Template:Team:Groningen/CONTENT/LOGBOOK/Overproduce TasA in B. subtilis ComI

Overproduce TasA in B. subtilis ComI

A biofilm is held together by a variety of different extracellular proteins, one of which is TasA. The TasA protein is encoded by the tasA gene. When expressed, the protein forms amyloid-like fibers in the extracellular matrix and provides stability and structural integrity of the biofilm.

Our goal is to overexpress TasA by adding a second copy of the tasA gene under the control of a salt inducible promoter. This overexpression will lead to a stronger biofilm when the biofilm is exposed to salt water.

Malesuada a nisi ac...

<a class="postscriptum protocol" href="https://2015.igem.org/Team:Groningen/Protocols_and_Protocols/No_protocol">Finibus sapien, nec cursus...</a>

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

Primers were designed for tasA in B. subtilis 168.

5

tasA for

CGGAATTCGCGGCCGCTTCTAGAGCAATAAAAGGGGAGCTTACCATG

6

tasA rev

CGCTGCAGCGGCCGCTACTAGTATTATTAATTTTTATCCTCGCTATGC

7

tasA EcoRI overlap for

GATTTATCTGCTAAAGAGAACTCAGCGAGTGTGAAC

8

tasA EcoRI overlap rev

GTTCACACTCGCTGAGTTCTCTTTAGCAGATAAATC

9

tasA PstI overlap for

GAGTTTAGGAGTTGCTTCAGCAGCACTAGGATTAG

10

tasA PstI overlap rev

CTAATCCTAGTGCTGCTGAAGCAACTCCTAAACTC

Primers for tasA in B. subtilis 168.

Primers for tasA.

Atze van Stralen

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

PCR 1

B. subtilis 168 DNA was used.

dNTP Mastermix (MM) was prepared with a final concentration of 2 mM of each dNTP by adding water, see Table 1.

100 mM from each dNTP (dATP, dTTP, dCTP, dGTP)

20 µL

\( \mathrm{H_2O}\)

920 µL

Preparation Mastermix.

The Mastermix with 2 mM of each dNTP is added to buffer, phusion polymerase and water in the amounts indicated in Table 2.

5x buffer

33 µL

dNTP MM (2 mM)

16.5 µL

Phusion polymerase

1.65 µL

\( \mathrm{H_2O}\)

112.2 µL

Mastermix for PCR 1.

The resulting solution was combined with template DNA and primers. See Table 3.

Mastermix PCR 1

30 µL

Template DNA

0.3 µL

Each primer

0.3 µL

Components per sample.

The following samples were made. See Table 4.

3

5 tasA for

10 PstI overlap rev

B. subtilis 168

4

9 PstI overlap for

8 EcoRI overlap rev

B. subtilis 168

5

7 EcoRI overlap

6 tasA rev

B. subtilis 168

Components per sample.

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 step 2 (30x)

6

Final extension

72 °C

5:00

PCR Thermocycle.

The samples were loaded on 1% agarose gel with EtBr to check for PCR products. The results are listed in Table 6.

3

± 100 bp

4

± 150 bp

5

± 700 bp

PCR Results.

As the product at ± 700 bp was not that clearly visible, the PCR was repeated. PCR purification products of samples 3, 4 and 5 were stored in freezer at -20 °C as 1.3, 1.4 and 1.5.

Harm Ruesink

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

PCR 2

B. subtilis 168 DNA was used.

The Mastermix (MM) with 2 mM of each dNTP is added to buffer, phusion polymerase and water in the amounts indicated in Table 7.

5x buffer

33 µL

dNTP MM (2 mM)

16.5 µL

Phusion polymerase

1.65 µL

\( \mathrm{H_2O}\)

112.2 µL

Mastermix for PCR 2.

The resulting solution was combined with template DNA and primers. See Table 8.

Mastermix PCR 1

30 µL

Template DNA

0.3 µL

Each primer

0.3 µL

Components per sample.

5

7 tasA EcoRI overlap for

6 tasA rev

B. subtilis 168

6

5 tasA for

8 tasA EcoRI overlap rev

PCR product 1.3 + 1.4

7

9 tasA PstI overlap for

6 tasA rev

PCR product 1.4 + 1.5

8

5 tasA for

8 tasA EcoRI overlap rev

PCR product 1.3 + 1.4 + 1.5

1

5 tasA for

8 tasA EcoRI overlap rev

PCR product 1.3

Components per sample.

Sample 1 is a control to check for genomic DNA of PCR 1.

1

Initial denaturation

98 °C

3:00

2

Denaturation

98 °C

0:30

3

Annealing

62 °C

0:30

4

Extension

72 °C

1:00

5

Go back to step 2 (3x)

6

Denaturation

98 °C

0:30

7

Annealing

60 °C

0:30

8

Extension

72 °C

1:00

9

Go back to step 6 (3x)

10

Denaturation

98 °C

0:30

11

Annealing

58 °C

0:30

12

Extension

72 °C

1:00

13

Go back to step 10 (3x)

14

Denaturation

98 °C

0:30

15

Annealing

56 °C

0:30

16

Extension

72 °C

1:00

17

Go back to step 14 (3x)

18

Denaturation

98 °C

0:30

19

Annealing

54 °C

0:30

20

Extension

72 °C

1:00

21

Go back to step 18 (3x)

22

Denaturation

98 °C

0:30

23

Annealing

54 °C

0:30

24

Extension

72 °C

1:00

25

Go back to step 22 (30x)

26

Final extension

72 °C

10:00

PCR Thermocycle.

5

± 700 bp

6

± 250 bp

7

no clear product

8

no clear product

1

± 250 bp

PCR Results.

Sample 7 should have showed a band at ± 850 bp and sample 8 at ± 950 bp, but as this was not observed, the product was discarded. In sample 1 still genomic DNA was present. For further PCR experiments, a gelpurification of the PCR product was done to get rid of the genomic DNA. Sample 1 was discarded, as it had no use in further experiments.

Harm Ruesink

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

Purification of PCR 2 product 5 and 6 with gelpurification on 1% agarose gel with EtBr. Clean up sample with PCR cleanup kit.

PCR product 6 contains part 3 and 4 of TasA.

PCR product 5 contains part 5 of TasA.

Stored in freezer at -20 °C as 2.5 and 2.6.

PCR 3

5x buffer

28 µL

dNTP MM (2 mM)

14 µL

Phusion polymerase

1.4 µL

\( \mathrm{H_2O}\)

95.2 µL

Mastermix for PCR 3.

Mastermix PCR 3

30 µL

Template DNA

0.3 µL

Each primer

0.3 µL

Components per sample.

3

5 tasA for

6 tasA rev

PCR product 2.5 + 2.6

4

5 tasA for

6 tasA rev

PCR product 1.3 + 1.4 + 2.5

Components per sample.

1

Initial denaturation

98 °C

3:00

2

Denaturation

98 °C

0:30

3

Annealing

56 °C

0:30

4

Extension

72 °C

1:00

5

Go back to step 2 (15x)

6

Denaturation

98 °C

0:30

7

Annealing

70 °C

0:30

8

Extension

72 °C

1:00

9

Go back to step 6 (repeat 15x)

10

Final extension

72 °C

10:00

PCR Thermocycle.

The PCR product was stored in the fridge at 4 °C.

Harm Ruesink

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

Run a gel with PCR 3 products on 1% agarose gel with EtBr.

On the gel, for sample 3 two bands were visible, indicating that the PCR assembly of fragement 2.5 and 2.6 did not work. Sample 4 had no product, so also the PCR assembly of product 1.3, 1.4 and 1.5 did not work.

Gibson Assembly

For the Gibson assembly, DNA was used with the concentrations indicated in Table 1. The concentration in the second column is the concentration of DNA based on the gel. The concentrations listed in column 3 are obtained by using Nanodrop.

1.3 (100 bp)

250 ng/µL

37 ng/µL

1.4 (150 bp)

170 ng/µL

27.1 ng/µL

2.5 (700 bp)

200 ng/µL

67.7 ng/µL

2.6 (250 bp)

40 ng/µL

42.1 ng/µL

DNA concentration of samples.

Two Gibson reaction samples were made.

1

PCR product 1.3 (100 bp)

0.588 µL

PCR product 1.4 (150 bp)

0.260 µL

PCR product 2.5 (700 bp)

0.143 µL

\( \mathrm{H_2O}\)

4.00 µL

2

PCR product 2.5 (700 bp)

0.588 µL

PCR product 2.6 (250 bp)

0.893 µL

\( \mathrm{H_2O}\)

3.59 µL

Gibson reaction samples.

15 µL of Gibson reaction mixture (Clement) was added to a total volume of 20 µL and incubated for 1h at 50 °C. The Gibson assembly products were purified with the PCR purification kit and stored as tasA DNA 1 and tasA DNA 2 in freezer at -20 °C.

PCR 4

5x buffer

14 µL

dNTP MM (2 mM)

7 µL

Phusion polymerase

0.7 µL

\( \mathrm{H_2O}\)

47.6 µL

Mastermix for PCR 4.

Mastermix PCR 4

30 µL

Template DNA

0.3 µL

Each primer

0.3 µL

Components per sample.

1

5 tasA for

6 tasA rev

tasA DNA 1

2

5 tasA for

6 tasA rev

tasA DNA 2

Components per sample.

1

Initial denaturation

98 °C

3:00

2

Denaturation

98 °C

0:30

3

Annealing

55 °C

0:30

4

Extension

72 °C

0:30

5

Go back to step 2 (30x)

6

Final extension

72 °C

10:00

PCR Thermocycle.

The product was stored in the freezer at -20 °C.

Harm Ruesink

00:00, 20 May 2015 - 00:00, 20 May 2015

PCR 4 was purified with the PCR purification kit. The products were stored as tasA DNA 1 & tasA DNA 2 and PCR 4 prod 20-5-2015 (side). On gel it was checked if the PCR had worked out. A gel was made of 1% agarose with EtBr. The loaded samples consisted of the components listed below. The samples were compared with 3 µL of DNA ladder.

Sample:

1.5 µL 6x loading buffer

2.5 µL \( \mathrm{H_2O}\)

2 µL sample

Ladder:

3 µL GeneRuler™ 1 kb DNA Ladder

On the gel there were no bands visible for sample 1. Apparently the tasA product had not formed. The gel of sample 2 did show a product at ± 950 bp. This fragment was checked with restriction enzymes to find out if the restriction sites were absent in the tasA gene. This product, PCR product 4.2, was digested with the biobrick assembly kit digestion enzymes (NEB) using EcoRI and PstI. The following samples were made. See Table 2.

1

Buffer 10x

2 µL

EcoRI

1 µL

PCR prod 4.2

2 µL

\( \mathrm{H_2O}\)

15 µL

2

Buffer 10x

2 µL

PstI

1 µL

PCR prod 4.2

2 µL

\( \mathrm{H_2O}\)

15 µL

Components used for digestion of PCR product 4.2.

The following digestion protocol was followed. See Table 3.

1

Digestion

37 °C

30:00

2

Enzyme inactivation

80 °C

10:00

Digestion protocol.

A gel was loaded with digestion product of sample 1 and 2 on 1% agarose with EtBr. The following components were added to each well.

Sample:

1.5 µL 6x loading buffer

5 µL digestion product

Ladder:

3 µL GeneRuler™ 100 bp DNA Ladder

On the gel there was one band visible for sample 1 at ± 950 bp. Apparently EcoRI did not cut tasA. A mutation in the EcoRI restriction site of tasA was possibly present. The gel of sample 2 showed a product at ± 950 bp, so pstI did not cut tasA. So a mutation in the PstI restriction site of tasA might have been present.

For the ligation of tasA into the "RFP backbone", first the digestion of tasA (PCR prod 4.2) and "RFP backbone" was carried out.

Sample (PCR prod 4.2)

10 µL

EcoRI

1 µL

PstI

1 µL

Buffer 10x

3 µL

\( \mathrm{H_2O}\)

15 µL

Components used for digestion of PCR product 4.2.

Sample ("RFP backbone")

5 µL

EcoRI

1 µL

PstI

1 µL

Buffer 10x

3 µL

\( \mathrm{H_2O}\)

20 µL

Components used for digestion of plasmid ("RFP backbone").

The samples were digested for 2 hours at 37 °C. Afterwards the DNA was cleaned up with the PCR purification kit, and stored at -20 °C. The DNA was stored in 20 µL elution buffer.

Plasmid

ligate RFP BB, digest prod 20-5-15

tasA

ligate tasA 4.2, digest prod 20-5-15

Storage information of purified PCR product.

NOTE: The next time a gel extraction must be done after digestion, as the RFP insert is too large to get rid of with the PCR purification kit.

Harm Ruesink

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

The ligation of the RFP backbone with tasA (4.2) was performed. First a gel extract was made on 1% agarose gel with EtBr to get rid of the RFP insert.

Sample:

4 µL 6x buffer

20 µL sample (RFP backbone)

Ladder:

3 µL GeneRuler™ 1 kb DNA Ladder

The gel was run for ± 45 minutes at 100 V. Afterwards the band for the RFP backbone was cut out to obtain purified RFP backbone without the RFP insert. The RFP backbone DNA was extracted from the gel with the PCR purification kit. For this binding buffer was added to the cut out gel in the ratio 2:1 binding buffer:gel weight. It was stored as BB1, purified backbone (50 µL elution buffer). Afterwards the ligation of tasA in BB1 was carried out. For that the following components were combined.

15 µL BB1

2 µL 10x buffer (2.1)

1 µL tasA DNA (4.2)

2 µL T4 ligase

The mixture was incubated at room temperature for 2 hours. During this time ampicillin plates were poured (100 µg/mL). Afterwards the heat shock transformation was carried out using the following components.

10 µL ligation mixture in competent DH5 alpha

Competent cells Harm (E.coli (1)) (CompH)

Competent cells Clement (CompC)

The transformed cells were plated on LB+amp plates.

90% CompH

10% CompH

90% CompC

10% CompC

The plates were put in the incubator overnight at 37 °C.

Harm Ruesink

00:00, 22 May 2015 - 00:00, 22 May 2015

The transformation was checked. There were 5 colonies visible on the LB+amp plates. 4 colonies on 90% compH and one colony on 10% CompH. Both plates were stored in the fridge at 4 °C.

Harm Ruesink

00:00, 26 May 2015 - 00:00, 26 May 2015

The five transformed colonies were inoculated overnight in liquid LB+amp (30 µL amp per 3 mL LB (1/100)). This should have been 3µL per 3mL amp, so also this concentration was made and used. The culture of the colony from the 10% plate was labeled sample 1 and the cultures of the four colonies from the 90% plate were labeled sample 2,3,4 and 5. The cultures were incubated at 37 °C, at 250 rpm.

Harm Ruesink

00:00, 27 May 2015 - 00:00, 27 May 2015

All cultures grew, of both the 1/100 and the 1/1000 amp concentrations. The cultures were miniprepped overnight according to the miniprep kit manual. The cultures were stored as tubes 1-5. DNA concentrations were determined using Nanodrop.

1

168.4

2

516.7

3

420.1

4

551.5

5

523.9

DNA concentrations.

The miniprep product was digested with EcoRI and PstI to check for insert.

Harm Ruesink

00:00, 27 May 2015 - 00:00, 27 May 2015

All cultures grew, of both the 1/100 and the 1/1000 amp concentrations. The cultures were miniprepped overnight according to the miniprep kit manual. The cultures were stored as tubes 1-5. DNA concentrations were determined using Nanodrop.

\( \mathrm{H_2O}\)

14 µL

15 µL

EcoRI

1 µL

PstI

1 µL

Sample DNA

2 µL

1 µL

10x buffer (2.1)

2 µL

DNA concentrations.

The samples were incubated at 37 °C for 10 minutes. Afterwards a gel was run with 1% agarose and EtBr.

For each sample:

2 µL miniprep sample

1 µL 6x buffer

3 µL \( \mathrm{H_2O}\)

Ladder:

3 µL GeneRuler™ 1 kb DNA Ladder

On the gel, multiple bands were visible per plasmid, so the plasmid seems to be wrong. Again tasA was gelpurified and ligated into the backbone. Also the backbone (BB1) was checked by the next steps. The following combinations were made:

EcoRI + PstI + RFP plasmid

EcoRI + SpeI + RFP plasmid

XbaI + PstI + RFP plasmid

XbaI + SpeI + RFP plasmid

uncut RFP plasmid

Digestion Mastermix:

12 µL 10x buffer (2.1)

9 µL DNA (RFP plasmid)

99µL \( \mathrm{H_2O}\)

Per sample:

28 µL Mastermix

2 µL restriction enzymes

The samples were incubated for 30 minutes at 37 °C. Afterwards the samples were loaded on gel, 1% agarose with EtBr.

For each sample:

5 µL sample

1 µL 6x buffer

Ladder:

2 µL GeneRuler™ 1 kb DNA Ladder

On the gel it was seen that BB1 is probably not good, as it still has RFP in it. This was checked with the restriction enzymes of Molgen.

10x buffer (2.1)

6.6 µL

EcoRI

2.2 µL

PstI

2.2 µL

\( \mathrm{H_2O}\)

50.6 µL

Components for Mastermix digestion.

RFP plasmid DNA

2 µL

10x buffer

3 µL

EcoRI (Molgen)

1 µL

PstI(Molgen)

1 µL

\( \mathrm{H_2O}\)

23 µL

Components for digestion.

The mixture was incubated at 37 °C for 2 hours. After 2 hours the sample was loaded on a 1% agarose gel with EtBr gel with uncut plasmid. On the gel it was visible that the band of the digested plasmid is lower, but there was no insert visible. Do not use this backbone for further ligations and transformations.

Harm Ruesink

00:00, 2 June 2015 - 00:00, 2 June 2015

The backbones BBa_K823023 and BBa_K823024 were digested and ligated with tasA by the following steps.

1

Mastermix

28 µL

DNA (BBa_K823023)

2 µL

2

Mastermix

28 µL

DNA (BBa_K823024)

2 µL

Digestion mixtures.

The samples were digested for 2 hours at 37 °C. Afterwards they were loaded on 1% agarose gel with SERVA DNA stain G. Also tasA was loaded on the gel for gel purification. 10 µL NEB DNA ladder log was used.

1

DNA (tasA 4.2)

20 µL

6x buffer

4 µL

2

DNA (BBa_K823023)

25 µL

6x buffer

5 µL

3

DNA (BBa_K823024)

25 µL

6x buffer

5 µL

Samples for on the gel.

The gel was run on 100 V for ± 1 hour and the following bands were visible:

tasA at ±800-900 bp

K823023 (±5000 bp) including a band of its RFP insert (±1100 bp)

K823024 (±6000 bp) including a band of its RFP insert (±1100 bp)

These band sizes match the in silico sizes. The bands were cut out of the gel and purified with the PCR purification kit.

tasA

77

144

K823023

181

362

K823024

133

266

Amounts of DNA and binding buffer added.

The samples were eluted with 20 µL elution buffer and stored at -20 °C as:

tasA DNA ligate

K823023 ligate

K823024 ligate

Harm Ruesink

00:00, 3 June 2015 - 00:00, 3 June 2015

For the ligation of tasA in BBa_K823023, the following components were used.

Vector DNA (K823023)

2 µL

Insert DNA (tasA)

2 µL

10x buffer

2 µL

T4 ligase

1 µL

\( \mathrm{H_2O}\)

13 µL

Components used for ligation.

The ligation mixture was left 2 hours at room temperature. Afterwards 10 µL of the mixture was put in 100 µL competent E.coli (1). Two mixtures were streaked out on an LB+amp plate:

90% plate (90 µL)

10% plate (10 µL)

Harm Ruesink

00:00, 4 June 2015 - 00:00, 4 June 2015

The transformation was checked. No colonies were visible on either plate so the transformation failed.

Harm Ruesink

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

PCR 5

In this experiment it was attempted to increase the amount of tasA. For this first a Mastermix was made, to which primer and DNA were added.

10x buffer

10 µL

dNTP MM (2 mM)

10 µL

Dreamtaq polymerase

1 µL

\( \mathrm{H_2O}\)

76,5 µL

Mastermix PCR 5.

Mastermix PCR 5

30 µL

tasA DNA (4.2)

0.3 µL

5 tasA for

0.3 µL

6 tasA rev

0.3 µL

Components PCR 5 sample.

The following PCR cycle was used.

1

Initial denaturation

98 °C

3:00

2

Denaturation

98 °C

0:30

3

Annealing

60 °C

0:30

4

Extension

72 °C

1:00

5

Go back to step 2 (repeat 30x)

6

Final extension

72 °C

10:00

PCR thermocycle.

The PCR prduct was loaded on a 1% agarose gel with DNA stain G (1:50000) and run for 1 hour on 100 V with the following samples.

Sample:

30 µL DNA (tasA).

10 µL 6x buffer.

Ladder:

10 µL NEB ladder.

The band of tasA was hardly visible. The band was cut out and purified with the PCR purification kit.

tasA

270

540

Amounts of DNA and binding buffer added.

The purified DNA was stored as tasA DNA (7.3 ng/µL Nanodrop value).

Harm Ruesink

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

For the digestion of tasA and the plasmids (BBa_K823023 and BBa_K823024), a Mastermix was made, which was added to the DNA.

10x buffer (2.1)

6.6 µL

EcoRI

3.3 µL

PstI

3.3 µL

\( \mathrm{H_2O}\)

52.8 µL

Components Mastermix digestion.

1

Mastermix

15 µL

DNA (tasA DNA)

10 µL

2

Mastermix

15 µL

DNA (K823023)

10 µL

3

Mastermix

15 µL

DNA (K823024)

10 µL

Components digestion samples.

The samples were incubated for 2 hours at 37 °C. Afterwards they were cleaned up with the PCR purification kit and eluted in 20 µL elution buffer. The purified samples were stored at -20 °C as:

tasA dig, 9-6-15.

23 dig, 9-6-15.

24 dig, 9-6-15.

The ligation of tasA in the plasmids (BBa_K823023 & BBa_K823024) was performed by combining the following components.

23 lig

insert (tasA dig)

10 µL

vector(23 dig)

5 µL

10x buffer

2 µL

T4 ligase

0.5 µL

\( \mathrm{H_2O}\)

2.5 µL

24 lig

insert (tasA dig)

10 µL

vector(24 dig)

5 µL

10x buffer

2 µL

T4 ligase

0.5 µL

\( \mathrm{H_2O}\)

2.5 µL

Components of ligation samples.

The mixtures were ligated for 2 hours at room temperature. Afterwards competent E.coli was transformed with the ligation products. A heat shock transformation was used. The cultures were plated on LB+amp plates and incubated overnight at 37°C. They were labeled as:

100% comp E. coli with 23 lig

100% comp E. coli with 24 lig

Harm Ruesink

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

The transformation was checked. One white colony was visible on the 23 lig plate and 7 colonies were visible on the 24 lig plate. The colonies were grown overnight in LB+amp at 37 °C, 200 rpm.

BBa_K823023 + tasA (possible)

BBa_K823023 + RFP

BBa_K823024 + tasA 7x (possible)

BBa_K823024 + RFP

Harm Ruesink

00:00, 11 June 2015 - 00:00, 11 June 2015

The colonies that were grown overnight were miniprepped, by eluting them in 50 µL and stored as 23RFP, 23TA, 24RFP, 24TA (1,2,3,4,5,6&7). All tasA samples were digested to check for the insert. For this a Mastermix for digestion was made.

10x buffer (2.1)

22 µL

EcoRI

9 µL

PstI

9 µL

\( \mathrm{H_2O}\)

167 µL

Mastermix for digestion.

Mastermix

18 µL

DNA

2 µL

Sample for digestion.

The samples were incubated at 37 °C for 2 hours and 15 minutes. Afterwards they were put on a 1% agarose gel with 1:50000 DNA stain G. The gel was run on 120 V.

Sample:

5 µL sample

1 µL 6x buffer

Ladder:

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

On the gel there was not much visible, so the procedure was repeated with more DNA.

Harm Ruesink

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

Digested tasA from backbone using EcoRI and PstI to check ligation. For this, a Mastermix was made for 13 samples (± 300 µL).

10x buffer (2.1)

30 µL

EcoRI

10 µL

PstI

10 µL

\( \mathrm{H_2O}\)

185 µL

Mastermix digestion.

DNA

5 µL

Mastermix

15 µL

Components per digestion sample.

The samples were incubated for 2 hours at 37 °C. Afterwards they were loaded on 1% agarose gel with 1:50000 DNA stain G.

Sample:

20 µL digestion sample

4 µL 6x buffer

Ladder:

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

There was nothing visible on the gel. The gel was probably too hot when adding the DNA stain G.

Harm Ruesink

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

The digestion of tasA of 15 June 2015 was repeated. The samples were digested for 1.5 hours at 37 °C. Afterwards the samples were loaded on gel.

Sample:

10 µL digestion sample

2 µL 6x buffer

Ladder:

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

A gel was run at 100 V for 1 hour. On the gel 24TA 3 seemed to have the correct insert.

Harm Ruesink

00:00, 18 June 2015 - 00:00, 18 June 2015

24TA 3 was send to Macrogen for sequencing:

5µL 1/20 primer (5 pmol/µL)

5µL plasmid DNA (24TA 3)

Sequencing code Macrogen:

Forward primer (5 tasA for): 1790ZAB067

Reverse primer (6 tasA rev): 1790ZAB068

Harm Ruesink

00:00, 19 June 2015 - 00:00, 19 June 2015

The seqencing results showed the tasA insert without restriction sites. The restriction sites are gone.

Harm Ruesink

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

In this experiment tasA was ligated into pSB1C3. For this first a Mastermix was made for the digestion of 24TA 3 (BBa_K823024 + tasA insert) and pSB1C3 EcoRI and PstI.

10x buffer (2.1)

6.6 µL

EcoRI

3.3 µL

PstI

3.3 µL

\( \mathrm{H_2O}\)

23.4 µL

Mastermix digestion.

1

24TA 3

10 µL

Mastermix

10 µL

2

pSB1C3

10 µL

Mastermix

10 µL

Components digestion sample.

The sample was digested for one hour at 37 °C. A PCR purification kit was used to clean up the DNA. Afterwards tasA was ligated in pSB1C3.

tasA DNA

10 µL

pSB1C3 DNA

5 µL

T4 ligase

0.5 µL

T4 ligase buffer

2 µL

\( \mathrm{H_2O}\)

2.5 µL

Components used for ligation.

The sample was ligated at room temperature for 2 hours. Afterwards a heat shock transformation was performed with 100 µL NEB competent cells + ligation product.

90% plate (90µL)

10% plate (10µL)

The plates were incubated overnight at 37 °C.

Harm Ruesink

00:00, 9 July 2015 - 00:00, 9 July 2015

The transformation was checked. On both 10% and 90% plates a large amount of colonies had grown, so the transformation was successful. 4 colonies from the 10% plate was grown overnight in 3 mL LB+cm (6 µL 1/500).

Harm Ruesink

00:00, 10 July 2015 - 00:00, 10 July 2015

The samples were miniprepped and digested to check for insert. It was seen that indeed tasA was inserted successfully into pSB1C3. The insert was stored as T2.

Harm Ruesink

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

The salt-promoter and tasA were ligated into BBa_K823023. The salt promoter from Marieke was used. For the digest the following combinations were made.

1

Psalt

EcoRI + SpeI

2

tasA (T2, 10-7-15)

XbaI + PstI

3

BBa_K823023

EcoRI + PstI

Digestion.

1

DNA (Psalt)

6 µL

EcoRI

1 µL

SpeI

1 µL

10x buffer (2.1)

2 µL

\( \mathrm{H_2O}\)

10 µL

2

DNA (T2)

3 µL

XbaI

1 µL

PstI

1 µL

10x buffer (2.1)

2 µL

\( \mathrm{H_2O}\)

13 µL

3

DNA (K823023)

6 µL

EcoRI

1 µL

PstI

1 µL

10x buffer (2.1)

2 µL

\( \mathrm{H_2O}\)

10 µL

Components samples.

The samples were incubated for one hour at 37 °C. The samples were purified using the PCR purification kit, using 20 µL elution buffer. The samples were stored as 1 and 2, with on the side 16-7-15. The samples 1, 2 and 3 were ligated using the following amounts.

DNA 1 (Psalt)

5 µL

DNA 2 (T2)

5 µL

DNA 3 (K823023)

2.5 µL

T4 ligase

0.5 µL

10x buffer

2 µL

\( \mathrm{H_2O}\)

5 µL

Components ligation sample.

The sample was ligated at room temperature for 30 minutes. Afterwards the heat shock transformation from the protocol book was performed. For this, 10 µL competent NEB cells with 10 µL ligation product was used. The resulting cells were plated on LB+amp plates and grown overnight at 37 °C.

Harm Ruesink

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

The transformation of the ligation product of the salt promoter with tasA was checked. On the plates a few white colonies were seen. Miniprep and colony PCR were performed to check whether the insert size was the right one. The colony PCR showed possible tasA with Psalt insert in one sample. This colony was grown overnight in LB+amp.

Harm Ruesink

00:00, 18 July 2015 - 00:00, 18 July 2015

The culture of the colony that seemed to have the right insert at 17 July, was miniprepped and stored as 33 (17-1-15).

Wiebrand de Boer

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

Sample 33 was send for sequencing with the prefix and suffix primers (no. 19 and 20) to Macrogen in the following amounts:

1/20 primer (5 pmol/µL)

5 µL

plasmid DNA (sample 33)

5 µL

Amount of primers and DNA sent for sequencing.

prefix primer (forward)

17C4ZAD518

suffix primer (reverse)

17C4ZAD519

Sequencing codes Macrogen.

Harm Ruesink

09:24, 20 July 2015 - 16:30, 20 July 2015

Sample 33 was transformed into Bacillus subtilis 3610 comI using Elrike's transformation protocol. For this a comI colony was taken from a plate and put in a tube with 2 mL MC 1x. The tube was put in the shaking incubator for ∼5 hours. After 5 hours of growing the OD at 600 nm was seen to be 0.15. 400 µL of culture was added to a new tube, and 5 µL of plasmid DNA (sample 33) was added. The new tube was placed in the shaking incubator at 37 °C.

Harm Ruesink

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

The sequencing results showed that the insert into the K823023 backbone was not the correct one. RFP was inserted instead of tasA with the salt promoter. The transformed Bacillus was discarded.

Harm Ruesink

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

Psalt, tasA and BBa_K823023 were ligated again. For this to be possible, first the salt promoter (Psalt) had to be digested using EcoRI and SpeI as restriction enzymes. tasA and BBA_K823023 were digested before already.

DNA (Psalt)

10 µL

EcoRI

1 µL

SpeI

1 µL

10x buffer (2.1)

2 µL

\( \mathrm{H_2O}\)

6 µL

Components for digestion salt promoter.

The sample was digested for one hour at 37 °C. Afterwards the sample was purified using the PCR purification kit. For this 20 µL elution buffer was used. The subsequent ligation was performed with the folowing components.

DNA 1 (Psalt)

5 µL

DNA 2 (tasA)

5 µL

DNA 3 (K823023)

5 µL

T4 ligase

1 µL

10x buffer

2 µL

\( \mathrm{H_2O}\)

2 µL

Components ligation Psalt, tasA and backbone.

The sample was ligated at room temperature for 2 hours. Afterwards the heat shock transformation form the protocol book was performed and the culture was plated on LB+amp plates and incubated overnight at 37 °C.

Harm Ruesink

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

The transformation of 23 July was checked. One white colony was visible on the plate, but this colony did not have the insert. So the ligation failed.

Harm Ruesink

09:30, 28 July 2015 - 16:00, 28 July 2015

The ligation of the T2 sample of 10 July with two promoters was performed, both with the Pveg promoter from the iGEM distribution kit 2015 (plate 1, 20G) and the Psalt promoter (from Marieke). For the promoters EcoRI and SpeI were used as restriction enzymes, and XbaI and EcoRI for the backbone. The following samples were made.

1

T2 DNA

5 µL

10x buffer

2 µL

EcoRI

1 µL

XbaI

1 µL

\( \mathrm{H_2O}\)

11 µL

2

Pveg DNA

10 µL

10x buffer

2 µL

EcoRI

1 µL

SpeI

1 µL

\( \mathrm{H_2O}\)

6 µL

4

Psalt DNA

5 µL

10x buffer

2 µL

EcoRI

1 µL

SpeI

1 µL

\( \mathrm{H_2O}\)

11 µL

Components digestion samples.

The samples were digested at 37 °C for 2 hours. Afterwards a 1% agarose gel with stain G 1:50000 was run on 100 V for ∼45 minutes. 20 µL of DNA samples were mixed with 4 µL 6x buffer. As a ladder the 10 µL 2log NEB ladder was used. The right bands were cut out, weighed and dissolved in twice as much binding buffer. The samples were purified with the PCR purification kit (Genejet #K0701, lot 00265976) using 25 µL elution buffer. The concentrations were determined with Nanodrop.

T2 dig (2)

4.5 ng/µL

Pveg (3)

1.4 ng/µL

Psalt (4)

5.0 ng/µL

Concentrations of samples determined by Nanodrop.

After the digestion, the promoters were ligated into the backbones. For this a ligation Mastermix was made for four samples.

10x buffer

12 µL

T4 ligase

4 µL

\( \mathrm{H_2O}\)

8 µL

Ligation Mastermix.

3

T2 dig (2)

12 µL

Pveg (3)

12 µL

Mastermix

6 µL

4

T2 dig (2)

12 µL

Psalt (4)

12 µL

Mastermix

6 µL

Components for ligation samples.

The samples were ligated overnight at room temperature.

Harm Ruesink

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

With the ligation product of 28 July, the heat shock transformation was performed according to the protocol book and the culture was plated on an LB+cm plate and incubated overnight at 37 °C.

Harm Ruesink

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

The transformation of 29 July was checked. No colonies were visible on the LB+cm plate. T2 was send to Macrogen for sequencing.

1/20 primer (5 pmol/µL)

5 µL

plasmid DNA (T2)

5 µL

Amount of primers and DNA sent for sequencing.

prefix primer (forward)

17C4ZAD658

suffix primer (reverse)

17C4ZAD659

Sequencing codes Macrogen.

Harm Ruesink

13:22, 6 August 2015 - 18:40, 6 August 2015

PliaG was digested and ligated with T2. Restriction enzymes were used that create a scar. For the digestion the restriction enzymes EcoRI and SpeI were used for the promoter and XbaI and EcoRI for T2. Below are listed the amounts that were added of each component.

1

PliaG DNA

5 µL

10x buffer

2 µL

EcoRI

1 µL

SpeI

1 µL

\( \mathrm{H_2O}\)

11 µL

2

T2 DNA

5 µL

10x buffer

2 µL

XbaI

1 µL

EcoRI

1 µL

\( \mathrm{H_2O}\)

11 µL

Components for digestion.

The sample was digested for 2 hours at 37 °C. Afterwards it was loaded on 1% agarose gel with DNA stain G 1:50000. On the gel were loaded 20 µL of the sample DNA with 4 µL buffer. As a ladder 10 µL of the 2log NEB ladder was used. The gel was run for ∼30 minutes at 100 V. The right bands were cut from the gel and twice as much binding buffer was added. The samples were purificated using the Thermo Scientific GeneJET PCR Purification Kit and their concentrations were checked using Nanodrop.

PliaG dig

4.6 ng/µL

T2 dig

4.9 ng/µL

Concentrations of samples determined with Nanodrop.

The ligation was carried out overnight at room temperature using the following components.

PliaG DNA

5 µL

T2 DNA

10 µL

10x T4 buffer

2 µL

T4 ligase

0.5 µL

\( \mathrm{H_2O}\)

2.5 µL

Components for ligation of PliaG with T2.

Harm Ruesink

11:30, 7 August 2015 - 13:30, 7 August 2015

A heat shock transformation was performed by adding 20 µL of DNA (ligation product of T2 with PliaG) to 10 µL competent NEB cells. After 2 hours the culture was plated on LB+cm and incubated overnight at 37 °C.

Harm Ruesink

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

The transformation of 7 August was checked. No colonies were visible.

Harm Ruesink

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

The transformation of 7 August was checked. Some white colonies were visible. With these colonies a colony PCR was carried out. After the PCR the samples were loaded on a 1% agarose gel and it was seen that sample 3 and 7 possibly have the insert. Cultures were made of sample 3 and 7 by putting some of the bacteria in a tube with 3 mL LB+cm. The tubes were put in the shaking incubator at 37 °C with 200 rpm.

Harm Ruesink

12:00, 11 August 2015 - 15:30, 11 August 2015

The cultures of sample 3 and 7 were miniprepped and loaded on 1% agarose gel with DNA stain G 1:50000. For each DNA sample 1 µL was added to the gel, with 4 µL \( \mathrm{H_2O}\) and 1 µL 6x buffer. As a ladder 10 µL of the Quick-Load® Purple 2-Log DNA Ladder (0.1 - 10.0 kb) was used. The gel was run for ∼30 minutes at 100 V. On the gel afterwards nothing was visible, so the DNA concentration needed to be checked again.

Harm Ruesink

10:30, 13 August 2015 - 20:15, 13 August 2015

The concentrations of the samples 3 and 7 were checked again and were found to be the following.

3

3.3 ng/µL

7

9.3 ng/µL

Concentrations of samples determined with Nanodrop.

The samples were loaded on 1% agarose gel + DNA stain G 1:50000.

3

Sample DNA

10 µL

6x buffer

2 µL

7

Sample DNA

3 µL

6x buffer

1 µL

\( \mathrm{H_2O}\)

2 µL

T2

Sample DNA

1 µL

6x buffer

1 µL

\( \mathrm{H_2O}\)

4 µL

Samples on gel.

The gel was run for 30 minutes at 100 V. On the gel no bands were visible. So the digestion was carried out again. For the promoter PliaG, the restriction enzymes SpeI and PstI were used, and for T2, XbaI and PstI. The following digestion samples were prepared.

1

PliaG DNA

10 µL

10x buffer (2.1)

2 µL

SpeI

1 µL

PstI

1 µL

\( \mathrm{H_2O}\)

6 µL

2

T2 DNA

10 µL

10x buffer (2.1)

2 µL

XbaI

1 µL

PstI

1 µL

\( \mathrm{H_2O}\)

6 µL

Components for digestion.

The samples were incubated at 37 °C for 3.5 hours. Afterwards they were loaded on a 1% agarose gel with DNA stain G 1:50000. For the DNA samples 20 µL was used with 4 µL 6x buffer. As a ladder 1 µL of the GeneRuler™ 1 kb DNA Ladder was used. The gel was run for ∼45 minutes at 100 V. The bands with the right sizes were cut from the gel and dissolved in twice as much elution buffer. The GeneJet PCR purification kit was used to extract the DNA, using 21µL elution buffer. With Nanodrop the concentrations were determined.

1

4.0 ng/µL

2

5.1 ng/µL

Concentrations of samples determined with Nanodrop.

The samples were ligated overnight at room temperature.

DNA sample 1

20 µL

DNA sample 2

20 µL

10x T4 buffer

5 µL

T4 ligase

1 µL

Components for ligation of PliaG with T2.

Harm Ruesink

00:00, 17 August 2015 - 00:00, 17 August 2015

A colony PCR with 6 colonies of the T2+PliaG plate was done. Each colony was taken from the plate and resuspended in 20 µL sterile MQ water. A Mastermix for 16 samples was made.

Phusion polymerase

4.5 µL

5x phusion buffer

80 µL

DNTP MM (2 mM)

35 µL

\( \mathrm{H_2O}\)

240 µL

VR + VF2 primers

1 µL each

Components Mastermix.

For the PCR samples, 18 µL of Mastermix was added to 4 µL of colony suspension. The following PCR thermocycle was used.

1

Initial denaturation

99 °C

10:00

2

Denaturation

95 °C

0:30

3

Annealing

58 °C

0:30

4

Extension

72 °C

1:30

5

Go back to step 2 (repeat 30x)

6

Final extension

72 °C

10:00

PCR thermocycle.

The samples were run on a 1% agarose gel with DNA stain G 1:50000. 5 µL of sample DNA was mixed with 1 µL 6x buffer. As a ladder 1 µL of the GeneRuler™ 1 kb DNA Ladder was used. The gel was run for ∼30 minutes at 100 V. No correct PCR product was visible on the gel. All bands are at the same height as the control (T2 without promoter).

Harm Ruesink

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

A new colony PCR was performed with 8 colonies from the T2+PliaG plate and control (T2). Each colony was resuspended in 20 µL MQ water. A Mastermix was made for 9 samples.

Phusion polymerase

2 µL

5x phusion buffer

36 µL

DNTP MM (2 mM)

18 µL

\( \mathrm{H_2O}\)

120 µL

Primers (pSB1C3 colony PCR)

3 µL each

Components Mastermix.

For the PCR samples, 18 µL of Mastermix was added to 4 µL of colony suspension. The following PCR thermocycle was used.

1

Initial denaturation

98 °C

10:00

2

Denaturation

95 °C

0:30

3

Annealing

55 °C

0:30

4

Extension

72 °C

1:30

5

Go back to step 2 (repeat 30x)

6

Final extension

72 °C

10:00

PCR thermocycle.

The samples were loaded on a 1% agarose gel with DNA stain G 1:50000. On the gel it was seen that the insert did not have the correct size, meaning that the promoter (PliaG) did not ligate in T2.

Harm Ruesink