Template:Team:Groningen/CONTENT/EXPERIMENTS/Overproduce BslA in B. subtilis ComI

00:00, 16 April 2015 - 00:00, 16 April 2015

Primers.

The following primers were designed for bslA (the gene responsible for the production of BslA) in B. subtilis 168.

1

bslA for

GCGAATTCGCGGCCGCTTCTAGAGCATTTTTTAGGGGGAATTTTGTTATG

2

bslA overlap for

GATTTGTTAGGAGCTGGCGAATTTAAATTAAAACTG

3

bslA overlap rev

CAGTTTTAATTTAAATTCGCCAGCTCCTAACAAATC

4

bslA rev

CGTACTAGTAGCGGCCGCTGCAGTTATTAGTTGCAACCGCAAGGCTGAG

Primer design for bslA.

Atze van Stralen

00:00, 13 May 2015 - 00:00, 13 May 2015

PCR 1

Prepare dNTP Mastermix with final concentration of 2 mM of each dNTP.

100 mM dATP

20 µL

100 mM dTTP

20 µL

100 mM dCTP

20 µL

100 mM dGTP

20 µL

\( \mathrm{H_2 O} \)

920 µL

dNTP Mastermix.

Harm Ruesink

00:00, 13 May 2015 - 00:00, 13 May 2015

Per sample:

Mastermix PCR 1

30 µL

template DNA (B. subtilis 168 DNA)

0.3 µL

bslA for

0.3 µL

bslA overlap for

0.3 µL

bslA overlap rev

0.3 µL

bslA rev

0.3 µL

Mastermix PCR 1.

Samples

1

yuaB for + yuaB overlap rev

2

yuaB overlap for + yuaB rev

Samples.

PCR thermocycle

1

Initial denaturation

98 °C

2:00

2

Denaturation

98 °C

0:10

3

Annealing

60 °C

0:20

4

Extension

72 °C

0:30

5

Go back to 2 (repeat 30x)

6

Final extension

72 °C

5:00

Recommended thermocycle.

Gel results

Sample 1 formed no product. Sample 2 formed a product of ±200bp visible on gel. Sample 2 was purified using PCR and Stored in -20°C as "1.2".

Harm Ruesink

00:00, 15 May 2015 - 00:00, 15 May 2015

PCR 2

The following samples were prepared using B. subtilis 168 DNA.

5x buffer

40 µL

dNTP MM (2mM)

2 µL

Phusion polymerase

20 µL

\( \mathrm{\mathrm{H_2 O}} \)

136 µL

Mastermix PCR 2.

Per sample:

Mastermix PCR 2

30 µL

Per template DNA

2 µL

Mix per sample.

A sample marked "4" was prepared with the following primers: yuaB for + yuaB overlap rev. Template DNA was B. subtilis 168 DNA. The following thermocycle was performed.

1

Initial denaturation

98 °C

3:00

2

Denaturation

98 °C

0:30

3

Annealing

62 °C

0:30

4

Extention

72°C

1:00

5

Go back to step 2 (repeat 3x)

6

Denaturation

98 °C

0:30

7

Annealing

60 °C

0:30

8

Extention

72 °C

1:00

9

Go back to step 6 (repeat 3x)

10

Denaturation

98 °C

0:30

11

Annaeling

58°C

0:30

12

Extention

72 °C

1:00

13

Go back to step 10 (repeat 3x)

14

Denaturation

98 °C

0:30

15

Annaaling

56 °C

0:30

16

Extention

72 °C

1:00

17

Go back to step 14 (repeat 3x)

18

Denaturation

98 °C

0:30

19

Annealing

54 °C

0:30

20

Extention

72 °C

1:00

21

Go back to step 18 (repeat 3x)

22

Denaturation

98 °C

0:30

23

Annealing

54 °C

0:30

24

Extention

72 °C

1:00

25

Go back to step 22 (repeat 30x)

26

Final Extention

72 °C

10:00

Recommended thermocycle.

Harm Ruesink

00:00, 18 May 2015 - 00:00, 18 May 2015

PCR 3

5x buffer

28 µL

dNTP MM (2mM)

14 µL

Phusion polymerase

1.4 µL

\( \mathrm{H_2 O} \)

95.2 µL

Mastermix PCR 3.

Per sample:

Mastermix PCR 3

30 µL

Per template DNA

0.3 µL

Per primer

0.3 µL

Mix per sample.

The following samples were prepared: 1. primers: yuaB for + yuaB rev, template DNA: B. subtilis 168 DNA 2. primers: yuaB for + yuaB overlap rev, template DNA: B. subtilis 168 DNA

1

yuaB for + yuaB rev

B. subtilis 168 DNA

2

yuaB for + yuaB overlap rev

B. subtilis 168 DNA

1

Initial denaturation

98 °C

3:00

2

Denaturation

98 °C

0:30

3

Annealing

56 °C

0:30

4

Extention

72 °C

1:00

5

Go back to step 6(repeat 15x)

6

Denaturation

98 °C

0:30

7

Annealing

70 °C

0:30

8

Extention

72 °C

1:00

9

Go back to step 6 (repeat 15x)

10

Final Extention

72 °C

10:00

Recommended thermocycle.

Harm Ruesink

00:00, 19 May 2015 - 00:00, 19 May 2015

Ran a gel with PCR 3 products on 1% agarose gel with EtBr. Both sample 1 and sample 2 show no bands visible. Probably because yuaB forward primer doesn’t work.

Harm Ruesink

00:00, 21 May 2015 - 00:00, 21 May 2015

Ordered new primers.

yuaB forward 2 (with RBS)

GCGAATTCGCGGCCGCTTCTAGAGTTAGGGGGAATTTTGTTATGAAACGC

yuaB forward 3 (no RBS)

GCGAATTCGCGGCCGCTTCTAGAGATGAAACGCAAATTATTATCTTCTTTGG

New primers.

Harm Ruesink

00:00, 8 June 2015 - 00:00, 8 June 2015

PCR 5

PCR'd part 1 of yuaB using primers yuaB forward 2 & yuaB forward 3 and genomic DNA from B. subtilis 168.

10x buffer

10 µL

dNTP MM (2mM)

10 µL

Dreamtaq polymerase

1 µL

\( \mathrm{H_2 O} \)

76.5 µL

PCR 5

The following mix was used for each sample.

Mastermix PCR 5

0,3 µL per template DNA

Per primer

0,3 µL

Sample contents.

With this mix the following samples were made:

2. primers: yuaB forward 2 + yuaB overlap rev, template DNA: B. subtilis 168 DNA

3. primers: yuaB forward 3 + yuaB overlap rev, template DNA: B. subtilis 168 DNA

The following PCR thermocycle was performed.

7

Initial Denaturation

98 °C

3:00

8

Denaturation

98 °C

0:30

9

Annealing

60 °C

0:30

10

Extention

72 °C

1:00

11

Go back to step 2 (repeat 30x)

12

Final Extension

72 °C

10:00

Recommended thermocycle.

Loaded PCR product on 1% agarose gel with DNA stain G (1:50000) and ran for 1 hour on 100V. Also loaded sample 1.2 (yuaB part 2 of length 209 bp). The following samples were prepared.

30 µL DNA(tasA)

6 µL 6x buffer

10µL of Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) was used.

2

bands visible of ~450 bp

3

bands visible of ~450 bp

1.2

bands visible of ~200 bp

PCR results

Bands were cut from the gel and purified using a PCR purification kit.

2 (1.2)

253 mg

506 µL

3 (1.3)

193 mg

386 µL

1.2 (2)

270 mg

540 µL

Gels.

Samples were stored as

1.2 (49,9 ng/µL Nanodrop value)

1.3 (56,5 ng/µL Nanodrop value)

2 (19,2 ng/µL Nanodrop value)

Harm Ruesink

00:00, 9 June 2015 - 00:00, 9 June 2015

Assembly PCR

Part 1 (450 bp) and part 2 (209 bp) were combined to get the whole bslA gene.

10x buffer

10 µL

dNTP MM (2 mM)

10 µL

Dreamtaq polymerase

1 µL

\( \mathrm{H_2 O}\)

78 µL

PCR Mastermix.

Per sample the following mix was made.

30 µL of mastermix

1 µL per template DNA

0.3 µL per primer

Using this mix two samples were prepared.

yuab forward 2 + yuaB rev

1.3 + 2

yuaB forward 3 + yuaB rev

1.3 + 2

Samples.

1

Denaturation

98 °C

3:00

2

Denaturation

98 °C

0:30

3

Annaeling

60 °C

0:30

4

Extention

72 °C

1:00

5

Go back to step 2 (repeat 30x)

6

Final Extension

72 °C

10:00

Recommended thermocycle.

PCR product was loaded on 1% agarose gel with DNA stain G (1:50000) and ran for one hour at 100 V. The following samples were analyzed.

30 µL of DNA (tasA)

6 µL of 6x buffer

10 µL of Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) was used. Sample 1 showed bands at ~ 650 bp. Sample 2 showed bands at ~650 bp. Band were cut from the gel and purified using a PCR purification kit.

1

176 mg

352 µL

2

248 mg

496 µL

Purification.

This was stored (20 µL of elution buffer) as follows.

bslA + RBS, 9-6-15

bsLA - RBS, 96-15

Harm Ruesink

00:00, 10 June 2015 - 00:00, 10 June 2015

Digestion

yuaB was digested using the following mix.

DNA (yuaB + RBS)

2 µL

10x buffer (2.1)

2 µL

EcoRI

1 µL

Pstl

1 µL

\( \mathrm{H_2 O} \)

14 µL

Digestion mix.

The mix was incubated for two hours at 37 °C. A 1% agarose + DNA strain G 1:50000 was ran at 120 V.

DNA

20 µL

6x buffer

4 µL

Sample.

10 µL of Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) was used. A band was visible around 600 bp. This means that the restriction site within the yuaB gene is gone.

DNA was extracted from the gel using a PCR purification kit.

yuaB

225 mg

450 µL

Purification.

Eluted in 10 µL elution buffer and stored as "yuaB dig".

Ligation of yuaB in BBa_K823023 was performed using the following ligation mix.

insert DNA (yuaB dig)

10 µL

vector DNA (BBa_K823023)

5 µL

10x buffer

2 µL

T4 ligase

0.5 µL

\(\mathrm{H_2 O}\)

2.5 µL

Ligation mix

This was ligated for 2 hours at room temperature. The DNA was then transformed into competent E. coli using heat shock transformation. 2 hours later it was plated on LB ampicilin plates and left in 37 °C overnight.

Harm Ruesink

16:00, 11 June 2015 - 16:30, 11 June 2015

Two white colonies and a lot of red colonies visible on plate. Inoculated 2 white colonies and one red colony in liquid LB with ampicilin overnight.

Harm Ruesink

00:00, 12 June 2015 - 00:00, 12 June 2015

Miniprepped overnight cultures. Stored as

23 yuaB 1

23 yuab 2

23 RFP

Harm Ruesink

00:00, 15 June 2015 - 00:00, 15 June 2015

Digested yuaB from backbone to check ligation using EcoRI and Pstl. The following mastermix was prepared for 13 samples.

10x buffer (2.1)

30 µL

EcorI

10 µL

Pslt

10 µL

\(\mathrm{H_2 O}\)

185 µL

Digestion mastermix.

Per sample the following mix was prepared.

DNA

15 µL

Mastermix

15 µL

Sample contents.

The samples were incubated at 37 °C for two hours. They were then loaded on 1% agarose gel with 1:50000 DNA stain G. To 20 µL of digestion sample 4 µL of 6x buffer was added. 10µL of Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) was used for each sample.

Nothing was seen on the gel. The gel may have been too hot when adding the DNA stain G.

Harm Ruesink

00:00, 16 June 2015 - 00:00, 16 June 2015

Repeated digest of tasA from 15-6-15. Digest for 1.5h at 37 °C.

10 µL of digestion sample was added to 2 L of 6x buffer. This was run at 100 V for 1 hour with 10 µL of 2log ladder.

No insert was seen on gel. yuaB probably wasn't ligated in the backbone.

Harm Ruesink

00:00, 8 July 2015 - 00:00, 8 July 2015

Ligation of yuaB into pSB1C3

yuaB + RBS with pSB1C3 was digested using EcoRI and Pstl.

10x buffer (2.1)

6.6 µL

EcoRI

3.3 µL

Pstl

3.3 µL

\(\mathrm{H_2 O}\)

23.4 µL

Ligation mastermix.

1

yuaB + RBS

10 µL

Mastermix

10 µL

2

pSB1C3

10 µL

Mastermix

10 µL

Samples.

This was digested for 1 h at 37 °C. The DNA was then purified using a PCR purification kit. yuaB was then ligated in pSB1C3.

2

bslA DNA

10 µL

pSB1C3 DNA

5 µL

T4 ligase

0.5 µL

T4 ligase buffer

2 µL

\(\mathrm{H_2 O}\)

2.5 µL

Sample.

The sample was ligated at room temperature for two hours. The ligation product was then transformed into 100 µL of NED competent cells using heat shock transformation. Two plates were made.

90% plate (90 µL)

10% plate (10 µL)

Plates were incubated overnight at 37 °C.

Harm Ruesink

00:00, 9 Juli 2015 - 00:00, 9 Juli 2015

Transformation results

Transformation was a success, both plates show a large number of colonies. Four colonies from the 10% plate were grown overnight in 3 mL LB with 6 µL of 1/500 chloramphenicol

Harm Ruesink

00:00, 16 July 2015 - 00:00, 16 July 2015

Ligation of a salt promotor and yuaB into BBa_K823023 was performed.

           Psalt

           EcoRI + Spel

           bslA (y4, 10-7-15)

           Xbal + PstI

           BBa_K823023

           EcoRI + PstI

Digest.

           DNA (Psalt)

           6 µL

           EcoRI

           1 µL

           Spel

           1 µL

           10x buffer (2.1)

           2 µL

           \(\mathrm{H_2 O}\)

           10 µL

           DNA (Y4)

           3 µL

           Xbal

           1 µL

           PstI

           1 µL

           10x buffer (2.1)

           2 µL

           \(\mathrm{H_2 O}\)

           13 µL

           DNA (K823023)

           3 µL

           EcoRI

           1 µL

           PstI

           1 µL

           10x buffer (2.1)

           2 µL

           \(\mathrm{H_2 O}\)

           10 µL

       Samples.

Samples were incubated for one hour at 37 °C. They were then

purified using a PCR purification kit (20 µL elution). Samples were stored as "1

& 2" with "16-7-15" on the side.

Samples 1 + 2 + 3 were ligated.

           DNA 1

           5 µL

           DNA 2

           5 µL

           T4 Ligase

           0.5 µL

           10x buffer

           2 µL

           \(\mathrm{H_2 O}\)

           5 µL

Ligation mix.

Ligated at room temperature for 30 minutes. Transformed into

10 µL of NEB competent cells with 10 µL of ligation product using heat shock transformation. The result was plated on LB ampicilin plates and left at 37 °C

overnight.

Harm Ruesink

00:00, 17 July 2015 - 00:00, 17 July 2015

Transformation results

A few white colonies were seen on the plate. A colony PCR was performed to check the size of the insert (which is supposed to be bslA + Psalt). The colony PCR showed possible bslA + Psalt insert in one sample. Colony was grown overnight in LB + ampicilin.

Harm Ruesink

00:00, 18 August 2015 - 00:00, 18 August 2015

Miniprep was performed and stored as "22 (17-7-15)".

Harm Ruesink

00:00, 20 July 2015 - 00:00, 20 July 2015

Send in sample 22 for sequencing with prefix (19) and suffix (20) primers.

Send sample 22 to Macrogen for sequencing.

1/20 primer (5 pM/µL)

5 µL

plasmid DNA (22)

5 µL

Samples.

Forward primer (prefix primer): 17C4ZAD516

Reverse primer (suffix primer): 17C4ZAD517

Harm Ruesink

00:00, 21 July 2015 - 00:00, 21 July 2015

<hmtl>

Transformed sample 22 into B. subtilis. Took B.

subtilis 3610 ComI colony from plate (13-7-15 in fridge) and put it in MC 1x for 5 hours. Optical density at 600 nm was measured to be 0.15. Then 5 µL of plasmid DNA (22) was added to 400 µL of cell culture and left in 37 °C

shaking incubator for a further two hours. The culture was then plated on LB

chloramphenicol plates and left overnight at 37 °C.

</html>

Harm Ruesink

00:00, 22 July 2015 - 00:00, 22 July 2015

Sequencing results

Results don't show correct insert. Insert is an RFP. Discarded transformed Bacillus subtilis due to incorrect insert.

Harm Ruesink

00:00, 23 July 2015 - 00:00, 23 July 2015

Psalt was digsted at for one huur at 37 °C using the following mix.

DNA (Psalt)

10 µL

EcoRI

1 µL

Spel

1 µL

10x buffer (2.1)

2 µL

\(\mathrm{H_2 O}\)

6 µL

Digestion mix

The digestion sample was purified using a PCR purification kit. Eluted with 20 µL. It was then ligated for two hours at room temperature using the following mix.

DNA 1 (Psalt)

5 µL

DNA 2 (bslA)

5 µL

DNA 3 (K23)

5 µL

T4 Ligase

1 µL

10x buffer

2 µL

\(\mathrm{H_2 O}\)

2 µL

Ligation mix.

A heat shock transformation was then performed. The result was plated on LB ampicilin and incubated overnight at 37 °C.

Harm Ruesink

00:00, 24 July 2015 - 00:00, 24 July 2015

Transformation Results

No white colonies were visible on the plate.

Harm Ruesink

00:00, 28 July 2015 - 00:00, 28 July 2015

Adding promoters to Y4 (10-7-15).

Pveg (iGEM distribution kit 2015, plate 1, 20 G)

For the promoters the restriction enzymes EcoRI & Spel were used. For the backbone Xbal & EcoRI were used.

1

Y4 DNA

5 µL

10x buffer

2 µL

EcoRI

1 µL

Xbal

1 µL

\(\mathrm{H_2 O}\)

11 µL

3

Pveg DNA

10 µL

10x buffer

2 µL

EcoRI

1 µL

Spel

1 µL

\(\mathrm{H_2 O}\)

11 µL

Digestion samples

These samples were digested at 37 °C for two hours. They were then on a gel at 100 V for 45 minutes. 1% agarose with DNA stain G 1:50000 was used. For each sample, 20 µL of DNA was mixed with 4 µL of 6x buffer. 10 µL of Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) was used.

1

156 mg

316 µL

3

223 mg

446 µL

4

300 mg

600 µL

Gel cuts.

The DNA was purified using a PCR purification kit (Genejet #K0701, lot 00265976). 25 µL elution buffer was used. The concentrations were checked using Nanodrop.

Y4 dig (1)

2 ng/µL

Pveg (3)

1.4 ng/µL

Psalt (4)

5 ng/µL

Sample concentrations.

Ligation of promoters in backbones

10x buffer

12 µL

T4 Ligase

4 µL

\(\mathrm{H_2 O}\)

8 µL

Ligation mastermix.

1

Y4 dig (1)

12 µL

Pveg (3)

12 µL

Mastermix

6 µL

2

Y4 dig (1)

12 µL

Psalt (4)

12 µL

Mastermix

6 µL

Ligation samples.

Samples were ligated overnight.

Harm Ruesink

10:00, 29 July 2015 - 12:00, 29 July 2015

Heat shock transformation was performed. Samples were plated on LB chloramphenicol and incubated overnight at 37 °C.

Harm Ruesink

00:00, 30 July 2015 - 00:00, 30 July 2015

Transformation results

No colonies visible on LB chloramphenicol plate.

Y4 was send to Macrogen for sequencing.

5 µL of 1/20 primer (5 pmole/µL)

5 µL of plasmid DNA (Y4)

Sequencing codes Macrogen

Forward primer (prefix (19)): 17C4ZAD660

Reverse primer (suffix (20)): 17C4ZAD661

Harm Ruesink

00:00, 6 August 2015 - 00:00, 6 August 2015

Digestion and ligation of Pliag in front of Y4 was performed. For the promoters EcoRI and Spel were used. For Y4 Xbal and EcoRI were used. These enzymes leave a scar.

1

Pliag DNA

5 µL

10x buffer

2 µL

EcoRI

1 µL

Spel

1 µL

\(\mathrm{H_2 O}\) (MQ)

11 µL

2

Y4 DNA

5 µL

10x buffer

2 µL

Xbal

1 µL

EcoRI

1 µL

\(\mathrm{H_2 O}\) (MQ)

11 µL

Samples.

Samples were digested for 2 hours at 37 °C. Samples were then loaded on 1% agarose gel with DNA stain G 1:50000

sample DNA

20 µL

6x buffer

4 µL

Samples.

10 µL of NED 2log ladder. Gel was run on 100 V for 30 minutes. It was then cut.

1

0.300 g

600 µL

2

0.118 g

236 µL

Gel cuts.

The samples were purified using a Thermo Scientific GeneJET PCR Purification Kit. Concentrations were checked using Nanodrop.

Pliag dig

4,6 ng/µL

Y4 dig

4,7ng/µL

Sample concentrations.

Samples were ligated overnight.

Pliag DNA

5 µL

Y4 DNA

10 µL

10x T4 buffer

2 µL

T4 Ligase

0.5 µL

\(\mathrm{H_2 O}\)

0.5 µL

Ligation mix.

Harm Ruesink

00:00, 7 August 2015 - 00:00, 7 August 2015

A heat shock transformation was performed. 20 µL of (Y4 + Pliag) DNA was added to 10 µL of competent NEB cells. This was plated on LB chloramphenicol plates and incubated overnight at 37 °C.

Harm Ruesink

00:00, 8 August 2015 - 00:00, 8 August 2015

Transformation results

No colonies were visible.

Harm Ruesink

00:00, 9 August 2015 - 00:00, 9 August 2015

Transformation results

White colonies were visible. A colony PCR was performed. PCR showed possible insert in sample 9 and 13. Cultures were grown overnight to check for the insert. The overnight cultures were grown in LB + chloramphenicol (3mL) at 37 °C, 200rpm.

Harm Ruesink

00:00, 11 August 2015 - 00:00, 11 August 2015

Cultures 9 and 13 were miniprepped and loaded on 1% agarose gel with DNA stain G 1:50000.

Sample DNA

1 µL

\(\mathrm{H_2 O}\)

4 µL

6x buffer

1 µL

Mix.

10µL of Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) was used. The gel was run at 100 V for 30 minutes. Nothing was visible on the gel. DNA concentration should be checked.

Harm Ruesink

00:00, 13 August 2015 - 00:00, 13 August 2015

Concentrations were checked using nanodrop.

9

Sample DNA

4 µL

6x Buffer

1 µL

\(\mathrm{H_2 O}\)

1 µL

13

Sample DNA

5 µL

6x Buffer

1 µL

Y4

Sample DNA

1 µL

6x Buffer

1 µL

\(\mathrm{H_2 O}\)

4 µL

Gel Samples.

Gel was run at 100 V for 30 minutes. The gel showed no bands.

Harm Ruesink