Team:Freiburg/NotebookCloning

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Our Protocols
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.

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.

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.

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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

4) 3 A Assembly

The 3 A Assembly was carried out according to the neb protocol.

3 A Assembly with pBS1C

component concentration volume[µL]
pBS1C 102ng/µl 50ng
T4 ligase Buffer 10x 2µl
T4 ligase 20u/µl 1µl
PcotYZ 20ng/µl 5ng
pIG16_38/47/48/50/51/54 differentng/µl 25ng/µl
ultrapure water - 20.0

3 A Assembly with pBS4S

component concentration volume[µL]
pBS4S 102ng/µl 50ng
T4 ligase Buffer 10x 2µl
T4 ligase 20u/µl 1µl
PcotYZ 20ng/µl 5ng
pIG16_38/47/48/50/51/54 differentng/µl 25ng/µl
ultrapure water - 20.0

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

3) Linearization #1

3 µg of the following plasmids were linearized using the appropriate enzyme in a reaction volume of 50 µL. The digestion was loaded on a TAE agarose gel and subjected to electrophoresis.

The linearized plasmids were extracted from the gel using the Qiagen gel extraction kit and subsequently transformed into competent Bacillus subtilis.

4) Linearization #2

Digestion of pIG16_77/81/82/100/101

component concentration volume[µL]
pIG16_77/81/82/100/101 2000ng 4,5µl
Cutsmart 10x 5µl
XhoI 20u/µl 0.5µl
ultra pure water - ad50.0

Digestion of pIG16_104/105/117

component concentration volume[µL]
pIG16_104/105/117 2000ng 4,5µl
CS Buffer 10x 5µl
EcoRV-HF 20u/µl 0.5µl
ultrapure water - ad50.0

→Linearization was followed by gel extraction for transformation in Bacillus subtilis.

4) Inoculation of 3 A Assemblies colonies.

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.

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.

6) Testdigestion of 3 A assembly from 20.9

Digestion 3 A assembly

component concentration volume[µL]
pIG16_ 500ng 4µl
2.1 Buffer 10x 5µl
EcoRI-HF 20u/µl 0.1µl
PstI 20u/µl 0.1µl
ultrapure water - ad20.0

→Colonies were sent to sequencing.

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.

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, pIG16_120(BBa_J18932) were treated with 5 unit of XbaI+PstI and analyzed by gel electrophoresis.

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.

Digestion 3 A assembly

component concentration volume[µL]
pIG16_60 6000ng 10µl
2.1 Buffer 10x 2µl
EcoRI-HF 20u/µl 1.2µl
SpeI 20u/µl 1.2µl
ultrapure water - ad20.0

Digestion 3 A assembly

component concentration volume[µL]
pBS1C/4S/E2 2000ng 4µl
CS Buffer 10x 2µl
EcoRI-HF 20u/µl 0.4µl
PstI 20u/µl 0.4µl
ultrapure water - ad20.0

Digestion 3 A assembly

component concentration volume[µL]
pIG16_46/47/49/56/65/mCherry 1000ng 4µl
3.1 Buffer 10x 2µl
XbaI 20u/µl 0.5µl
PstI 20u/µl 0.5µl
ultrapure water - ad20.0

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.

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

5) 3 A assembly

3 A Assembly with pBS1C

component concentration volume[µL]
pBS1C 102ng/µl 50ng
T4 ligase Buffer 10x 2µl
T4 ligase 20u/µl 1µl
PcotYZ 20ng/µl 9.5ng
pIG16_46/49/53/56/65/mCherry different ng/µl 41ng/µl

3 A Assembly with pBS2E

component concentration volume[µL]
pBS2E 80ng/µl 50ng
T4 ligase Buffer 10x 2µl
T4 ligase 20u/µl 1µl
PcotYZ 20ng/µl 9.5ng
pIG16_mCherry different ng/µl 28ng/µl

3 A Assembly with pBS4S

component concentration volume[µL]
pBS4S 75ng/µl 50ng
T4 ligase Buffer 10x 2µl
T4 ligase 20u/µl 1µl
PcotYZ 20ng/µl 12.5ng
pIG16_46/49/mCherry different ng/µl 55ng/µl

28.08.16

1) Inoculation

Colonies from the 3 A assembly (27.08) were picked.

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

3 A Assembly with pBS1C

component concentration volume[µL]
pBS1C 102ng/µl 50ng
T4 ligase Buffer 10x 2µl
T4 ligase 20u/µl 1µl
PcotYZ 20ng/µl 9.5ng
pIG16_58/59/66 different ng/µl 55ng/µl

3 A Assembly with pBS4S

component concentration volume[µL]
pBS4S 75ng/µl 50ng
T4 ligase Buffer 10x 2µl
T4 ligase 20u/µl 1µl
PcotYZ 20ng/µl 12.5ng
pIG16_66 different ng/µl 55ng/µl

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

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

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.

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: NIKLAS???

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 further colonies 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.

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.

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
I 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.
II Further colonies of pIG16_043, 052, 126 were picked and inoculated.

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

After linearization 5 µL of the reaction was analyzed by agarose gel electrophoresis. The remaining volume was purified using the QiaGen PCR purification kit. The eluted DNA was used for transformation of B. subtilis.

2) MiniPrep
The plasmids from the following inoculations were prepared using the ZymoResearch Zyppy Prep kit:
pIG16_043 052 067 068 070 073 074 075 076 126 (colonies #5-9 each).

3) Testdigestion
500 ng of the prepared plasmids were treated with 5 units of XbaI and PstI in 1X NEBuffer 2.1 in a total reaction volume of 20 µL for 2 hours at 37 °C. The Digestion was analyzed by agarose gel electrophoresis.

12.09.16

13.09.16

1) PCR
Amplification of the Sporovector pSBBS4S-Sporo (pIG16_017) in order to introduce an alpha helical linker to the MCS by Gibson assembly.

template Oligos oIG16_
pIG16_017 079+082
pIG16_017 081+080

Reaction conditions

component conc. volume
Q5 MasterMix 2x 25µL
Primer fw 10 µM 2.5
Primer rv 10 µM 2.5
template - 0.1
dH20 - 20µL

Cycling
Touchdown PCR 60 and 67

2) Gibson Assembly
The two amplified fragments were mixed at a ratio of 1:1 (50 ng each) and incuabted in NEB HiFi assembly mix at 50 °C for 1 hour. Transformation of 2µL into chemically competent E.coli and spreaded on LB agar plates supplemented with ampicilin.

14.09.16

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

After linearization 5 µL of the reaction was analyzed by agarose gel electrophoresis. The remaining volume was purified using the QiaGen PCR purification kit. The eluted DNA was used for transformation of B. subtilis.

update: Gel image

15.09.16

1) PCR
Amplification of anti-GFP nanobody and mCherry to introduce additional overhangs containing restriction site.

template Oligos Restriction site
1 pIG16_023 oIG16_083 + 084 EcoRI + XbaI
2 pIG16_023 oIG16_085 + 086 NcoI + NheI
3 pIG16_020 (mCherry) oIG16_087 + 088 XhoI + XbaI

Reaction conditions

component conc volume [µL]
Q5 MasterMix 2x 25
Primer 1 10 µM 2.5
Primer 2 10 µM 2.5
template - 0.1
dH2O - 20

Cycling
pIG16_023: Touchdown68
pIG16_020: Touchdown66

After amplification the reaction was analyzed by agarose gel electrophoresis.

2) Transformation
BBa_K180000 (Lac regulated taq promoter) from the iGEM distribution kit was dissolved in 10 µL of ultra pure water. 2 µL were used for the transformation of chemically competent E.coli DH5alpha and spread on an LB agar plate supplemented with chloramphenicol.

3) Oligo design
The PCotYZ-Promoter (BBa_K823030) did not contain a ribosome binding site. An additional RBS is introduced by primers containing an additional insert.

16.09.16

1) Gel extraction
The amplified samples from the previous day were extracted from the gel and purified using the QiaGen gel extraction kit.

2) Digestion
1µg of the amplified nanobody/mCherry were digested with the 10 unit of the appropriate enzymes in 1X Reaction buffer in a total reaction volume of 30 µL in order to clone them into their respective backbone.

DNA oligos Restriction enzymes
aGFPnano 83 + 84 EcoRI + XbaI
aGFPnano 85 + 86 NcoI-HF + NheI-HF
mCherry 87 + XhoI + XbaI
pIG16_033 - NcoI-HF + NheI-HF
pIG16_034 - XhoI + XbaI-HF
pIG16_035 - EcoRI-HF + XbaI-HF

The amplified inserts were directly purified using the Qiagen PCR purification kit. The digested backbones (pIG16_033, 034, 035) were loaded on an agarose gel. After electrophoresis the bands corresponding to the expected sizes were extracted.

3) T4-Ligation
The digested inserts and backbones were ligated at a molar ration of 1:3 (Backbone:Insert) using T4 ligase in 1X T4 ligation buffer in a total reaction volume of 20 µL. The reaction was incubated at RT for 20 min.

No. Insert Backbone Resulting construct
1 EcoRI-aGFPnano-XbaI EcoRI-pIG16_035-XbaI pIG16_128
2 NcoI-aGFPnano-NheI NcoI-pIG16_033-NheI pIG16_127
3 XhoI-mCherry-XbaI XhoI-pIG16_034-XbaI pIG16_129

5 µL of the reaction mix was transformed into chemically competent E.coli DH5alpha and spread on LB agar plates supplemented with ampicilin. Incubation over night at 37 °C.

4) Inoculation
5 colonies of pIG16_130 were inoculated in 5 mL of LB medium supplemented with chloramphenicol.

17.09.16

1) MiniPrep
The plasmids from the transformed E.coli containing pIG16_130#1-5 were prepared using the QiaGen MiniPrep kit. The DNA was eluted in 30 µL of ultra pure water.

2) Testdigestion
500 ng of pIG16_130 #1-5 was treated with 4 units of EcoRI-HF and SpeI-HF in 1X cutsmart buffer in a total reaction volume of 20 µL for 2 hours at 37 °C.

Gel image: NIKLAS???

3) Digestion
5 µg of pIG16_130 #2 was treated with 20 units of EcoRI-HF and SpeI-HF in 1X cutsmart buffer in a total reaction volume of 50 µL for 2 hours at 37 °C in order to release the biobrick part (Lac inducible promoter). The digestion was loaded on an agarose gel and subjected to electrophoresis. The band corresponding to the size of the promoter was extracted using the QiaGen gel extraction kit.

18.09.16

1) Restriction digestion
1.5 µg of pIG16_033 and 035 were treated with 5 unit of NcoI-HF+NheI-HF and EcoRI-HF+XbaI, respectively. The reaction was performed in 1X cutsmart buffer in a total reaction volume of 50 µL for 2 hours at 37 °C. After digestion the linearized plasmids were purified using the QiaGen PCR purification kit.

2) T4-Ligation

The digested inserts and backbones were ligated at a molar ration of 1:3 (Backbone:Insert) using T4 ligase in 1X T4 ligation buffer in a total reaction volume of 20 µL. The reaction was incubated at RT for 20 min.

Backbone Insert Resulting construct
NcoI-pIG16_033-NheI NcoI-aGFPnano-NheI pIG16_127
EcoRI-pIG16_035-XbaI EcoRI-aGFPnano-XbaI pIG16_128

2 µL of the ligation mix was used for transformation of chemically competent E.coli DH5alpha.

19.09.16

1) Inoculation
5 colonies of each plate containing the ligations from the previous day were picked and inoculated in 5 mL of LB medium supplemented with ampicilin. Incubation o/n at 37 °C and 200 rpm.

20.09.16

1) MiniPrep
Plasmid preparation of colonies pIG16_127#1-5 and pIG16_128#1-5 using the QiaGen MiniPrep kit. The DNA was eluted in 30 µL of ultra pure water.

2) Testdigestion
I 500 ng of pIG16_127 was treated with 4 units of NcoI-HF and NheI-HF in 1X Cutsmart buffer for 90 min at 37 °C. II 500 ng of pIG16_128 was treated with 4 units of EcoRI-HF and XbaI-HF. The digested DNA was analyzed by agarose gel electrophoresis.

3) PCR
Amplification of additional promoters for the expression of fusion consructs.

No. Template Oligos comment Annealing temperature [°C]
1 pSB1C3 (pIG16_020) oIG16_093+094 pTrpC-promoter from RFP-cassette 62
2 pIG16_017 oIG16_091+092 PCotYZ-RBS 54
3 pIG16_022 oIG16_095+096 BamHI-GST-XbaI 64

Reaction conditions

component conc volume [µL]
Q5 MasterMix 2X 25
template - 0.1
Primer 1 10 µM 2.5
Primer 2 10 µM 2.5
dH2O - 20 µL

After amplification the samples were loaded on an agarose gel and extracted from the gel using the Qiagen gel extraction kit.

4) Digestions
I 5 µg of integration vector DNA was treated with 20 units of BamHI-HF and XbaI in 1X cutsmart buffer in a total reaction volume of 20 µL for 2 hours at 37 °C.

No. Vector Enzymes
1 pCgeA-C(pIG16_034) BamHI-HF + XbaI
2 pCotZ-C(pIG16_032) BamHI-HF + XbaI
3 GST(amplified from pIG16_022) BamHI-HF + XbaI

II For digestion of the amplified promoters 3 µg of extracted DNA was treated with the appropriate restriction enzymes in 1X reaction buffer in a total volume of 50 µL for 1 h at 37 °C.

Template Promoter Restriction enzymes
pIG16_017 PCotYZ-RBS EcoRI-HF + SpeI-HF
pIG16_020 PTrpC EcoRI-HF + SpeI-HF

The digested backbones were loaded on an agarose gel and the bands corresponding to the expected size were extracted using the QiaGen gel extraction kit.

5) 3A Assembly
The constructs for surface display were assembled using various promoters for expression in E.coli and B.subtilis. 3A assembly of a promoter and a fusion construct into an integration vector. All inserts were treated with the appropriate restriction enzymes from previous assemblies and stored at -20 °C. The ligation was performed at a molar ratio of 1:3 (vector:inserts) using 50 ng of vector DNA. The reaction was incubated at RT for 20 min and subsequently transformed into chemically competent E. coli DH5alpha and spread on LB-agar plates supplemented with the appropriate antibiotic.
I pLac promoter (inducible, BBa_K180000)

No. Fusion construct Result
1 pIG16_047 pIG16_134
2 pIG16_048 pIG16_135
3 pIG16_049 pIG16_136
4 pIG16_066 pIG16_138

IIpTrpC promoter (consitutive promoter, amplified from RFP-cassette)

No. Fusion construct Result
1 pIG16_042 pIG16_143
2 pIG16_047 pIG16_144
3 pIG16_048 pIG16_145
4 pIG16_049 pIG16_146
5 pIG16_059 pIG16_147
6 pIG16_066 pIG16_148
7 pIG16_039 pIG16_149

IIIpCotYZ-RBS (B. subtilis CotYZ-promoter with ribosome binding site)

No. Fusion construct Result
1 pIG16_039 pIG16_150
2 pIG16_042 pIG16_151
3 pIG16_047 pIG16_152
4 pIG16_048 pIG16_153
5 pIG16_049 pIG16_154
6 pIG16_059 pIG16_155
7 pIG16_066 pIG16_156
8 pIG16_056 pIG16_157
9 pIG16_058 pIG16_158
10 pIG16_054 pIG16_159
11 pIG16_051 pIG16_160

IV Further ligations (Ligation of GST into surface display vectors)

No. Insert Backbone Result
1 BamHI-pIG16_032-XbaI BamHI-GST-XbaI pIG16_131
2 BamHI-pIG16_034-XbaI BamHI-GST-XbaI pIG16_142

21.09.16

1) Transformation
The transformations of the T4-ligations of the previous day were repeated.

2) Inoculation
Inoculation of the following colonies transformed on the previous day:
I PTrpC: pIG16_143#1 148#1 149#1-2
II PLac: pIG16_134#1-2 135#5 138#1-5
III PCotYZ: pIG16_150#1 159#1-4 157#1-4 155#1
IV pIG16_131 #1-2
Inoculation in 5 mL of LB medium supplemented with the appropriate antibiotic.

22.09.16

1) MiniPrep
Plasmid preparation of the inoculated colonies from the previous day using the zymo research zyppy kit. I PTrpC: pIG16_143#1 148#1 149#1-2
II PLac: pIG16_134#1-2 135#5 138#1-5
III PCotYZ-RBS: pIG16_150#1 159#1-4 157#1-4 155#1
IV pIG16_131 #1-2

2) Testdigestion
500 ng of the prepared plasmids were treated with 4 units of XbaI and PstI in 1X NEBuffer 2.1 for 1 hour at 37 °C. The digested DNA was analyzed by agarose gel electrophoresis.

3) Sequencing
the Following plasmids were sent so sequencing: pIG16_131#1 138#3 149#2 143#1 150#1 159#2+3 155#11 158#4+5

4) Inoculation
Inoculation of further constructs with the new promoters:
I PTrpC: pIG16_147#1-2 148#2 149#2-3 142#2 145#1-3
II PLac: pIG16_134#1-5 136#1-3
III PCotYZ-RBS: pIG16_160#1 157#1-5 155#2 150#2-4 159#1 151#1-2
IV pIG16_131 #3-5 142#1

23.09.16

1) MiniPrep
The the plasmids of inoculated colonies from the previous day were prepared using the Zyppy Miniprep kit:

2) Testdigestion
500 ng of prepared DNA was treated with 4 units of XbaI and PstI for 1 hour at 37 °C. The digestion was analyzed by agarose gel electrophoresis.

3) Sequencing
Positive colonies were sent to sequencing.

4) Inoculation
Inoculation of colonies for preparation of glycerol stocks:
I PLac: 132#1 137#1 138#3 133#2
II PTrpC: 143#1 149#2
III PCotYZ-RBS: 150#1 158#5 159#3 151#1 160#1
IV 129#1 142#1 131#1 126#1 127#3

24.09.16

1) MiniPrep & Glycerl stocks The plasmids from the inoculated colonies of the previous day were prepared using the Zyppy MiniPrep kit.
1 mL of the inoculated colonies was used for the preparation of 15 % glycerol stocks.

2) Linearization
5 µg of the following plasmids was linearized using 25 units of the appropriate enzyme in 1X reaction buffer for 2 hours at 37 °C:

No Plasmid pIG16_ Enzyme
1 150 XhoI
2 151 XhoI
3 158 XhoI
4 159 XhoI
5 160 XhoI
6 126 ScaI
7 128 ScaI
8 129 ScaI
9 141 PstI
10 131 PstI

After linearization the DNA was purified using the QiaGen PCR purification kit.

3) PCR
The anti-GFP nanobody was amplified in order to introduce BamHI and XbaI restrictions sites:

Component Conc. Vol [µL]
DNA template . 0.1
oIG16_97 10 µM 2.5
oIG16_98 10 µM 2.5
Q5 MasterMix 2X 25
dH2O . 20

Amplification using the Touchdown67 program. The PCR product was loaded on a 1% TAE agarose gel and subjected to electrophoresis. The band corresponding to the expected size was extracted and purified from the gel using the QiaGen Gelextraction kit.

4) Digestion
2 µg of the extracted PCR product was treated with 10 units of BamHI and XbaI for 1 hour at 37 °C. The DNA was purified using the QiaGen PCR purification kit.

5) T4 ligations

No. Insert Backbone Resulting plasmid
1 BamHI - aGFPnano - XbaI BamHI - pCgeA-C - XbaI pIG16_161
2 BamHI - aGFPnano - XbaI BamHI - pCotZ-C - XbaI pIG16_141

The DNA was ligated using T4 ligase at a molar ratio of 1:3 (Vector : Insert) in 1X T4 ligation buffer in a total volume of 20 µL for 30 min at RT. 5 µL of the ligation mix were transformed into chemically competent E. coli DH5alpha and plated on LB agar plates supplemented with ampicilin. Incubation o/n at 37 °C.

25.09.16

1) Glycerol stocks
Preparation of 15 % glycerol stocks with the following strains:
pIG16_134 143 149 150 151 157 159 160 138 137 134 133 132 126 127 129 131 141

26.09.16

1) MiniPrep
The Plasmids from the following strains were prepared using the Zyppy MiniPrep kit:
pIG16_142#1,2 136#1-3 131#1 141#1 126#1

2) Testdigestion
500 ng of the prepared DNA (pIG16_142 and 136) was treated with 5 units of the appropriate enzyme in 1X reaction buffer in a total volume of 20 µL for 1 h at 37 °C.

Plasmid Restriction enzymes
pIG16_142 XbaI + NcoI-HF
pIG16_136 XbaI + PstI

Analysis by agarose gel electrophoresis.

3) Linearization
3.5 µg of the following plasmids were linearized with 20 units of the appropriate enzyme in 1X reaction buffer in a total reaction volume of 50 µL for 1 hour at 37 °C.

Plasmid enzyme
131 PstI
141 PstI
126 ScaI
142 PstI
034 ScaI

5 µL of the digestion was analyzed by gel electrophoresis. The remaining sample was purified using the QiaGen PCR purification kit.

4) Inoculation Inoculation of further colonies for test digestion: pIG16_161#1-5

27.09.16

1) MiniPrep
The plasmid DNA of the inoculated colonies of pIG16_161#1-5 were prepared using the QiaGen MiniPrep kit.

2) Testdigestion
500 ng of plasmid DNA was treated with 4 units of SpeI-HF and XbaI in 1XCutsmart buffer in a total reaction volume of 20 µL for 1 hour at 37 °C. The samples were analyzed by gel electrophoresis. Colony #1 was sent to sequencing.

28.09.16

29.09.16

30.09.16

01.10.16

02.10.16

03.10.16

04.10.16

1.) Digestion for 3 A Assembly

05.10.16

06.10.16

1.) Testdigestion

Digestion mixture:

Component concentration volume [µL]
DNA 500ng/µL 4-6µl
PstI-HF 20u/µL 0.1
XbaI 20u/µL 0.1
NEBuffer CS 10X 2
ultra pure water - ad 20 µL

Posted by: iGEM Freiburg

Nanocillus - 'cause spore is more!