligase	10X	2
ultra pure water	-	ad 20 µL

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.

09.08.16

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

No.	Template	Primer oIG16_	Annealing temp [°C]	Resulting construct
1	pGEX6P1	036 + 054	64	GST_HA_G4S
2	pGEX6P1	041 + 055	64	G4S_HA_GST
3	pJET_cotZ	006 + 008	58	HA_G4S_CotZ
4	pJET_cotZ	003 + 004	58	CotZ_G4S_HA
5	pJET_cotG	014 + 016	62	HA_G4S_CotG
6	pJET_cotG	011 + 012	62	CotG_G4S_HA
7	pJET_cotB	022 + 024	56	HA_G4S_CotB
8	pJET_cotB	019 + 020	56	CotB_G4S_HA

Reaction conditions

Component	volume
2X Q5 MasterMix	25 µL
Primer1	2.5 µL
Primer2	2.5 µL
Template	0.1 µL
water	19.9 µL

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.

10.08.16

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:

Component	concentration	volume [µL]
DNA	500ng/µL	1-3
EcoRI	20u/µL	0.1
SpeI	20u/µL	0.1
NEBuffer Cut Smart	10X	2
ultra pure water	-	ad 20 µL

Culture #1 was sent to sequencing.

11.08.16

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.

12.08.16

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:

Component	concentration	volume [µL]
DNA	2500ng	5µl
XbaI	20u/µL	0.5
PstI	20u/µL	0.5
NEBuffer 3.1	10X	2
ultra pure water	-	ad 20 µL

Digestion mixture PcotYZ:

Component	concentration	volume [µL]
DNA	5000ng	10
EcoRI	20u/µL	1
SpeI	20u/µL	1
NEBuffer Cut Smart	10X	5
ultra pure water	-	ad 50 µL

4) 3A Assembly

Ligation mixture of plG_38/39 #1:

Component	concentration	volume [µL]
pBS1C	50ng	1
plG_39-41	20ng	1
plG_42	25ng/µL	11
PcotYZ	5.7ng	0.5
T-4 Ligase	20u/µl	1µl
NEBuffer T-4 Ligase	10X	2
ultra pure water	-	ad 20 µL

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

No.	Fragment 1	Fragment 2	digested backbone	resulting construct
1	GST oIG16_036+054	CotG oIG16_014+016	pSB1C3	pIG16_046
2	GST oIG16_036+054	CgeA oIG16_046+045	pSB1C3	pIG16_047
3	GST oIG16_036+054	CotZ 006+008	pSB1C3	pIG16_048
4	GST oIG16_036+054	CotB 022+024	pSB1C3	pIG16_049
5	GST oIG16_041+055	CgeA 050+052	pSB1C3	pIG16_050
6	GST oIG16_041+055	CotZ 003+004	pSB1C3	pIG16_051
7	GST oIG16_041+055	CotB 019+020	pSB1C3	pIG16_052
8	aGFPnano oIG16_030+047	CotG 014+016	pSB1C3	pIG16_053
9	aGFPnano oIG16_030+047	CotZ 006+008	pSB1C3	pIG16_054
10	aGFPnano oIG16_030+047	CotB 022+024	pSB1C3	pIG16_055
11	positive control	positive control	postive control	positive control from NEB HiFi Assembly kit

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.

13.08.16

1) Extension PCR

template	Primer	Resulting construct
pJET1.2_CotZ	oIG16_007+008	HA_alphaHelix_CotZ_pSB1C3-OH
pJET1.2_CotZ	oIG16_003+005	pSB1C3-OH_CotZ_alphaHelix_HA

Reaction conditions

component	volume
2XMM	25 µL
Primer1	2.5 µL
Primer2	2.5 µL
template	0.1 µL
dH2O	19.9 µL

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

No.	Fragment1	Fragment2	Digested Backbone	resulting construct
1	pJet1.2_cotZ oIG16_007+008	aGFPnano oIG16_30+31	pSB1C3	pIG16_056
2	pJet1.2_cotZ oIG16_006+008	aGFPnano oIG16_30+47	pSB1C3	pIG16_057
3	pJet1.2_cotZ oIG16_003+005	aGFPnano oIG16_51+42	pSB1C3	pIG16_058
4	pJet1.2_cotZ oIG16_003+004	aGFPnano oIG16_53+42	pSB1C3	pIG16_059
5	positive control	positive control	positive control	C+ from NEB

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.

14.08.16

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

15.08.16

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

16.08.16

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

17.08.16

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

18.08.16

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

Sample	Sequencing Primer
pIG16_046#7	oIG16_039
pIG16_046#7	oIG16_040
pIG16_049#6	oIG16_039
pIG16_049#6	oIG16_040
pIG16_052#10	oIG16_039
pIG16_052#10	oIG16_040
pIG16_058#2	oIG16_039
pIG16_058#2	oIG16_040
pIG16_059#5	oIG16_039
pIG16_059#5	oIG16_040
pIG16_062#3	oIG16_028
pIG16_062#3	oIG16_029
pIG16_063#2	oIG16_028
pIG16_063#2	oIG16_029
pIG16_064#3	oIG16_028
pIG16_064#3	oIG16_029
pIG16_065#5	oIG16_028
pIG16_065#5	oIG16_029

19.08.16

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.

20.08.16

1) Extension PCR

Template	Primer	Resulting construct
pJet1.2_CotG	oIG056+012	pSB1C3-OH_CotG_G4S_HA
pJet1.2_CotG	oIG16_056+013	pSB1C3-OH_CotG_aHelix_HA

Reaction conditions

component	concentration	volume[µL]
Q5 MasterMix	2X	25
Primer1	10 µM	2.5
Primer2	10 µM	2.5
ultrapure water	-	20.0
template DNA		0.1

Thermal Cycling
Touchdown 62

2) Gibson Assembly

Number	Fragment 1	Fragment 2	Linearized backbone	Resulting construct
1	CotG oIG16_056+013	aGFPnano oIG16_051+042	pSB1C3 XbaI+SpeI	pIG16_043
2	CotG oIG16_056+012	aGFPnano oIG16_053+042	pSB1C3 XbaI+SpeI	pIG16_065
3	CotG oIG16_056+013	GST oIG16_041+055	pSB1C3 XbaI+SpeI	pIG16_066
4	CotG oIG16_014+016	aGFPnano oIG16_030+047	pSB1C3 XbaI+SpeI	pIG16_053

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

component	concentration	volume[µL]
PcotYZ	6000ng	20µl
CS Buffer	10x	5µl
EcoRI-HF	20u/µl	1.2µl
SpeI-HF	20u/µl	1.2µl
ultrapure water	-	50.0

Digestion of pBS1C and pBS4S

component	concentration	volume[µL]
pBS1C and pBS4S	5000ng	2,5µl
2.1 Buffer	10x	5µl
EcoRI-HF	20u/µl	1.2µl
PstI	20u/µl	1.2µl
ultrapure water	-	50.0

Digestion of pIG16_38 47 48 50 51 54

component	concentration	volume[µL]
pIG16_#	3000ng	20µl
3.1 Buffer	10x	5µl
XbaI	20u/µl	0.6µl
PstI	20u/µl	0.6µl
ultrapure water	-	50.0

21.08.16

1) Inoculation
5 colonies per plate were inoculated in 5 mL of LB medium supplemented with chloramphenicol from the Gibson assembly from the previous day. Incubation o/n at 37 °C and 250 rpm.

Reinoculation of pIG16_046#7, 049#6, 052#10, 058#2, 059#5, 062#3

2) Glycerol stocks

Preparation of 15 % glycerol stocks for:
pIG16_045#3, 062#2, 063#3, 064#5

22.08.16

1) Plasmid preparation
The plasmids of the following strains were extracted using the QiaQuick MiniPrep kit:
pIG16_043 053 065 066
500 ng of DNA was digested with 5 units of XbaI and PstI and analyzed by gel electrophoresis.

Gel pic

2) Glycerol stocks Glycerol stocks of pIG16_045 062 063 064 46 were prepared and stored at -80 °C.

3) Sequencing pIG16_058#3 and pIG16_058#4 were sent to sequencing.

4) Inoculation new colonies picked of pIG16_049 #11 - 16 and pIG16_052 #11-16 were picked

5) Transformation
The Biobrick BBa_J18932 from the Registry distribution kit was dissolved in 10 µL of ultrapure water. 2 µL were used for transformation of chemically competent E.coli DH5alpha and spread on a LB agar plate supplemented with chloramphenicol.

23.08.16

1) Plasmid preparation
Miniprep of pIG16_049#11-16 and pIG16_052#11-16. Testdigestion: 500 ng of DNA was treated with 5 unit of XbaI and PstI and analyzed by gel electrophoresis.

Gel image

2) Preparation of Plasmids for transformation of B.subtilis
MiniPrep of pIG16_045 + 062\ Linearization of 2µg of DNA with a 10 unit of ScaI. The DNA was purified with the QiaGen PCR purification kit and used for transformation of competent B. subtilis.

24.08.16

1) Sequencing
The following samples were sent to sequencing:
pIG16-044, 059#4, 058#3, 077, 082

2) Plasmid preparation
The DNA of pIG16_043#6-11 was extracted using the QiaQuick Miniprep kit.

3) Test digestion
500ng of pIG16_043, 049, 052, pIG16_120(BBa_J18932) were treated with 5 unit of XbaI+PstI and analyzed by gel electrophoresis.

Gel image

4) Sequencing
The following plasmids were sent to sequencing:
pSB1C3_mCherry#2 (BBa_J18932), pIG16_044#1, 049#12, 058#3, 059#4, 077#1, 082#1, 101#1, 104#1, 105#1, 117#5

25.08.16

1) Inoculations
Inoculations of the following samples for subsequent plasmid preparations and glycerol stocks: pIG16_045, 062, 063, 064, 046#7, 049#12, 059#4, 056#1, 058#3, 101#1, 044#1, 082#2, 104#1, 077#1, 081#1,105#2, 100#1, 117#4

26.08.16

1) Plasmid preparation
The plasmids from the previous inoculation were extracted using the Qiagen Plasmid MiniPrep kit. The DNA was eluted in 30 µL of ultra pure water and the DNA concentration was determined by NanoDrop.

2) Sequencing
The following plasmids were sent to sequencing: pIG16_081#1, 100#1, 117#4

3) Preparation of Plasmids for Transformation of B.subtilis
2 µg of the following plasmids were linearized by treatment with ScaI-HF in 1X cutsmart buffer at a total volume of 50 µL.
pIG16_045, 062, 063, 064, 117, 104, 044, 077, 081, 082, 100, 101, 105.
The linearized plasmids were loaded on a 1% TAE agarose gel and extracted after electrophoresis and used for transformation of competent B. subtilis.

27.08.16

1) Digestion for 3A assembly
Digestion 1 µg of DNA of pIG16_046, 049, 053, 059, 065, 066, 053 with 10 units of XbaI and PstI in 1XCutSmart buffer in a reaction volume of 50 µL. The digested samples were loaded on an 1 % agarose TAE gel and subjected to electrophoresis.

Gel image

After separation the appropriate fragments were extracted from the gel using the QiaGen gel extraction kit.

2) Extension PCR

No.	Template	Oligos oIG16_	Resulting construct
1	pIG16_22(GST)	37+55	aHelix_HA_GST_pSB1C3-OH
2	pIG16_22(GST)	36+38	pSB1C3-OH_GST_HA_aHelix
3	pIG16_030(CotB)	23+24	HA_aHelix_CotB_pSB1C3-OH
4	pIG16_030(CotB)	19+21	pSB1C3-OH_CotB_aHelix_HA

Reaction conditions

component	volume[µL]
2X Q5 MasterMix	25
Primer1 10 M	2.5
Primer2 10 M	2.5
template	<0.1
dH2O	20

The samples were loaded on an 1 % Agarose TAE gel and subjected to electrophoresis.

Gel image

After separation the appropriate fragments were extracted using the QiaGen gel extraction kit.

3) Gibson Master Mix
Preparation of 1.33x Gibson Master Mix.

4) Inoculation

4 colonies of each plate were picked from the previous transformation: pIG16_079, 085, 088, 096, 108, 112, 121, 122, 123

28.08.16

29.08.16

1)Sequencing The following plasmids were sent to sequencing: pIG16_032 033 034 035. (Integration vectors sent from Dr. Hinc, University of Gdansk)

2) 3A assembly by Niklas

NIKLAS update: 3A assembly

30.08.16

1) Sequencing
The following plasmids were sent to sequencing: pIG16_112#3, 121#3, 122#2, 079#1, 085#3, 088#4, 096#1

2) Inoculation
E.coli DH5alpha containing the following plasmids were inoculated in 5 mL LB medium supplemented with the appropriate antibiotics. Incubation o/n at 37 °C and 200 rpm. pIG16_100, 117, 123, 081, 104, 077, 082, 122, 121, 101, 045, 062, 063, 064, 032, 033, 034, 035

3) Gibson assembly

The Fragments for Gibson assembly were adjusted to a concentration of 50 ng/µL.

Number	Fragment1 amplified with oIG16_	Fragment2 amplified with oIG16_	Linearized backbone	resulting construct
1	CotZ oIG16_007 + 008	GST 036 + 038	pSB1C3	pIG16_067
2	CotZ 003 + 005	GST 037 + 055	pSB1C3	pIG16_068
3	CotG 056 + 013	aGFPnano 051 + 042	pSB1C3	pIG16_043
4	CotG 015 + 015	GST 036 + 038	pSB1C3	pIG16_069
5	CotG 056 + 013	GST 037 + 055	pSB1C3	pIG16_070
6	CgeA 044 + 045	GST 036 + 038	pSB1C3	pIG16_075
7	CgeA 050 + 035	GST 037 + 055	pSB1C3	pIG16_076
8	CotB 019 + 020	GST 037 + 055	pSB1C3	pIG16_052
9	CotB 022 + 024	aGFPnano 030 + 047	pSB1C3	pIG16_055
10	CotB 023 024	GST 036 + 038	pSB1C3	pIG16_073
11	CotB 019 + 021	GST 037 + 055	pSB1C3	pIG16_074
12	positive control	positive control	positive control	-

4) Linearization
Linearization of integration vectors for transformation of B.subtilis: 2 µg of the DNA was treated with 20 units of an appropriate enzyme in 1X cutsmart buffer at a total reaction volume of 50 µL for 2 hours at 37 °C.

Vector pIG16_	Containing coat gene	Insert locus	Enzyme for linearization
077	CotZ	AmyE	XhoI
081	CotZ	AmyE	XhoI
082	CotZ	AmyE	XhoI
100	CgeA	AmyE	XhoI
045	CgeA	AmyE	XhoI
062	CgeA	AmyE	XhoI
063	CgeA	AmyE	XhoI
064	CotG	AmyE	XhoI
108	CotG	thrC	NcoI
123	mCherry	thrC	ScaI
104	CotZ	thrC	NcoI
105	CotZ	thrC	NcoI
117	CgeA	thrC	NcoI
101	CgeA	AmyE	XhoI

Gel image

After linearization the DNA was purified using the QiaGen PCR purification kit. The DNA was used for transformation of B. subtilis.

31.08.16

1) Glycerolstocks
15 % Glycerol stocks of pIG16_032 033 034 035 were prepared and stored at -80 °C.

2) Sequencing
The following samples were sent to sequencing by GATC labtech pIG16_044#4, 078#4 080#3 086#2 089#4 109#4

3) Plasmid preparation
The following plasmids were prepared using the QiaQuick Miniprep kit: pIG16_032 33 34 35 45 62 63 64 123 117 81 82 100 117 123 121 122 101

4) Gibson assembly
The fragments were adjusted to a concentration of 50 ng/µL

No	amplified Fragment 1	amplified Fragment 2	Backbone	Resulting plasmids pIG16_
1	CotG oIG16_056+013	aGFPnano 51 + 42	pSB1C3	043
2	CotZ oIG16_7+8	GST_37+55	pSB1C3	067
3	CotG 056+013	aGFPnano 051 + 042	pSB1C3	052
4	CotG 015 + 016	GST 036 + 038	pSB1C3	055
5	cotG 056 + 013	GST 037 + 055	pSB1C3	070
6	cgeA 044 + 045	GST 036 + 038	pSB1C3	068
7	cgeA 050 + 035	GST 037 + 055	pSB1C3	069
8	cotB 019 + 020	GST 037 + 055	pSB1C3	073
9	cotB 002 + 024	aGFPnano 030 + 047	pSB1C3	074
10	cotB 023 + 024	GST 036 + 038	pSB1C3	075
11	cotB 019 + 021	GST 037 + 055	pSB1C3	076

0.5 µL of each fragment were mixed alongside with 0.5 µL of the backbone. The volume was adjusted to 2.5 µL and added to 7.5 µL of 1.33X Gibson Master Mix. The Reaction was incubated for 1h at 50 min and transformed into competent E. coli DH5alpha. The transformed cells were plated on LB agar plates supplemented with chloramphenicol.

01.09.16

1) Inoculation
Inoculation of the colonies from the transformation of the Gibson assembly: pIG16_043 067 052 055 070 068 069 073 074 075 076. 5 colonies were picked and inoculated in 5 mL of LB medium with chloramphenicol. Incubation o/n at 37°C and 200 rpm.

Inoculation of pIG16_043 070 074 067 068 075 073 055 076 052 069 in 5 mL LB medium supplemented with ampicilin.

02.09.16

1) Plasmid Preparation
The following samples were prepared from E.coli DH5alpha by a MiniPrep:
I) From Gibson assembly (Previous day): pIG16_043 067 052 055 070 068 069 073 074 075 076

II) For Transformation of B.subtilis: pIG16_077 81 82 100 45 62 63 64 108 123 104 105 117 101 122

2) Linearization of plasmids for transformation of B.subtilis
Linearization of integration vectors for transformation of B.subtilis: 2 µg of the DNA was treated with 20 units of an appropriate enzyme in 1X cutsmart buffer at a total reaction volume of 50 µL for 2 hours at 37 °C.

Vector pIG16_	Containing coat gene	Insert locus	Enzyme for linearization
077	CotZ	AmyE	XhoI
081	CotZ	AmyE	XhoI
082	CotZ	AmyE	XhoI
100	CgeA	AmyE	XhoI
045	CgeA	AmyE	XhoI
062	CgeA	AmyE	XhoI
063	CgeA	AmyE	XhoI
064	CotG	AmyE	XhoI
108	CotG	thrC	NcoI
123	mCherry	thrC	ScaI
104	CotZ	thrC	NcoI
105	CotZ	thrC	NcoI
117	CgeA	thrC	NcoI
101	CgeA	AmyE	XhoI
122	mCherry	LacA	NgoMIV

Gel image

After linearization the DNA was purified using the QiaGen PCR purification kit. The DNA was used for transformation of B. subtilis.

03.09.16

1) Test digestion
For the verification of the proper sizes 500 ng the following plasmids were treated with 5 U of XbaI and PstI and analyzed by gel electrophoresis on a 1 % Agarose TAE gel:
pIG16_043 067 052 055 070 068 069 073 074 075 076
The expected bands were not visible.

Gel image

04.09.16

1) Annealed oligo cloning
In order to insert an alpha helical linker into the Sporovector from the Munich iGEM team 2012 the oligos oIG16_079 and oIG16_080 were annealed at a molar ratio of 1:1, heated at 100 °C for 30 min and slowly cooled down. The annealed oligos contained NgoMIV corresponding sticky ends and were stored at 4 °C. 2 µg of the vector pIG16_017 (Sporovector) was linearized with NgoMIV and purified. The linearized backbone and the annealed oligos were ligated at molar ratios of 1:1, 1:2 and 1:3 using 1 µL of T4 ligase (from NEB) in 1X T4 buffer in a total reaction volume of 20 µL. The reaction was incubated for 30 min at RT and transformed into chemically competent E.coli. The cells were plated on LB agar plates supplemented with ampicilin.

05.09.16

1)Inoculation
Inoculation of E.coli containing integration vectors from glycerol stocks: pIG16_077 81 82 100 45 62 63 64 108 123 104 105 117 101 122. Incubation in 5 mL LB medium supplemented with ampicilin. Incubation o/n at 37 °C and 200 rpm.

Inoculation of colonies from the annealed oligo cloning. 5 colonies per plate were inoculated in 5 mL of LB medium with ampicilin. Incubation o/n at 37 °C and 200 rpm.

06.09.16

1) Plasmid preparation
MiniPrep of integration vectors: pIG16_077 81 82 100 45 62 63 64 108 123 104 105 117 101 122

pIG16_124 (at molar ratios 1:1, 1:2, 1:3,each #1-5).

Gel image

2) Sequencing
pIG16_124 1:1 #2
pIG16_124 1:2 #3
pIG16_124 1:3 #1

07.09.16

1) Plasmid Preparation
MiniPrep of pIG16_043 067 052 055 070 068 069 073 074 075 076

2) Testdigestion 500 ng of DNA was treated with 5 units of XbaI + PstI in 1X NEBuffer 2.1 in a total reaction volume of 20 µL. Incubation for 1 h at 37 °C. The reaction was analyzed by gel electrophoresis.

Gel image

08.09.16

1) Sequencing
Gibson assembly reactions were sent to sequencing:
pIG16_043 067 052 055 070 068 069 073 074 075 076

2) Digestion
I pIG16_017: 2 µg of plasmid DNA was treated with 10 unit of XbaI and NgoMIV in 1X Cutsmart buffer in a total reaction volume of 50 µL for 2 hours at 37 °C. For further assembly by Freiburg Standard RFC25.
II pIG16_020: 2 µg of plasmid DNA was treated with 10 unit of XbaI and SpeI-HF in 1X Cutsmart buffer in a total reaction volume of 50 µL for 2 hours at 37 °C for further gibson assembliy reactions.

3) PCR
The biobrick BBa_J18932 (pIG16_120) containing mCherry was amplified in include additional restriction site for Freiburg standard assembly RFC25.
Reaction conditions

Component	concentration	volume in µL
Q5 Master Mix	2X	37.5
DNA template	-	1 µL
oIG16_075	10 µM	3.75
oIG16_076	10 µM	3.75
dH2O	-	29

Amplified by Touchdown-PCR. The annealing temperature was determined by the NEB online tool Tm-calculator.

09.09.16

1) Gel extraction
The digestions and PCR reaction from the previous day were extracted from an agarose gel accorind the the protocol of the manufacturer.

Gel image 17 20 mCherry

2) Digestion
1 µg of the amplified mCherry (pIG16_120 + oIG16_075+076) was treated with AgeI and XbaI in 1X cutsmart buffer in a total reaction volume of 50 µL for 90 min at 37 °C. The DNA was directly purified using the Qiagen PCR purification kit according to the instructions of the manufacturer.

3) T4 Ligation
Ligation of digested mCherry (Insert) into the digested pIG16_017 (Sporovector) at a molar ratio of 1:3 (Vector : Insert) in 1X T4-ligation buffer at a total reaction volume of 20 µL. The ligation was incubated for 30 min at RT and transformed into chemically competent E.coli.

4) Gibson Assembly

No	amplified Fragment 1	amplified Fragment 2	Backbone	Resulting plasmids pIG16_
1	CotZ oIG16_7+8	GST_37+55	pSB1C3	067
2	cgeA 044 + 045	GST 036 + 038	pSB1C3	068
3	cotB 019 + 020	GST 037 + 055	pSB1C3	073
4	cotB 002 + 024	aGFPnano 030 + 047	pSB1C3	074
5	cotB 023 + 024	GST 036 + 038	pSB1C3	075
6	cotB 019 + 021	GST 037 + 055	pSB1C3	076
7	pos. control	positive control	positive control	-

The reaction was incubated for 1 h at 50 °C and tranformed into chemically competent E.coli DH5 alpha.

10.09.16

1) Inoculation
From each plate with E.coli transformed with the gibson assemblies 5 colonies were picked and inoculated in 5 mL of LB medium supplemented with chloramphenicol.

11.09.16

1) Linearization
3.5 µg of the following plasmids were linearized with the listed restriction enzyme in the appropriate buffer for transformation into B. subtilis:

Plasmid pIG16_	coat gene	Insert Locus/Selection marker	Enzyme
044	cgeA	1C	XhoI
045	cgeA	1C	XhoI
062	cgeA	1C	XhoI
063	cgeA	1C	XhoI
064	cotG	1C	XhoI
077	cotZ	1C	XhoI
078	cotZ	1C	XhoI
079	cotZ	1C	XhoI
080	cotZ	1C	XhoI
081	cotZ	1C	XhoI
082	cotZ	1C	XhoI
085	cotG	1C	XhoI
086	cotG	1C	XhoI
088	cotG	1C	XhoI
089	cotG	1C	XhoI
096	cotB	1C	XhoI
100	cgeA	1C	XhoI
101	cgeA	1C	XhoI
104	cotZ	4S	NcoI
105	cotZ	4S	NcoI
108	cotG	4S	NcoI
109	cotG	4S	NcoI
117	cgeA	4S	NcoI
122	mCherry	2E	NgoMIV
123	mCherry	4S	ScaI

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