• piGEM010 = 22ng/µl
• piGEM013 = 46 ng/µl
• piGEM 009 = 13 ng/µl

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

The miniprep was performed accoding 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

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

Starting from 1 mM 8 dilutions have been prepared

We received the synthetized DARPin-gene in a pEC-A2 vector with spe1 restriction sites at the 5’- and 3’-site at an amount of 2,7µg plasmid in the tube.

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

Concentration of stock solution = 100 ng/µl

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

Concentration of stock dilution = 10 ng/µl.

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

3 x6ml LB-Amp were inoculated with clones from the successful transformed plate (18.2) and incubated at 37°C overnight.

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. 24h at 37°C shaking (Viktor AG Waldminghaus)

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 plan to repeat the transformation after checking the growth of the Bacillus subtilis Wildtype 3610 on LB-CM plate with 5 µg/ml CM.

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

Antibiotics and chemicals:

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

For MLS selection plates Erythromycin with 1mg/ml were needed. Therefore the 4 mg/mL stock solution was diluted 1:4 by mixing 400 µl Eryhtomycin 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

About 120 plates were stored at the 4°C room at AG Graumann labs.

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

In order to exclude the false making of plates new ones were made with new CM-stocks with 25 mg/ml and 5 mg/ml.

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

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

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

Incubation at 37°C overnight.

The flagellin construct was amplificated with Primer 95 and 54 from Florian Altegoer which allow the amplification of the construct with cup1-1 without flanks (just Hag) including Nco1 & BamH1 restriction sites.
Template dilution 1:10:
1µL pet24d-cup1-1 was mixed with 9 µL to an 1:10 dilution of 16,3 ng/µL
Primer dilution 1:10:
Primers 95 & 54 from Florian Altegoer were diluted 1:10 by mixing 10µL of primers with 90µL milipore water each.

PCR was run overnight.

Prepped pet24d was cut with Nco1 and BamH1 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 Nco1/ BamH1 restriction sites for that purpose as well.

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

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)

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.

The with Spe1 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

The DARPin gene with Spe1 sites was used as insert for the ligation into linearized pMAD (from 18.5) which contains the designed Spe1 site. The ligation attempt was done twofold and calculated by the Ligation Calculator from the iGEM-Team Texas in 2011:

Incubation was performed overnight at room temperature.

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 Bacillus CM threshold is.

Incubation at 30°C over the weekend.

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

The whole 20µL attempt from the overnight ligations were transformed into E.Coli XL1-Blue and plated on LB-Amp plates.  Additionally a negative control was created by transforming 1µL Spe1 cut piGEM-005 into E.Coli XL1-Blue as well.

The with Spe1 digested pEX-A2 was purified via electrophoresis with an 1% agarose gel like in 18.6. The band 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: 10 ng/µl

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.

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.

The cut Spe1 cut piGEM-005 was dephosporylated at the 5’-end with Antarctic phosphatase to prevent the vector from relegation.

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

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.

Incubation 3h at room temperature

The whole 20µL attempt from the ligations (18.15) were transformed into E.Coli XL1-Blue and plated on LB-Amp plates.  Additionally a negative control was created by transforming 1µL Spe1 cut piGEM-005 into E.Coli XL1-Blue as well.

Result: Bacillus subtilis Wildtype 3610 did not grow on the 25 ng/µLCM 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.

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.

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.

Because of the ligation via 2 Spe1 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 the plasmids have to be amplified by minipreps.

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

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

We saw that Bacillus grew on almost every CM concentration 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 from Daniel Schindler.

Additionally WT from Felix Dempwolff which was made competent was also transformed with piGEM-004 was 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.

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

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.

The PCR products from 18.21 were digested with Nco1 and BamH1 to create restriction sites in order to ligate the fragments into Nco/Bam cut pet24d.

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

The Nco/cut Flagellin PCR products from 18.22 (from clone 7 and piGEM-005) were ligated with Nco1 and BamH1 cut pet24d.

Incubation overnight at room temperature.

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

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

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

The whole 20µL attempt was transformed into E.Coli X1-Blue, plated out on LB-Can plates and incubated at 37°C overnight.

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

pMAD-Hag-Flank-DARPin and the hormone receptor clone 1 were given away 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:

Competent WT from Felix Dempwolff was transformed with piGEM-002, -003 and -004 by incubating the aliquods with 5 µL plasmid for 30 min at 37°C. After half an hour the whole attempt was plated out on LB-Cm (5 ng/µL).

Incubation at 30°C for 2 days.

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

Because of the unsuccessful ligation from 18.23 new vector (pet24d) was cut with Nco1 and BamH1 according to the following scheme:

Incubation at 37°C for 2h.

The DARPin-Hag constructs contain a Pst1 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 cut with Pst1.

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

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

Incubation at 37°C for 2h.

piGEM-018 & -005 were used as template for a PCR with the primers from Florian Altegoer 54 and 56. The products could be used for further cloning after Nco/Bam digest.

The fragments were analysed on an 1% agarose gel.

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

Referring to the gel analysis the amplification was successful.

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

Then 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.

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

Spe1 cut pIGEM-005 (148 ng/µL) dilution 1:2

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.

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 10-6 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. The plates were incubated at 42°C overnight.

Unfortunately the plate was empty, no colonies. The vector was cut newly in order to repeat the assembly.

In order to repeat the Gibson assembly the piGEM-005 was cut with spe1 freshly.

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

From the dilution plates was one WHITE clone picked and transferred on a Master X-Gal Plate as well as on a MLS plate so that 18 clones were proven for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C overnight.

As insert cup1-1 PCR product from 15.55 and Spe1 cut piGEM-005 from 15.55 were used for a new Gibson assembly.

PCR fragment cup1-1 (464 ng/µL)

Spe1 cut pIGEM-005 (53 ng/µL)

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.

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

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 picked 18 B.s. clones were cooked in 10 µL PBS for 5 min at 95°C.

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

4x 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.

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-Bamh1 fw) was performed. The picked colonies were the same which wre used to inoculate minipreps from 15.57. The fragment was supposed to e ca. 1250 bp.

The analysis via 1% agarose gel eletrophoresis showed now bands. All 4 picked clones were prepped to use the plasmid as template.

This time the prepped plasmid DNA from the picked 6clones was used as a template with different primer pairs. First of all tie PCR attempts 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 6 attempts were also driven 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.

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

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

The clones with piGEM-005 seem to be positive all 9. 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.

piGEM-018 & -005 were used as template for a PCR with the primers from Florian Altegoer 54 and 56. The products could be used for further cloning after Nco/Bam digest.

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.

Because of the low amount of right clones new vector (pet24d) was cut with Nco1 and BamH1 according to the following scheme:

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

Endconcentration: 32 ng/µL

The PCR products from 18.x were digested with Nco1 and BamH1 to create restriction sites in order to ligate the fragments into Nco/Bam cut pet24d in case of negative clones refering to the cPCR from 18.x

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

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 applicated on the gel as well.

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.

The plasmids are digested with Nco1 and BamH1. 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.

The Nco/cut Flagellin PCR products from 18.x (from clone 7 and piGEM-005) were ligated with Nco1 and BamH1 cut pet24d.

The attempts were carried out in double and incubated at room temperature -One attempt for 1h  followed by transformation into E.Coli XL1-Blue and the second one overnight.

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

Run overnight

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 with PBS. The transformation efficiency seemed to be very low so clones from the new ligation attempts should be analysed.

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.

For checking the insertion of the Hag/-DARPin into pet24 the clones from the transformation plate were transferred to another LB-Can plate  and cooked for 10 min in 30µL PBS at 95°C. At the same time the colonies were used to inoculate LB-Can for minipreps.

Run overnight

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

The picked clones from the ligation attempts 18.38 were prepped. In order to check the insertion of the Hag/ Hag-DARPin domain the plasmids were digested with Nco1/ BamH1 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 Nco/Bam in order to receive a marker band for the pet24d backbone.

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

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

The cut pet24d Nco/Bam which was used for the previous ligations was not purified and contained an 1 kb insert. For a new cealn ligation the attempt was repreated with pet24d cut by Carina Knauer .The Nco/cut Flagellin PCR products (from clone 7 and piGEM-005) were ligated with Nco1 and BamH1 cut pet24d.

The attempts were carried out in double. The attempts 1 & 2 were incubated 1h at room temperature and finally transformed into E. Coli XL1-Blue after inactivation at 65 for 10 min. The attempts 1.1 and 2.1 were incubated overnight at room temperature. 1 µL of cut vector was transformed as a negative control in order to check the amount of religants.

The ligation between pet24d and the Hag-/Hag-DARPin fragments which were both cut Nco/Bam was successful. The ligation plates showed over 20 clones each whereby on the negative control  grew a few clones as well which were very small and sticking together. Bad plate out might be the reason for that. 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 overnight at 37°C.

Plasmid from prepped 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.

The plasmids were digested with Nco1 and BamH1. 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.

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 exclude sequencing mistakes the PCR attempts were purified via gel extraction of the 1,5 kb band in case of the Hag-DARPin construct.

The purified cPCR product from 20.x has to be amplified again in order to get a higher concentration for sequencing.

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

As insert cup1-1 PCR product from 15.55 and Spe1 cut piGEM-016 from 18.44 were used for a new Gibson assembly.
PCR fragment cup1-1 (464 ng/µL)
Spe1 cut pIGEM-005 ( ng/µL)

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.

In order to screen the clones for the right insertion of cup1-1 4 clones were picked from the LB-Can plate and transferred to a LB-Can Master 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 domain fragment.

The PCR showed no bands. The colonies were used for minipreps so that the isolated plasmid can the used for analysis of the 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.

No bands were shown on the gel. The PCR was repeated with Q5 mastermix to exclude the disfunction of the polymerase.

2x6 mL of LB were inoculated with Bacillus WT3610 from an LB plate and incubated at 37°C overnight.