Team:Marburg:Project:Notebook:June

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Notebook: June

02.06.2014

13.56 Making glycerine stocks of the following strains

Aim: Storage of strains

  • Dh5α piGEM010 (p003 + Tag 21)
  • Dh5α piGEM015 (p004 + Tag 23)
  • Dh5α piGEM014 (p003 + Tag 22)
  • Dh5α piGEM008 (p002 + Tag 22)
  • Dh5α piGEM007 (p002 + Tag 21)
  • Dh5α piGEM011 (p003 + Tag 22)
  • Dh5α piGEM012 (p003 + Tag 23)
13.57 Mini Prep of cultures inoculated on Friday 30.05.14 containing the nose plasmids
  • piGEM010 = 22ng/µl
  • piGEM013 = 46 ng/µl
  • piGEM 009 = 13 ng/µl

low concentration: new transformation to obtain more plasmid DNA
Strain: new competent E. coli XL1-blue

15.50 Miniprep of pMAD-fla-cup1-1 for miniprep

The miniprep was performed according to the miniprep protocol.

Concentration 40 ng/µL
low concentration: new transformation to gain more plasmid DNA should be done
Strain: new competent E. coli XL1-blue

13.58 Bacillus Trafo with all Nose Plasmids containing one/no Degradation Tag

Aim: Create Bacillus subtilis strain 3610 containing the following plasmids: piGEM002 to 004 and piGEM007 to 016

Add 5µl plasmid DNA and incubate for 30 min at 37°C, LB plate with 5 ng/µL Cm (prepared freshly)

13.59 Inoculation of Bacillus subtilis wildtype (3610), gfp and silver nose

Aim: perform MTA to test the Ag concentrations where cells are still viable, since 1 mM until 10 µM were too high and the cells died

Starting from 1 mM, eight serial dilutions have been prepared

14.38 Purification of PheA-Arc1p-C-1x

Aim: Purify PheA-Arc1p-C-1x in three steps

The resuspended cells from 14.37 were lysed using a french press. The lysate was incubated on ice with 5 µg/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C) to pellet cell debris. The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-1x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-1x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.

18.1 Tumor Terminator - Dilution of Plasmid

Aim: Creating a stock solution

We received the synthetized DARPin-gene in a pEC-A2 vector with SpeI restriction sites at the 5' - and 3' -site with an amount of 2,7 µg plasmid per tube.

A stock solution was created by adding 27 µl water to the tube.

Concentration of stock solution = 100 ng/µl

A 1:10 dilution was created by using 1 µl of stock solution adding 9µL of water to an end concentration of 10ng/µl.

Concentration of stock dilution = 10 ng/µl.

18.2 Transformation of E.coli XL1-Blue with pEX-A2-DARPin

Aim: Transformation of E.coli XL1-Blue with pEX-A2-DARPin for amplification of the plasmid.

1µl of the 1:10 dilution was used for transformation of E. Coli XL1-Blue

03.06.2014

18.3 Miniprep of pEX-A2-DARPin from E.coli XL1-Blue

Aim: Receiving a higher amount of plasmid for further experiments.

3 x 6ml LB-Amp were inoculated with clones from the successful transformed plate (18.2) and incubated at 37°C overnight.

13.60 Microtiter plate assay with wildtype, gfp strain 186 and silver nose

Aim: perform MTA to test the Ag concentrations where cells are still viable, since 1mM until 10 µM was too high and the cells died

Starting from 1 mM 8 dilutions have been prepared.
In the beginning Minimalmedium S750 has been prepared with 0.5‰ Xylose
In the wells the following mix was added to a total volume of 150 µl:
135µl of MM (90‰) and Silver solution (10‰)
15µl of cells
Finally 70 µl are added to avoid evaporation of the medium

Incubation for appr. 24 hours at 37°C shaking (Viktor AG Waldminghaus)


No positive GFP signal could be detected for the B. subtilis strain with the possible Ag sensitive promoter. The strain was restreaked onto a LB-Cm plate and he did not grow, which proved it to be a wrong strain, which has not been transformed successfully.

13.61 Checking Bacillus Trafo with all Nose Plasmids containing one/no Degradation Tag

Every transformation plate showed a huge growth of bacillus without single colonies. After one day of incubation this seems to be an unrealistic growth, which is why we planned to repeat the transformation after checking the growth of the Bacillus subtilis wildtype 3610 on LB-CM plate with 5 µg/ml chloramphenicol.

13.62 Checking Bacillus Trafo Wildtype 3610

Aim: check if wildtype has a resistance against chloramphenicol or if the plates are faulty

Bacillus subtilis Wildtype 3610 was plated on LB-chloramphenicol plate with 5 µg/ml chloramphenicol and incubated overnight at 37°C.

15.51 Making selection plates for clean deletions - integration of pMAD-construct into Bacillus subtilis chromosome

Aim: For the integration of the constructs we cloned into pMAD many steps of a special selection are necessary.

Antibiotics and chemicals:

  • Erythromycin 4 mg/ml
  • Lincomycin 25mg/ml
  • X-Gal 100mg/ml

For MLS selection plates erythromycin with 1 mg/ml were needed. Therefore the 4 mg/ml stock solution was diluted 1:4 by mixing 400 µl eryhtromycin with 1200 µl of 70% ethanol.

3 types of plates were made:

  • 1. MLS selectin: 800 mL LB agar + 800 µl eryhtromycin + 800 µl lincomycin
  • 2. MLS X-Gal: 800 mL LB agar + 800 µl eryhtromycin + 800 µl lincomycin + 800 µl X-Gal
  • 3. X-Gal: 800 mL LB agar + 800 µL X-Gal

04.06.2014

18.4 Miniprep of pEX-A2-DARPin from E.coli XL1-Blue
Plasmid pEX-A2 + DARPinEc1 Concentration after prep (ng/µl)
Clone 1 398,7
Clone 2 340
Clone 3 309
15.52 Sequencing of pMAD-fla-cup1-1

Aim: Checking the correct insertion of the metallothionein-domain into the hag-flank construct

Making a pre-mix for Eurofins MWG prepairing the sequencing. Primers from Florian Altegoer were used:

  • Flo83- Hag11_fw 1:10 with 1µl primer and 9µl water
  • Flo86- Hag12_rv 1:10 with 1µl primer and 9µl water
  Pre-mix 1 (µL) Pre-mix 2 (µL)
piGEM-016 pMAD-cup1-1
40ng/µL
10 10
Flo83- Hag11_fw 2 -
Flo86- Hag12_rv - 2
Total Volume 12 12

Label Pre-mix 1 Primer
AGB0023375 Pre-mix 1 Flo83 – Hag11_fw
AGB0023374 Pre-mix 1 Flo86 - Hag12_rv
13.62 Checking Bacillus Trafo Wildtype 3610

Aim: check if wildtype has a resistance against chloramphenicol or if the plates are not effective/ made wrong

Result: Bacillus subtilis Wildtype 3610 grew on the chloramphenicol plate which shows us that there has to be a mistake in making the LB-chloramphenicol plates or the WT has a resistance.

New plates were made with new chloramphenicol-stocks with 25 mg/ml and 5 mg/ml.

3 aliquots of competent B.s. WT3610 were plated on new LB-chloramphenicol plates and incubated at 37°C.

18.5 SpeI restriction pEX-A2-DARPin & pMAD-hag-flank (piGEM-005)

Aim: Receiving iGEM-005 as linearized vector for cloning the DARPin gene with SpeI sites into pMAD-fla (iGEM-005)

Restriction pEX-A2-DARPin digest (µl) pMAD-Hag-Flank digest (µl) Spe1-Mastermix
pEX-A2+DARPin (398 µg/µL) 15 = ca. 6µg - -
pMAD-Fla (114 µg/µL) - 10= ca. 1,14 µg -
SpeI - - 1,5
CutSmart-Buffer (10x) - - 2
H2O - - 16,5
Mastermix 5 10 -
Total 20 20 20

Incubation at 37°C for 2h and heat shock at 81°C for 5 min. After that the pMAD-hag-flank-construct was purified with the OMEGA gel extraction kit according to the protocol.

c(digested piGEM-005)= 4 ng/µl

13.62 Checking Bacillus Trafo Wildtype 3610

Aim: check if wildtype has a resistance against chloramphenicol or if the plates are not effective/ made wrong

Result: Bacillus subtilis Wildtype 3610 grew on the chloramphenicol plate. We have already repeated making plates so it might be that the WT has got a resistance while making it competent. A new WT was plated on 2 chloramphenicol plates with 5 µg/µL (old and new one) and 2x LB plates from gly stocks.

Incubation at 37°C overnight.

14.39 Creating PheA-Arc1p-C-0x

Aim: Using Round-the-horn mutagenesis to create PheA-Arc1p-C-0x-pET28a

The PCR with 5' phosphorylated TG_0xGSSG FP und TG_0xGSSG RP should generate the 7491 bp fragment for ligation from the methylated template PheA-Arc1p-C-8x-pET28a from 14.5.

The used Primer concentration was 1 µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.

Content Volume [µL]
Water 29.5
5x GC Buffer 10
DMSO 2.5
TG_0xGSSG FP 2.5
TG_0xGSSG RP 2.5
PheA-Arc1p-C-8x-pET28a 1
dNTPs 1
Phusion-Polymerase 1
Total Volume 50

Step Temperature [°C] Time [min:sec]
1 98 2:00
2 add Polymerase  
3 98 0:10
4 67.5 0:30
5 72 4:00
6 go to 3 32x
7 72 10:00
8 4 hold

The amplified fragment was purified on an agarose gel on which it showed the correct size. The extracted linear template was then digested to degrade the original unmutated plasmid using DpnI.

Content Volume [µL]
10x CutSmart Buffer 3.6
Plasmid 30
DpnI 3
Total Volume 36.6

The reaction mixture was incubated at 37 °C and 300 rpm for 1 h and afterwards heated to 80 °C for 20 min.

In order to create the circular plasmid 100 ng of the linear template were mixed with 1 µL 10x T4-Ligasebuffer, 1 µL T4-Ligase and water to give a total volume of 10 µL. This mixture was incubated for 9 h at 16 °C. The resulting plasmid was dialysed with water to decrease the salt concentration for the following transformation of E. coli Top10 electrocompetent cells with the plasmid.

06.06.2014

19. Crystallization of flagellin

In order to crystallize flagellin with a domain it is necessary to produce the protein by E. coli in a high concentration. For that aim the Fla-construct will be amplified out of pET24d which includes a domain. For elimination of the flanks a PCR has to be performed which just amplifies hag with domain and creates NcoI and BamHI restriction sites. After purification and digest with Nco/Bam the fragment can be ligated into digested pET24d . After transformation the production can be induced by IPTG.

19.1 Elimination of flanks from fla-construct including cup1-1 from pET24d

Aim: Preparation for using pET24d as expression vector for flagellin

The flagellin construct was amplified with specifically designed primers (95 and 54 from Florian Altegoer), which allow the amplification of the construct with cup1-1 without flanks (just Hag) including NcoI & BamHI restriction sites.
Template dilution 1:10:
1 µL pET24d-cup1-1 was mixed with 9 µL to an 1:10 dilution of 16,3 ng/µL
Primer dilution 1:10:
The primers were diluted 1:10 by mixing 10 µL of primers with 90 µL millipore water each.

Mix [µl] reaction with phusion reaction with Q5 mastermix
pET24d-cup1-1
1:10 dilution
1 1
Primer 54Flo 1 1
Primer 56Flo 1 1
Phusion 1 -
Phusion Buffer 5x 10 -
dNTP Mix 1 -
Water 35 22
Q5 Mastermix - 25
Total Volume 50 20

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 2 min
5 Go To 2 30x
6 72 4min
7 8 infinite

The PCR was run over night.

19.2 Restriction digest of pET24d with NcoI and BamHI

Aim: Creating Nco/Bam restriction sites in pET24d for cloning PCR fragments amplified with primers flo56 and flo54 which contain these sites as well.

Isolated pET24d was cut with NcoI and BamHI in order to create restriction sites for ligating the Flagellin/ Hag-Domain fragments into the vector. The Hag-fragments which were amplified via PCR with primers flo54 and 56 contain NcoI/ BamHI restriction sites for that purpose as well.

Component Volume [µl]
pET24d (148 ng/µL) 7
CutSmart 10x 2
NcoI 0,5
BamHI 0,5
Water 10
Total Volume 20

Incubation for 1 hour at 37°C and heat shocked at 80°C.

15.52 Miniprep of different plasmids

Aim: Receiving more plasmid DNA for further experiments

Colonies were picked from plates and 8ml LB with necessary antibiotics each inoculated, followed by incubation at 37°C overnight.

Clones were picked from plates with plasmids:

  • pET24d-Fla-Cup1-1 (piGEM-006)
  • pMAD-Hag-Flank (piGEM-005)
  • pMAD-Hag-Flank -Cup1-1 (piGEM-016)
  • pEX-A2+DARPin (piGEM-017)

07.062014

15.53 Sequencing pMAD-fla-cup1-1

The sequencing showed the correct insertion of the cup-1-1 gene into the hag-flank construct but contained a stop codon, which was not removed by the used reverse primer. Unfortunately we used primers for amplification of the whole cup1-1 gene and not just for the truncated gene coding for the metallothionein domain only. Therefore new primers had to be designed an the Gibson assembly for integration of the metallothionein domain into pMAD and pET24d has to be repeated.

18.6 gel extraction & purification of DARPin gene

Aim: Receiving DARPin gene with SpeI sites for ligation into linearized piGEM-005

The with SpeI digested pEX-A2 was purified via electrophoresis with an 1% agarose gel in order to divide pEX-A2 backbone and the DARPin gene (489 bp). The digest attempt was splitted and loaded into two pockets to be sure not to overload the gel. The correct bands were cut out and the DNA fragment was rescued according to the protocol of our OMEGA gel extraction kit.

Expected bands:
DARPin gene: 489 bp
pEX-A2 backbone: 1961bp
The concentration after gel extraction: 9 ng/µl

18.7 Ligation of DARPin gene and linearized piGEM-005

Aim: Ligation of DARPin- (from 18.6) into Spe1 digested piGEM-005 from 18.5 for integration of the DARPin-Flagellin construct into Bacillus subtilis chromosome

The DARPin gene with SpeI sites was used as insert for the ligation into linearized pMAD (from 18.5) which contains the designed restriction site. The ligation attempt was done twofold and calculated by the Ligation Calculator from the iGEM-Team Texas in 2011:

Component Volume [µl]
Insert (DARPin, c= 9 ng/µL) 2
Vector (piGEM-005, c=4ng/µL) 10
Ligase-Buffer 10x 2
T4 Ligase 1
Milipore water 5
Total Volume 20

Incubation was performed overnight at room temperature.

13.63 checking Bacillus WT 3610 growth on 5µg/µL CM

Aim: check if wildtype has a resistance against chloramphenicol (CM)

Result: Bacillus subtilis Wildtype 3610 grew on the CM plate.

In order to make out if a selection is possible WT3610 was plated out from an LB plate (13.62)  on 25 ng/ µL CM to check were the threshold of CM for B. subtilis is.

Incubation at 30°C over two days.

08.06.2014

18.8 new SpeI restriction of DARPin-gene in pEX-A2 & pMAD-hag-flank (piGEM-005)

Aim: Receiving new insert and vector in case of an unsuccessful ligation for further attempts.

Restriction pEX-A2-DARPin digest (µl) pMAD-Hag-Flank digest (µl) SpeI-Mastermix
pEX-A2+DARPin (398 µg/µL) 10 = ca. 4µg - -
pMAD-Fla (150 µg/µL) - 7= ca. 1 µg -
SpeI - - 1
CutSmart-Buffer (10x) - - 2,5
Water - - 21,5
Mastermix 10 13 -
Total Volume 20 20 25

Incubation was performed for 2 h at 37°C and heat inactivated at 80°C for 5 minutes.

18.9 Transformation of ligation from 18.7

The whole 20µL attempt from the ligations were used to transform E.Coli XL1-Blue, which were plated on LB-Amp plates.  Additionally a negative control was created by transforming E.Coli XL1-Blue with 1 µL SpeI cut piGEM-005 as well.

18.10 Gel extraction for isolation of DARPin gene out of pEX-A2

Aim: Receiving new insert in case of an unsuccessful ligation for further attempts like in 18.6

The SpeI digested pEX-A2 was purified via electrophoresis with an 1% agarose gel like in 18.6. The correct bands were cut out and the DNA fragment was purified according to the protocol of our OMEGA gel extraction kit.

Expected bands:
DARPin gene: 489 bp
pEX-A2 backbone: 1961bp
The concentration after gel extraction: 10 ng/µl

09.06.2014

18.12 Checking transformation of overnight ligation from 18.7
Plate Amount of colonies
Negative control 6
Ligation attempt 1 7
Ligation attempt 2 0
18.13 Checking success of ligation via cPCR

Aim: A cPCR with the primers 54 and 56 shows the success of ligation by amplification of the Hag-construct.

In order to check the positive insertion of the DARPin gene into pMAD a PCR amplification with primers 54 and 56 for the flagellin should generate an ca. 1500 bp fragment on the gel. Negative clones would just show a 1000 bp band because of the amplification of the Hag-SpeI site construct. The picked clones were transferred on a new LB-amp plate.

Mix Master-Mix for 7 attempts (µL) Mix for PCR attempt (µL)
Colony - part of a colony
Primer Flo 54 3,5 -
Primer Flo 56 3,5 -
Phusion 7 -
Phusion Buffer 5x 28 -
dNTP Mix 3,5 -
Water 94,5 -
Mastermix - 20
Total Volume 140 20

Expected bands:
Hag-DARPin-construct: ca. 1500 bp

The gel shows bands at 1000 which means that the clones contain just relegated pMAD vector and are all negative. The ligation had to be repeated.

18.14 Dephosphorylation of linearized piGEM-005

Aim: Dephosphorylation of 5'-end with antarctic phosphatase in order to decrease the religation of the linearized piGEM-005 from 18.8

The cut SpeI cut piGEM-005 was dephosporylated at the 5'-end with antarctic phosphatase to prevent the vector from relegation.

Component Volume [µl]
Vector (piGEM-005, c=148 ng/µL) 20 (=ca. 2 µg)
Antarctic phos.-Buffer 10x 3
Antarctic phosphatase 1
Milipore Water 6
Total Volume 30

The sample was incubated for 1 h at 37°C at heat inactivated at 70°C for 10 minutes.

18. new ligation of linearized/ dephosphorylized piGEM-005 & DARPin gene

Aim: Dephosphorylation piGEM-005 was ligated with extracted DARPin gene again

Because of the negative colony PCR from 18.13 a new ligation with dephosphorylized piGEM-005 from 18.14 and new extracted DARPin gene from 18.10 was performed in 2 attempts.

Component Volume [µl]
Insert (DARPin, c= 10 ng/µL) 12,4
Vector (piGEM-005, c= 148 ng/µL) 4,6
Ligase-Buffer 10x 2
T4 Ligase 1
Milipore Water 5
Total Volume 20

Incubation 3h at room temperature

18.16 Transformation of overnight ligation from 18.15

The whole 20 µL reaction of the ligations (18.15) were used to transform E. coli XL1-Blue and plated on LB-Amp plates.  Additionally a negative control was created by transforming E.Coli XL1-Blue with 1µL SpeI digested piGEM-005 as well.

13.64 checking Bacillus WT 3610 growth on 5, 10 & 15 µg/µL CM

Aim: check if wildtype has a resistance against CM

Result: Bacillus subtilis Wildtype 3610 did not grow on the 25 ng/µl CM plate

In order to check at which concentration CM Bacillus cannot survive he was plated out from an LB plate on LB-CM plates with different CM concentrations (5, 10, 15 ng/mL).

Incubation at 30°C over night.

10.06.2014

18.17 Checking Transformation of ligation from 18.16
Plate Amount of colonies
Negative control 1
Ligation attempt 1 2
Ligation attempt 2 >80
18.18 Checking success of ligation via cPCR

Aim: A cPCR with the primers 54 and 56 shows the success of ligation by amplification of the Hag-construct.

In order to check the positive insertion of the DARPin gene into pMAD a PCR amplification with primers 54 and 56 for the flagellin should generate an ca. 1500 bp fragment on the gel. Negative clones would show still just a 1000 bp band because of the amplification of the Hag-Spe1 site construct. The picked clones were transferred on a new LB-amp plate. 12 clones were picked.

Mix Master-Mix for 14 attempts (µL) Mix for PCR attempt (µL)
Colony - part of a colony
Primer Flo 54 7 -
Primer Flo 56 7 -
Phusion 14 -
Phusion Buffer 5x 56 -
dNTP Mix 7 -
Water 189 -
Mastermix - 20
Total Volume 280 20

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 1min 45sec
4 72 1 min
5 Go To 2 30x
6 72 4min
7 8 infinite

Expected bands:
Hag-DARPin-construct: ca. 1500 bp

Clone 1, 2, 3, 4, 5, 7, 9, 11 and 12 were the positive clones at 1500 bp.

14.40 Overnight culture

Aim: Amplify Plasmid in an overnight culture

Six clones of the transformed E. coli Top10 cells from 14.39 were picked and used to inoculate overnight cultures (6 x 5 mL).

18.19 Miniprep of positive clones

Aim: In order to check the orientation of the insert in the vector of the positive clones minipreps were inoculated for further experiments

Because of the ligation via two SpeI sites, the chance is 50:50 that the clones contain the pMAD-Hag-Flank-DARPin construct in the right orientation (Startcodon on the Hag1-site). For test digests a higher amount of plasmid is needed, which is achieved by inoculation of minipreps.

The clones were used to inoculate 7 mL LB-Amp and incubated at 37°C over night.

13.64 checking Bacillus subtilis WT 3610 growth on 25 & 35 µg/µL CM

Aim: check if wildtype has a resistance against CM

Result: Bacillus subtilis Wildtype 3610 grew on the 5, 10 & 15µg/µl LB-CM plate.

We saw that the Bacillus grew on almost every CM concentrations except 25 µg/µL. So we transformed competent WT3610 with Nose plasmid piGEM-003 as a positive control and plated them out on LB-CM with 25 µg/µL and 35 µg/µL.

Additionally, competent wildtype 3610 cells were transformed with piGEM-004 and plated out on LB-CM with 5, 10 and 15 µg/mL CM to check, if these cells can be selected better.

Incubation at 30°C for 2 days.

11.06.2014

18.19 Miniprep of positive clones

The concentration of the isolated plasmids was between 100 and 150 ng/µL.

18.20 Test digest with EcoRV

Aim: Checking the right orientation of the insert in the isolated plasmids

The plasmids from the positive clones were digested with EcoRV so that specific fragments emerge when cutting into the DARPin gene and the vector backbone.

Component Volume [µl] Mastermix for 10 attempts [µl]
Plasmid of clone 2 (ca. 200ng) -
Cutsmart 10x - 8
EcoRV - 1
Millipore water - 71
Mastermix 8 -
Total Volume 10 80

Right orientation:    7900 – 1570 – 2500 – 55
Wrong orientation: 7900 – 1170 – 2500 – 460

Clone 1, 2, 3, 4, 7, 9 and 11 have the right orientation.

20.1 inoculation of preculture from Bacillus subtilis WT 3610

Aim: Preparation of for mainculture the next day

2 x 6 mL of LB were inoculated with Bacillus subtilis WT3610 from an LB plate and incubated at 37°C overnight.

14.41 Preparation of plasmids

Aim: Purify the amplified plasmids from overnight cultures

The plasmids were purified from the overnight cultures created in 14.40 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.

12.06.2014

18.21 PCR with pMAD-Hag-Flank-DARPin (piGEM-019) and piGEM-005

Aim: Amplification of Hag-SpeI-construct and Hag-DARPin construct with NcoI/BamHI sites for cloning into pET24d → crystallization of domains.

The templates (piGEM-005, prepped pasmid from clone 7 & 12 ) were diluted 1:10. piGEM-005 should function as a negative control. Clone 12 was picked in case the second orientation could be used for different experiments. 5 µL PCR product + 1 µL loading dye were analysed on an 1% agarose gel.

Mix Volume
Template 1:10 1
Primer Flo 54 1
Primer Flo 56 1
Phusion DNA-polymerase 1
Phusion Buffer 5x 10
dNTP Mix 1
Water 35
Total Volume 50

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 1min 45sec
4 72 2 min
5 Go To 2 30x
6 72 4min
7 8 infinite

Expected bands:
 clone 7 & 12 PCR product at ca. 1500bp – piGEM-005 PCR product at 1kb

14.42 Transformation of BL21 cells

Aim: Transform E. coli BL21(DE3) with PheA-Arc1p-C-0x-pET28a

40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-Arc1p-C-0x-pET28a from two different clones, incubated for 1 h at 37 °C and plated out on LB-Kan50 plates that were incubated overnight at 37 °C.

18.22 restriction digest of Hag PCR fragments from 18.21 with NcoI/BamHI

Aim: creating restriction sites for cloning into pET24d cut with NcoI/BamHI

The PCR products from 18.21 were digested with NcoI and BamHI to create restriction sites in order to ligate the fragments into NcoI/BamHI cut pET24d.

Component Volume [µl]
PCR products 45
Cutsmart 10x 5,2
NcoI 1
BamHI 1
Water 2,8
Total Volume 52

 Incubation for 2 hours at 37°C with heat shock at 80°C.

18.23 Ligation of pET24d and Hag PCR fragments from 18.22 cut with NcoI/BamHI

Aim: cloning inserts into pET24d for crystallization

The NcoI/BamHI digested flagellin PCR products from 18.22 (from clone 7 and piGEM-005) were ligated with NcoI/BamHI digested pET24d.

Component Pet-Hag attempt (µL) Pet-Hag-DARPin attempt (µL)
Insert (PCR fragments , c= ca. 400 ng/µL) 2 2
Vector (pet24d, c= 50ng/µL) 5 5
Ligase-Buffer 10x 2 2
T4 Ligase 1 1
Millipore Water 10 10
Total Volume 20 20

Incubation over night at room temperature.

20.2 Transformation of competent Bacillus subtilis

Aim: transformation of Bacillus subtilis WT3610 with piGEM005 and piGEM018

100 µL of overnight culture were added to 10 mL of MNGE-Medium and incubated to an OD600 of 1,1-1,3 at 37°C which could take 4-5 hours.

After reaching an OD of 1,1-1,3 2 x 400 µL of the culture were transformed with 1,5 µg piGEM-005 and -018. After one hour incubation at 37°C 100 µL expression mix were added and incubated for one hour as well.

In the end the 500 µL sample was plated out on MLS-X-Gal plates and incubated at 30 °C until colonies could be seen.

13.06.2014

18.24 Transformation of ligation

Aim: checking success of ligation and amplification of ligated construct

The whole 20 µL attempt was used to transform E. coli X1-Blue, plated out on LB-Can plates and incubated at 37°C overnight.

18.25 Miniprep of pMAD-Hag-Flank-DARPin (piGEM-018)

Aim: increase amount of plasmid for work and storage

2 x 6 mL of LB-Amp were inoculated with a colony from a piGEM-018 transformation plate and incubated at 37°C overnight.

18.26 Sequencing of constructs

Aim: checking the right insertion into the plasmids

pMAD-Hag-Flank-DARPin was sent in form of a premix with primers for sequencing. 10 µL with 50-100 ng/µL were mixed with 2 µL of primers for sequencing according to the following scheme:

Premix Label-Nr. Construct Primer
1 391 pMAD-Hag-Flank-DARPin Flo54 (Hag-F-Nco)
2 392 pMAD-Hag-Flank-DARPin Flo56 (Hag-R-Bam)
13.65 Checking growth of Bacillus subtilis WT3610

Aim: check which WT can be used for selection with Nose plasmids

Competent 3610 were transformed with piGEM-002, -003 and -004 by incubating the aliquots with 5 µL plasmid for 30 min at 37°C. After half an hour the whole sample was plated out on LB-Cm (5 ng/µL).

Incubation at 30°C for 2 days.

14.06.2014

18.27 miniprep of plasmids from 18.25

Aim: isolation of plasmids

The miniprep of pMAD-Hag-Flank-DARPin was done according to the protocol of the OMEGA DNA kit.

18.28 pET24d digest with Nco/Bam

Aim: preparing vector for new ligation attempts

Because of the unsuccessful ligation from 18.23 new vector (pET24d) was digested with NcoI and BamHI according to the following scheme:

Component Volume [µl]
pET24d (148 ng/µL) 15
Cutsmart 10x 3
NcoI 0,5
BamHI 0,5
Water 6
Total Volume 30

Incubation at 37°C for 2h.

18.29 PstI digestion of PCR fragments from piGEM-018 with Hag-primers

Aim: check if the correct inserts are used for ligation

The DARPin-Hag constructs contain a PstI restriction site which could be used to check if the DARPin gene was inserted into the Hag-construct correctly again. For that purpose PCR products based on piGEM-018 from clone 7 and 12 (right and wrong orientation) as a template were digested with PstI.

PCR products from clone 7: 658 bp and 781 bp fragment.

 PCR products from clone 12: 1000bp and 429 bp fragments.

Component Volume [µl]
PCR fragment (350 ng/µL) 3
Cutsmart 10x 1
PstI 0,5
Water 5,5
Total Volume 10

Incubation at 37°C for 2h.

18.30 PCR amplification of DARPin-Hag and Hag fragment

Aim: amplification of DARPin-Hag fragments for repeating the ligation

piGEM-018 & -005 were used as template for a PCR with the primers 54flo and 56flo. The products could be used for further cloning after NcoI/BamHI digest.

Mix Volume
Template (piGEM-005 & -018) 1
Primer Flo 54 1
Primer Flo 56 1
Phusion 1
Phusion Buffer 5x 10
dNTP Mix 1
Water 35
Total Volume 50

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 1min 45sec
4 72 2 min
5 Go To 2 30x
6 72 4min
7 8 infinite

The fragments were analysed on an 1% agarose gel.

15.54 new PCR amplification of cup1-1

Aim: amplification of cup1-1 domain for Gibson assembly with pMAD

The cup1-1 domain was amplified via PCR with new primers iGEM-024 and -025. The fragment should be ca. 160 bp.

Mix Volume
Template (cup1-1 gene) 1
Primer iGEM-024 1
Primer iGEM-025 1
Phusion DNA-polymerase 1
Phusion Buffer 5x 10
dNTP Mix 1
Water 35
Total Volume 50

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 30 sec
5 Go To 2 30x
6 72 4min
7 8 infinite


According to the gel analysis the amplification was successful.

15.06.2014

20.3 Overnight culture of positive clones

Aim: transformation of Bacillus subtilis WT3610 with plasmid

Colonies grew on the transformand plates (piGEM-005 & - 018). The blue/ white screening showed positive transformed blue clones.  Three clones per plate were picked and used for inoculation of LB-MLS (4 mL LB, 4 µL lincomycin, 4 µL erythromycin). Incubation was carried out over night at 30°C with the six cultures.

16.06.2014

14.43 Overnight culture

Aim: Inoculate Overnight culture of PheA-Arc1p-C-0x-pET28a

5 mL of LB-Kan50 medium were inoculated with a clone from 14.42 bearing the PheA-Arc1p-C-0x-pET28a plasmid.

17.06.2014

20.3 First temperature shift

Aim: integration of pMAD-Insert into Bacillus chromosome via flanks

The 6 overnight cultures were used to inoculate 10 mL LB MLS, which were incubated until the culture obtained an OD of 0,1. The cultures were incubated at 30°C for 2h.

Subsequently, the temperature was shifted to 42°C for 6h.

After the heat shock dilutions from 10-4 to 10-6 of each culture were plated out on MLS-X-Gal so that 18 plates could be incubated overnight at 42°C.

15.54 Gibson-assembly with PCR amplified cup1-1 and SpeI digested piGEM-005

Aim: insertion of cup1-1 into piGEM-005

The cup1-1 domain was amplified via PCR with new primers iGEM-024 and -025. Vector and template were diluted to a concentration under 75 ng/µL. Spe1 cut piGEM-005 was used from the cloning experiments with DARPin.

PCR fragment cup1-1 (464 ng/µL) dilution 1:10

SpeI digested pIGEM-005 (148 ng/µL) dilution 1:2

Component Volume [µl]
Gibson-Mix 15
Insert (cup1-1 58 ng/µL) 1,3
piGEM-005 1
Water 2,7
Total Volume 20

The mix was incubated 5 min at room temperature and then for 1h at 50°C.

In the end the whole mix was transformed into E.Coli XL1-Blue and plated out on LB-Amp. Incubation was done overnight at 37°C.

14.44 Test Expression

Aim: Test the expression of PheA-Arc1p-C-0x

Using the preculture from 14.43 60 mL of LB-Kan50 medium were inoculated with 600 µL of the preculture and incubated at 37 °C and 220 rpm until OD600 reached 0.5. The culture was cooled down to 18 °C and the protein production was induced with 12 µL of 500 mM IPTG. Over night the culture was incubated at 18 °C and 220 rpm. Afterwards the cell culture was centrifuged (17000 rpm, 20 min, 4 °C), the pellet resuspended in 3 mL buffer A and stored at -20 °C. A glycerolstock of the strain containing the PheA-Arc1p-C-0x-pET28a plasmid was stored at -80 °C.

18.06.2014

20.4 Second temperature shift

Aim: flip out of the pMAD backbone

One blue colony per diluted clone was used to inoculate 4 mL LB. The 6 cultures were incubated at 30°C for 6h and afterwards for 3h at 42°C.

Dilutions from 10-4 to 10-4 were plated out on 18 X-Gal plates without MLS selection. The positive clones should not contain the resistance inside the backbone as well as the galactosidase, resulting in normal colored colonies. The plates were incubated at 42°C overnight.

15.54 Checking Gibson assembly plate piGEM-005-cup1-1

Aim: insertion of cup1-1 into piGEM-005

Unfortunately the plate was empty: no colonies. The vector was digested again in order to repeat the assembly.

15.55 restriction digest of piGEM-005 with SpeI

Aim: linearizing plasmid for repeating the Gibson assembly

In order to repeat the Gibson assembly the piGEM-005 was cut with SpeI again.

Component Volume [µl]
piGEM-005 (108 ng/µL) 10
Cutsmart 10x 2
SpeI 0,5
Water 7,5
Total Volume 20

Incubation 1h at 37°C with following heat shock at 80°C

19.06.2014

20.5 Selection of positive clones

Aim: check the correct excision of the pMAD backbone

One white clone was picked from the dilution plates and transferred on a X-Gal Plate as well as on a MLS plate so that 18 clones were tested for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C over night.

15.56 new Gibson-assembly with cup1-1 and SpeI digested piGEM-005

Aim: insertion of cup1-1 into piGEM-005

As insert cup1-1 PCR product from 15.55 and SpeI digested piGEM-005 from 15.55 were used for a new Gibson assembly.

PCR fragment cup1-1 (464 ng/µL)

SpeI digested pIGEM-005 (53 ng/µL)

Component Volume [µl]
Gibson-Mix 15
Insert (cup1-1 58 ng/µL) 2,5
piGEM-005 2,5
Total Volume 20

The mix was incubated 5 min at room temperature and then for 1h at 50°C.

In the end the whole mix was used to transform E. coli XL1-Blue and plated out on LB-Amp. Incubation was done overnight at 37°C.

20.06.2014

20.5 selection of positive clones

Aim: checking the correct flip out of the pMAD backbone

The white colonies grew on the X-Gal Master plates but not on MLS plates so the transformation seemed to be successful. The Backbone with the MLS resistance flipped out of the genome.

20.6a cPCR with checked clones

Aim: checking integration of constructs Hag-Spe and Hag-Spe-DARPin into B. s. genome

No clone was growing on MLS plates so the picked clones seemed to be positive. In order to check the correct integration of the domain constructs, a cPCR with the picked clones on the X-Gal plates was done. The 18 picked B.subtilis clones were cooked in 10 µL PBS for 5 min at 95°C.

Mix Master-Mix for 18 attempts (µL) Mix for PCR attempt (µL)
Colony (picked clones 1-9 from Flagellin/-DARPin B.s. plates) - Part of a cooked colony
Primer Flo54 Hag-fw 9 -
Primer Flo56 Hag-rv 9 -
Phusion 18 -
Phusion Buffer 5x 72 -
dNTP Mix 9 -
Water 237 -
DMSO 6 -
Mastermix - 20
Total Volume 100 360

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 1 min 45 sec
4 72 2 min
5 Go To 2 30x
6 72 4min
7 8 infinite

The gel showed no bands. The PCR had to be repeated.

15.57 Miniprep of clones from Gibson assembly from 15.56

Aim: increase the amount of plasmid for further experiments

4 x 5 mL of LB-Amp was were inoculated with a picked colony from a transformation plate with XL1-Blue containing the Gibson-assembly product from 15.56.

Incubation at 37°C overnight.

21.06.2014

15.58 cPCR with colonies from Gibson-assembly of piGEM-005 + cup1-1 plate

Aim: checking correct insertion of cup1-1

In order to check the correct insertion of the cup1-1 domain into the Hag-Flank-construct in pMAD a cPCR with primers iGEM-025 (Cup1-1 rv) and Flo89 (pMAD-flank1-BamHI fw) was performed. The picked colonies were the same which were used to inoculate minipreps from 15.57. The fragment was supposed to have approx. 1250 bp.

Mix Master-Mix for 5 attempts (µL) Mix for PCR attempt (µL)
Colony (picked clones 1-4 for miniprep) - Part of a colony
Primer Flo89 2,5 -
Primer piGEM-025 2,5 -
Phusion DNA-polymerase 2,5 -
Phusion Buffer 5x 20 -
dNTP Mix 2,5 -
Water 70 -
Mastermix - 20
Total Volume 100 20

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min 30 sec
5 Go To 2 30x
6 72 4min
7 8 infinite

The analysis via 1% agarose gel eletrophoresis showed now bands. The plasmids of all four clones were isolated to use them as template.

15.59 PCR with isolated plasmid from Gibson piGEM-005 + cup1-1 plate

Aim: checking correct insertion of cup1-1

This time the isolated plasmid DNA from the picked six clones was used as a template with different primer pairs. First of all the PCR reactions were performed with primers Flo89 and Flo90 in order to receive the whole Hag-Flank-Cup1-1 construct with ca. 2100 bp length. As negative control also piGEM-005 was used as template producing a 2000kb fragment. Because of the small difference, all six reactions were also performed with the primers Flo89 and iGEM-025 (cup1-1 rv) in order to check the insertion of cup1-1 directly with a fragment of 1268 bp.

Mix Master-Mix 1 for  5 reactions (µL) Master-Mix 2 for  5 reactions (µL) Mix 1 for PCR reactions (µL) Mix 2for PCR reactions (µL)
Plasmid DNA from clone 1-4/ piGEM-005 - - 1 -
Primer Flo89 2,5 2,5 - -
Primer Flo90 2,5 - - -
Primer piGEM-025 - 2,5 - -
Phusion DNA-polymerase 2,5 2,5 - -
Phusion Buffer 5x 20 20 - -
dNTP-mix 2,5 2,5 - -
Water 70 70 - -
Master-Mix 1/ 2 - - 20 20
Total Volume 100 100 20 20

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 20 sec
5 Go To 2 30x
6 72 4min
7 8 infinite

The gel analysis shows that all 4 clones seem to be positive. An amplification of the cup1-1 fragment with the plasmid DNA as template could proof that.

20.6b Repeated cPCR with checked clones

Aim: checking integration of constructs Hag-SpeI and Hag-SpeI-DARPin into B. s. genome

Because of the unsuccessful cPCR before, the PCR was repeated. The 18 picked B.subtilis clones were cooked in 30 µL PBS for 10 min at 95°C.

Mix Master-Mix for 18 attempts (µL) Mix for PCR attempt (µL)
Colony (picked clones 1-9 from Flagellin/-DARPin B.s. plates) - Part of a cooked colony
Primer Flo54 Hag-fw 9 -
Primer Flo56 Hag-rv 9 -
Phusion DNA-polymerase 18 -
Phusion Buffer 5x 72 -
dNTP Mix 9 -
Water 237 -
DMSO 6 -
Mastermix - 20
Total Volume 100 360

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 1 min 45 sec
4 72 2 min
5 Go To 2 30x
6 72 4min
7 8 infinite


All nine clones with piGEM-005 seem to be positive. Clone 1, 3, 7, 8 and 9 of transformed piGEM-018.1 might be positive as well as clone 2-6 from piGEM-018.2. The sequencing of the PCR products should tell us if the insertion was successful.

22.06.2014

18.31 PCR amplification of DARPin-Hag and Hag fragment

Aim: amplification of DARPin-Hag fragments for repeating the ligation

piGEM-018 & -005 were used as template for a PCR with the primers Flo 54 and Flo 56. The products could be used for further cloning after NcoI/BamHI digest.

Content Volume [µl]
Template (piGEM-005 & -018) 1
Primer Flo 54 1
Primer Flo 56 1
Phusion 1
Phusion Buffer 5x 10
dNTP Mix 1
Water 35
Total Volume 50

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 30x
6 72 4min
7 8 infinite
18.32 cPCR with new clones

Aim: checking picked clones for correct Hag/ Hag-DARPin insert in pET24d

More clones from each plate were picked to analyse them via cPCR with the primers flo54 and flo56 (Hag-fw and Hag-rv). The result should be a ca. 1kb fragment for correct pET24d-Hag containing clones and a 1,5 kb fragment for pET24d-Hag-DARPin containing clones.

Content Volume (µL) MM for 14 attempts (µL

Colony

 (8-15 from pet-Hag,7-14 from pet-Hag-DARPin)
1 colony -
Primer Flo 54 - 7
Primer Flo 56 - 7
Phusion DNA-polymerase - 7
Phusion Buffer 5x - 56
dNTP Mix - 7
Water - 196
Mastermix 20 -
Total Volume 20 280

Step Temperature °C Time
1 98 10 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 35x
6 72 4min
7 8 infinite


Clone 1 from each pET24d-Hag-SpeI and pET24d-Hag-DARPin clones was positive and were digested with NcoI/BamI to check them again.

18.33 pET24d digest with NcoI/BamHI

Aim: preparing vector for new ligation attempts

Because of the low amount of right clones, new vector (pET24d) was digested with NcoI and BamHI according to the following scheme:

Component Volume (µL)
pET24d (50 ng/µL) 20
CutSmart 10x 3
NcoI 0,5
BamHI 0,5
Water 6
Total Volume 30

Incubation at 37°C for 2 hours with heat shock in the end at 80°C

Endconcentration: 32 ng/µL

18.34 Restriction digest of Hag PCR fragments from 18.31 with NcoI/BamHI

Aim: creating restriction sites for cloning into pet24d cut with NcoI/BamHI

The PCR products from 18.31 were digested with NcoI andBamHI to create restriction sites in order to ligate the fragments into NcoI/BamHI digested pet24d in case of negative clones refering to the cPCR from 18.x

Component PCR Hag-fragment (µL) PCR Hag-DARPin fragment (µL)
PCR products 45 45
Cutsmart 10x 5,2 5,2
NcoI 0,5 0,5
BamHI 0,5 0,5
Water 3,8 3,8
Total Volume 52 52

Incubation for 2h at 37°C with heat shock at 80°C.

Endconcentration:
digested Hag-fragment 260 ng/µL
digested Hag-fragment 268 ng/µL

15.60 PCR pMAD-Hag-Flank-cup1-1 (piGEM-016)

Aim: checking success of Gibson assembly with pMAD-Hag-Flank and cup1-1

Using the plasmids as template the cup1-1 fragment at 160 bp should be visible on the agarose gel. As positive control PCR products from the cup1-1 amplification used for the Gibson Assembly into piGEM-005 were applied to the gel as well.

Content Volume (µL) MM for 5 attempts (µL)
Template (Plasmid from clone 1-4) 0,5 -
Primer iGEM-024 - 2,5
Primer iGEM-025 - 2,5
Phusion DNA-polymerase - 2,5
Phusion Buffer 5x - 20
dNTP Mix - 2,5
Water - 70
Mastermix 19,5 -
Total Volume 20 100

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 20 sec
5 Go To 2 35x
6 72 4min
7 8 infinite

The bands under the 200 bp mark indicate the positive insertion of cup1-1 into piGEM-005. The plasmid has to be sequenced and is named iGEM-016.

18.35 Test digest with NcoI/BamHI - Checking insertion of Hag/-DARPin into pet24d

Aim: proving insertion of Hag/-DARPin fragment into pET24d

The plasmids are digested with NcoI and BamHI. In case of a correct insertion the pET24d backbone and the insert should be seen on the gel at 5300 bp and 1000/ 1500 bp. As control pET24d-Hag-Flank (piGEM-001) is used to check the mastermix.

Component Volume (µl) Mastermix
2x Plasmid from clone 6 5 -
CutSmart 10x - 5
NcoI - 0,5
BamHI - 0,5
Water - 19
Total Volume 10 25


The used marker was the GeneRuler 1 kb DNA ladder. The lower fragment of the digested pET24d-Hag was too big (2 kb) and pET24d-Hag-DARPin seemed only to be linearized with the exception of two very thin bands below the linear plasmid, which were all of the wrong size.

18.36 ligation of pET24d and PCR Hag/ Hag-DARPin fragments cut NcoI/BamHI

Aim: ligation of construct with pET24d for overproduction of flagellin and crystallization

The NcoI/BamHI digested flagellin PCR products from 18.22 (from clone 7 and piGEM-005) were ligated with NcoI/BamHI digested pET24d.

Component pET24d-Hag reaction (µL) pET24d-Hag-DARPin raction (µL)
Insert (PCR fragments) (ca. 260 ng/µL) 2 2
Vector (pET24d, c= 50ng/µL) 5 5
Ligase-Buffer 10x 2 2
T4 Ligase 1 10
Millipore Water 10 10
Total Volume 20 20

The reactions were prepared in double and were incubated at room temperature. One reaction was incubated for 1 hour followed by transformation of E. coli XL1-Blue and the second one over night.

18.37 Repetition of PCR with 3 clones of pET24d-Hag and pET24d-Hag-DARPin ligation

Aim: checking picked clones for correct Hag/ Hag-DARPin insert in pET24d

3 clones from ligation plates with pET-Hag and pET-Hag-DARPin were resuspended in 30 µL PBS and cooked at 95 °C for 10 min. After that procedure 1 µL of this suspension was used as PCR template.

Content Volume (µL) MM for 7 reactions (µL)
Template ( 1µL cooked suspension)
 (8-10 from pET-Hag, 7-9 from pET-Hag-DARPin)
1 -
Primer Flo 54 - 3,5
Primer Flo 56 - 3,5
Phusion DNA-polymerase - 3,5
Phusion Buffer 5x - 28
dNTP-mix - 3,5
Water - 98
Mastermix 19 -
Total Volume 20 140

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 35x
6 72 4min
7 8 infinite

Run over night

Transformation
Plasmid Concentration (ng/µL) Antibiotic Cells Volume (µL)
pET24d 148 Can DH5α 1
piGEM-018 160 Amp DH5α 0,5
piGEM-014 350 Amp DH5α 0,5
Gibson pMAD-cup1-1 clone 4 60 Amp DH5α 1
pET-Hag attempt from 18.36 - Can XL1-Blue 20
pET-Hag-DARPin attempt from 18.36 - Can XL1-Blue 20
Control pET24d cut NcoI/BamHI 32 Can XL1-Blue 1

23.06.2014

14.45 Purification of test expression

Aim: Purify PheA-Arc1p-C-0x on a small scale

Lysozyme was added to the cell suspension from 14.44 to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 °C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.

60 mL LB-Kan50 medium was inoculated from the glycerolstock prepared in 14.44 and incubated at 37 °C and 220 rpm over night.

Sequencing of constructs from 23.06.2014
Premix Label-Nr. Construct Primer
1 AGB0023402 piGEM-018 (pMAD-DARPin) Flo54 (Hag-F-Nco)
2 AGB0023403 piGEM-018 (pMAD-DARPin) Flo56 (Hag-R-Bam)
3 AGB0023404 pMAD-Hag-Flank-cup1-1 Flo54 (Hag-F-Nco)
4 AGB0023405 pMAD-Hag-Flank-cup1-1 Flo56 (Hag-R-Bam)
5 AGB0023406 piGEM-002 (Nose plasmid) TW259
18.37 Repetition of PCR with 3 clones of pET24d-Hag and pET24d-Hag-DARPin ligation

Aim: checking picked clones for correct Hag/ Hag-DARPin insert in pET24d

Gel electrophoresis:

The gel shows negative bands in case of the Hag-DARPin clones. The PCR for Hag 8 and 9 did not work although we cooked the clones in PBS. The transformation efficiency seemed to be very low, so clones from the new ligation attempts should be analysed.

18.38 Ligation checking success of transformed ligation attempts

On every plate grew a high amount of colonies. 6 clones of each plate were picked for inoculation of minipreps and for cPCR after cooking with PBS. The clones were transferred to another LB-Can plate.

18.39 cPCR with picked clones from 18.38 ligation attempt transformation

Aim: checking picked clones for correct Hag/ Hag-DARPin insert in pET24d

For checking the insertion of the Hag/-DARPin into pET24d the clones from the transformation plate were transferred to another LB-Can plate  and cooked for 10 min in 30 µL PBS at 95°C. At the same time the colonies were used to inoculate LB-Can for minipreps.

Content Volume (µL) MM for 14 attempts (µL)
Template ( 1µL cooked suspension)
 (1-6 from pet-Hag, 1-6 from pet-Hag-DARPin)
1 -
Primer Flo 54 - 7
Primer Flo 56 - 7
Phusion DNA-polymerase - 7
Phusion Buffer 5x - 198
dNTP-mix - 7
Water - 98
Mastermix 19 -
Total Volume 20 280

Step Temperature °C Time
1 98 5 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 35x
6 72 4min
7 8 infinite

Run over night

24.06.2014

14.46 Expression of PheA-Arc1p-C-0x

Aim: Express PheA-Arc1p-C-0x for further use

10 x 500 mL LB-Kan50 medium were inoculated with 5 mL of the overnight culture prepared in 14.45 and incubated at 37 °C and 220 rpm until OD600 reached 0.5. The flasks were cooled to 18 °C and protein production was induced by adding 100 µL 500 mM IPTG. The cultures were incubated over night at 18 °C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 °C), resuspended in buffer A and stored at -20 °C until further use.

25.06.2014

Sequencing results of constructs from 23.06.2014
Premix Label-Nr. Construct Result
1 AGB0023402 piGEM-018 (pMAD-DARPin) Correct insertion of DARPin
2 AGB0023403 piGEM-018 (pMAD-DARPin) Did not work
3 AGB0023404 pMAD-Hag-Flank-cup1-1 Correct insertion, point mutation refers to bad sequencing
4 AGB0023405 pMAD-Hag-Flank-cup1-1 Did not work
5 AGB0023406 piGEM-002 (Nose plasmid) Correct insertion of the CM resistance
18.39 Gel cPCR with picked clones from 18.38 ligation attempt transformation

Aim: checking picked clones for correct Hag/ Hag-DARPin insert in pET24d

Gel electrophoresis: no bands except for the controls were seen. The PCR has to be repeated with more colony in the PCR tubes or the prepped plasmids

18.40 Test digest with NcoI/BamHI of isolated plasmids from picked colonies

Aim: checking picked clones for correct Hag/ Hag-DARPin insert in pET24d

The picked clones from the ligation attempts 18.38 were isolated. In order to check the insertion of the Hag/ Hag-DARPin domain the plasmids were digested with NcoI/BamHI so that the gel should show the insert of 1 or 1,5 bp length and the pET24d backbone at 5300 bp. Because of an unavailable marker as a control the PCR amplified inserts which were used for the ligation will run  with the gel as well. piGEM-001 will also be cut NcoI/BamHI in order to receive a marker band for the pET24d backbone.

Component Volume (µL) Mastermix for 8 attempts
Plasmid (ca. 45 ng/µL 5 -
Cutsmart 10x - 4
NcoI - 0,5
BamHI - 0,5
Water - 35
Mastermix 5 -
Total Volume 10 40


The gel shows that the digested clones were all negative. The ligation had to be performed again.

20.6 Purification of cPCR samples with Gel extraction kit

Aim: purification of samples for sequencing

Purified samples:
clone 3 & 5 from transformed piGEM-005
clone 3 & 9 from transformed piGEM-018

Clone Concentration (ng/µl)
Hag 3 10
Hag 5 14
Hag-DARPin 3 9
Hag-DARPin 9 9

26.06.2014

20.7 PCR with purified cPCR samples from 20.6

Aim: PCR amplification of purified cPCR samples

The concentration of the cPCR samples after purification was too low for sequencing. Therefore the cPCR samples have to be amplified with PCR again.

Content Volume (µL) MM for 6 attempts (µL)
Purified cPCR product from 20.6
 (3 & 5 from pet-Hag, 3 & 9 from pet-Hag-DARPin)
1 -
Primer Flo 54 - 6
Primer Flo 56 - 6
Phusion DNA-polymerase - 6
Phusion Buffer 5x - 6
dNTP-mix - 60
Water - 216
Mastermix 19 -
Total Volume 20 300

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 35x
6 72 4min
7 8 infinite
20.8 Sequencing of purified cPCR samples from pMAD transformation
Premix Label-Nr. Construct Primer
1 AGB0023407 Hag-SpeI in B. subtilis genome clone 3 Hag-Nco-fw
2 AGB0023408 Hag-SpeI in B. subtilis genome clone 5 Hag-Nco-fw
3 AGB0023409 Hag-SpeI-DARPin in B. subtilis genome clone3 Hag-Nco-fw
4 AGB0023410 Hag-SpeI-DARPin in B. subtilis genome clone 9 Hag-Nco-fw
18.41 New ligation of pET24d and PCR Hag/ Hag-DARPin fragments cut NcoI/BamHI transformation

Aim: ligation of construct with pET24d for overproduction of flagellin and crystallization

The cut pET24d NcoI/BamHI which was used for the previous ligations was not purified and contained an 1 kb insert. For a new clean ligation the reaction was repeated with digested pET24d. The NcoI/BamHI digested flagellin PCR products (from clone 7 and piGEM-005) were ligated with NcoI/BamHI digested pET24d.

The reactions were carried out in double and were incubated 1h at room temperature. Finally they were used to transform E. coli XL1-Blue after inactivation at 65°C for 10 min. The attempts 1.1 and 2.1 were incubated over night at room temperature. 1 µL of digested vector was used for a transformation as a negative control in order to check the amount of religants.

Component pET-Hag attempt (µL) – 1 & 1.1 pET-Hag-DARPin attempt (µL) 2& 2.1
Insert (PCR fragments) (ca. 260 ng/µL) 3 3
Vector (pET24d, c= 86ng/µL) 5 5
Ligase-Buffer 10x 2 2
T4 Ligase 1 1
Millipore water 9 9
Total Volume 20 20

27.06.2014

18.42 Miniprep of ligation clones

The ligation between pET24d and the Hag-/Hag-DARPin fragments which were both cut NcoI/BamHI was successful. The ligation plates showed over 20 clones each. However, on the negative control  grew a few clones as well, which were very small and sticking together. In order to analyse the insert of the clones, 5 clones per ligation plate 1 and 2 were used to inoculate minipreps in 5 mL LB-Can. For the Ligation 1 pET-Hag clones 1-5 were picked, as well as clones 6-10 from Plate Ligation 2 pET-Hag-DARPin.

The cultures were inoculated over night at 37°C.

28.06.2014

18.42 Miniprep of ligation clones
Construct Clone Concentration (ng/µL)
pET24d-Hag 1 50
pET24d-Hag 2 87
pET24d-Hag 3 65
pET24d-Hag-DARPin 6 83
pET24d-Hag-DARPin 7 59
18.43 cPCR of clones from 18.41

Aim: checking picked clones for correct Hag/ Hag-DARPin insert in pET24d

Plasmid from isolated clones from each plate were analysed via cPCR with the primers flo54 and flo56 (Hag-fw and Hag-rv). The result should be a ca. 1kb fragment for correct pET24d-Hag containing clones and a 1,5 kb fragment for pET24d-Hag-DARPin containing clones.

Content Volume (µL) MM for 6 attempts (µL)
Picked clones from 18.42
(1-3 pET24d-Hag, 6-7 from pET24d-Hag-DARPin)
0,5 -
Primer Flo 54 - 3
Primer Flo 56 - 3
Phusion DNA-polymerase - 3
Phusion Buffer 5x - 6
dNTP-mix - 24
Water - 81
Mastermix 19,5 -
Total Volume 20 120

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 30x
6 72 4min
7 8 infinite

The samples can be seen on the gel under 18.44. The PCR for the Hag-fragment in pET24d seemed to be positive (PCR.H1 - H3), as well as for the Hag-DARPin fragment (PCR.D7)

18.44 test digest with NcoI/BamHI - Checking insertion of Hag/-DARPin into pET24d

Aim: proving insertion of Hag/-DARPin fragment into pET24d

The plasmids were digested with NcoI and BamHI. In case of a correct insertion the pet24d backbone and the insert should be seen on the gel at 5300 bp and 1000/ 1500 bp. As control pET24d-Hag-Flank (piGEM-001) is used to check the mastermix.

Component Volume (µL) Mastermix(6 attempts)
Plasmid from clone
( pET24d-Hag 1-3, pET24d-Hag-DARPin 6 & 7)
5 -
Cutsmart 10x - 3
NcoI - 0,5
BamHI - 0,5
Water - 26
Total Volume 10 30

20.9 Gel Purification of amplified cPCR products

Aim: Purification for more efficient sequencing

The PCR product of the amplification of cPCR products was not pure considering different bands on the gel next to the right one. In order to prevent sequencing mistakes the PCR attempts were purified via gel extraction of the 1,5 kb band in case of the Hag-DARPin construct.

20.10 PCR with purified Hag-DARPin cPCR products for sequencing

Aim: amplification of purified Hag-DARPin fragment for sequencing

The purified cPCR product from 20.9 has to be amplified again in order to get a higher concentration for sequencing.

Content Volume (µL) MM for 2 attempts (µL)
Purified PCR product from 20.x
(Hag-/ Hag-DARPin fragment)
1-2 -
Primer Flo 54 - 1
Primer Flo 56 - 1
Phusion DNA-polymerase - 1
Phusion Buffer 5x - 2
dNTP-mix - 20
Water - 75
Mastermix 19 -
Total Volume 20 100

Step Temperature °C Time
1 98 5 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 35x
6 72 4min
7 8 infinite
18.45 SpeI digest of pET24d-Hag (piGEM-019)

Aim: linearizing pET24d-Hag with SpeI site for a Gibson assembly with Cup1-1

Component Volume (µL)
piGEM-016 (ng/µL) 17
CutSmart 10x 2
SpeI 0,5
Water 0,5
Total Volume 20

Incubation 1 h at 37°C with following heat inactivation at 80°C

18.46 Gibson assembly with linearized pET24d-Hag (piGEM-019) and cup1-1

Aim: Insertion of cup1-1 domain into pET24d-Hag construct for crystallization

As insert cup1-1 PCR product from 15.55 and SpeI digested piGEM-016 from 18.44 were used for a new Gibson assembly.
PCR fragment cup1-1 (464 ng/µL)
SpeI digested pIGEM-005 ( ng/µL)

Component Volume (µl)
Gibson-mix 15
Insert (cup1-1 464 ng/µL) 2,5
piGEM-019 2,5
Total Volume 20

The mix was incubated 5 min at room temperature and then for 1h at 50°C.

In the end the whole mix was used to transform E. coli XL1-Blue, which were plated out on LB-Amp. Incubation was done over night at 37°C.

29.06.2014

18.47 cPCR of Gibson assembly clones of pET24d-Hag (piGEM-019) and cup1-1

Aim: screening clones for right insert

In order to screen the clones for the right insertion of cup1-1 four clones were picked from the LB-Can plate and transferred onto a LB-Can plate. Additionally the clones were used for inoculation of minipreps and also cooked in 50 µL dest. Water for 10 min at 95°C. The lysate was used as template.

The PCR with Flo54 and iGEM-025 should generate a 785 bp fragment and the one with iGEM-024 and -025 the 164bp cup domain fragment.

Content Volume (µL) MM 1 for 5 attempts (µL) MM 2 for 5 attempts (µL)

Cooked lysate solution from clones 1-4

3 -  
Primer Flo 54 - 2,5 -
Primer iGEM-024 - - 2,5
Primer iGEM-025 - - 2,5
dNTP-mix - 2,5 2,5
Phusion DNA-polymerase - 5 5
Phusion Buffer 5x - 20 20
Water - 67,5 67,5
Mastermix 17 - -
Total Volume 20 100 100

Step Temperature °C Time
1 98 10 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 35x
6 72 4min
7 8 infinite

The PCR showed no bands. Nevertheless, colonies were used to inoculate cultures for minipreps so that the isolated plasmid can be further analysed for the insert.

30.06.2014

Sequencing
Premix Label-Nr. Construct Primers
1 AGB0023411 pET24d-Hag-SpeI construct cl. 2 Hag-Nco-fw
2 AGB0023412 pET24d-Hag-DARPin construct cl. 7 Hag-Nco-fw
18.48a Miniprep of plasmids from Gibson assembly clones with pET24d-Hag (piGEM-019) and cup1-1

Aim: isolation of Plasmid DNA for further experiments

Construct Clone Concentration (ng/µL)
pET24d-Hag-Cup1-1 1 122
pET24d-Hag-Cup1-1 2 85
pET24d-Hag-Cup1-1 3 172
pET24d-Hag-Cup1-1 4 116
18.48b PCR of isolated plasmids from Gibson assembly clones (pET24d-Hag (piGEM-019) and cup1-1)

Aim: screening clones for right insert

The isolated plasmid 1:10 diluted from the picked clones 1-4 were used as PCR template.

The PCR with Flo54 and iGEM-025 should generate a 785 bp fragment and the one with iGEM-024 and -025 the 164bp domain fragment.

Content Volume (µL) MM 1 for 5 attempts (µL) MM 2 for 5 attempts (µL)

Cooked lysate solution from clones 1-4 (1:10)

3 -  
Primer Flo 54 - 2,5 -
Primer iGEM-024 - - 2,5
Primer iGEM-025 - 2,5 2,5
dNTP-mix - 2,5 2,5
Phusion - 5 5
Phusion Buffer 5x - 20 20
Water - 67,5 67,5
Mastermix 17 - -
Total Volume 20 100 100

Step Temperature °C Time
1 98 3 min
2 98 30 sec
3 55 30 sec
4 72 1 min 45 sec
5 Go To 2 35x
6 72 4min
7 8 infinite

No bands could be observed on the gel. The PCR was repeated with Q5 mastermix to exclude the disfunction of the polymerase.

20.9 Inoculation of a preculture from Bacillus subtilis WT 3610

Aim: Preparation for a mainculture the for the next day

2 x 6 mL of LB were inoculated with Bacillus subtilis WT3610 from an LB plate and incubated at 37°C over night.

14.47 Purification of PheA-Arc1p-C-0x

Aim: Purify PheA-Arc1p-C-0x in three steps

The resuspended cells from 14.46 were lysed using a french press. The lysate was incubated on ice with 5 µg/mL DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C) to pellet cell debris. The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-0x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C-0x were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.