<legend><a name="exp20.10">20.10 pMAD-Transformation of competent Bacillus subtilis  WT 3610</a></legend>

     <p>100 µL of overnight culture were added to 10 mL of MNGE-Medium and incubated till an  OD of 0,7 at 37°C which took  7h.</p>

     <p>After  reaching OD of  0,7  400 µL of the culture were transformed with  1,5 µg piGEM-021. After 1h incubation at 37°C 100 µL Expression mix was added  and incubated for 1h as well.</p>

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

     <td>Pet24d-Hag-Spe1 construct cl. 2</td>

     <td>Correct insertion of  Hag-SpeI</td>

     <td>Pet24d-Hag-DARPin  construct cl. 7</td>

     <legend><a name="exp19.3">19.3 Co-Transformation of piGEM-019 & -020 into E.Coli BL21(DE3) with FlaiS</a></legend>

     <p>piGEM-019  and -020 were transformed into E.Coli BL21 (DE3) together with a plasmid  containing the flagellin chaperone FlaS in order to express both proteins at  the same time after induction so that the flagellin monomer accumulates inside  the cells.</p>

     <p>The  cells were plated out on Can/Amp-LB plates and incubated overnight at 37°C.</p>

     <legend><a name="exp18.49">18.49 new PCR of prepped Gibson assembly clones ( Pet-Hag (piGEM-019) and Cup1-1)</a></legend>

     <th scope="col">Volume (µL)</th>

     <th scope="col">MM 1 for 5 attempts (µL)</th>

     <th scope="col">MM 2 for 5 attempts (µL)</th>

         <p>The gel’s second row shows the  amplified cup-fragment under the 200 bp ladder mark. The expected size is ca.  164 bp and fits to the gel result.</p>

         <p>Plasmid from clone 4 was taken,  transformed into <em>E.Coli DH5</em><em>a</em>and named piGEM-021.</p>

     <legend><a name="exp19.4">19.4 Expression-Test with transformed E.Coli BL21 (DE3)</a></legend>

     <p>Aim: Checking the overexpression of Hag-Spe1 and Hag-Spe1-DARPin</p>

     <p>In  order to check the expression of the proteins a single clone containing  piGEM-019/-20 was used to inoculate 20 mL of LB-Can/Amp. The culture was  incubated at 37°C until an OD of 0,7.</p>

     <p>A 1 mL  preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL).  After that the cultures were induced with 20 µL IPTG for 2h.</p>

     <p>An  induction sample (I) was taken (320 µL with an OD of 2,2; 350 µL with an OD of  2).</p>

     <p>PI and  I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and  the pellet resuspended in 60 µL water and 40 µL SDS-Buffer.</p>

     <p>The  four samples were analysed on an SDS-PAGE gel with 5 and 10 µL volume per sample.</p>

     <img src="https://static.igem.org/mediawiki/2014/0/0f/MR_2014-07-03_19.4.jpg" width="30%" />

     <legend><a name="exp19.a5">19.5 Protein overexpression with transformed E.Coli BL21 (DE3)</a></legend>

     <p>For  that purpose 2 x 1 L LB were inoculated with a solution of LB and every clone  on the cotransformation plate. All clones from the piGEM-019/ -020 + FlaS  cotransformation plates were picked and resuspended in 1 mL LB. 500 µL were  added t the 1 L culture flasks. Each flask was additionally mixed with 1 mL  Ampicillin (100 mg/ mL) and 1 mL Canamycin (50 mg/ mL).</p>

     <p>The  cultures were incubated overnight at 30°C.</p>

     <td>Pet24d-Hag-Spe1-Cup  constr. Cl. 4</td>

     <legend><a name="exp19.5b">19.5 Protein overexpression with transformed E.Coli BL21 (DE3)</a></legend>

     <p>1 mL of  culture was spinned down, mixed with 60 µL water, 40 µL SDS-PAGE loading buffer  and analysed on a SDS-PAGE gel.</p>

     <p> The overnight induced cells were raised till  an OD of 2-3  and spinned down at 4000  rpm.  The pellets were frozen in liquid  nitrogen and stored at -80°C.</p>

     <legend><a name="exp19.6a"> 19.6 Co-Transformation of piGEM-021 & with FlaiS  E.Coli BL21(DE3) </a></legend>

     <p>piGEM-021  and pFlaiS were transformed into <em>E.Coli BL21(DE3)</em> and plated out  on LB-Canamycin plates. The plate was incubated at 37°C overnight. </p>

     <legend><a name="exp19.7a">19.7 Expression-Test with transformed E.Coli BL21 (DE3) with piGEM-021</a></legend>

     <p>Aim: Checking the overexpression of Hag-Spe1-Cup1-1</p>

     <p>In  order to check the expression of the proteins a single clone containing  piGEM-021 was used to inoculate 20 mL of LB-Can/Amp. The culture was incubated  at 37°C until an OD of 0,7.</p>

     <p>A 1 mL  preinduction sample (PI) was taken (0,7 (favoured OD) : 0,7 (actual OD) = 1 mL).  After that the cultures were induced with 20 µL IPTG for 2h.</p>

     <p>An  induction sample (I) was taken (520 µL with an OD of 1,34).</p>

     <p>PI and  I were centrifuged at 14000 rpm for 1 min, the supernatant was discarded and  the pellet resuspended in 60 µL water and 40 µL SDS-Buffer.</p>

     <p>The  samples were analysed on an SDS-PAGE gel with 5 and 10 µL volume per sample.  Additionally PI and I from the expression test with transformed piGEM-021 were  loaded on the gel as well as a comparison.</p>

     <legend><a name="exp19.6b">19.6 Protein overexpression with transformed E.Coli BL21 (DE3) (piGEM-021)</a></legend>

     <p>For  that purpose 2 x 1 L LB were inoculated with a solution of LB and every clone  on the cotransformation plate. All clones from the piGEM-021 + FlaiS  cotransformation plates were picked and resuspended in 1 mL LB. 500 µL were  added t the 1 L culture flasks. Each flask was additionally mixed with 1 mL Ampicillin  (100 mg/ mL) and 1 mL Canamycin (50 mg/ mL).</p>

     <p>The  cultures were incubated overnight at 30°C.</p>

     <p>Aim: transformation of plasmid into Bacillus subtilis WT3610</p>

     <p>Colonies  were grown on the plates with transformed piGEM-021. The blue/ white screening  showed positive transformed blue clones.   3 clones of different morphology per plate were picked and used for  inoculation of LB-MLS (4 mL LB, 4 µL Lincomycin, 4 µL Erythromycin). Incubation  was carried out overnight at 30°C with the 3 cultures.<strong> </strong></p>

     <legend><a name="exp19.6">19.6 Protein overexpression with transformed E.Coli BL21 (DE3) (piGEM-021)</a></legend>

     <p>The  overnight induced cells were raised till an OD of 2-3  and spinned down at 4000 rpm.  The pellets were frozen in liquid nitrogen  and stored at -80°C.</p>

     <p>The 3  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.</p>

     <p>Then  the temperature was shifted to 42°C for 6h. Unfortunately the temperature  decreased to 33°C because of a wrong incubator setting. New overnight cultures  were inoculated with 50 µL from those used in the morning. The new cultures  were incubated overnight at 30°C.</p>

     <td>pet24d-Hag-Spe1-Cup  constr. Cl. 4</td>

     <p>The 3  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.</p>

     <p>Then  the temperature was shifted to 42°C for 6h.</p>

     <p>After  the heat shock 100 µL dilutions from 10-4 to 10-6 of each  culture were plated out on MLS-X-Gal so that 9 plates could be incubated  overnight at 42°C.</p>

     <legend><a name="exp19.7b">19.7 Purification of Flagellin-Hag-SpeI from frozen pellet</a></legend>

     <p>The  pellets which were stored at -80°C were resuspended in 20 mL buffer A and  cracked with the microfluidizer. The cell parts were spinned down at 20000 rpm  for 20 min at 4°C. The supernatand was transferred to a new 50 mL falcon and  stored on ice (!!!).</p>

     <li>taking 40 µL supernatant  (load)+ 10 µL SDS-Buffer - L-Sample</li>

     <li>taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample</li>

     <li>taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample</li>

     <li>hanging pipe on column, 6x2ml Evolutions - E 1-6</li>

     <li>taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-sample 1-6</li>

     <p>Meanwhile  the Elutions 1-6 were pooled (combined 12 mL) in a 50 mL falcon and transferred  in a concentrator column which was centrifuged until the volume in the filter  was 1,5 mL for injection into the gel filtration station. </p>

     <p>Parallel  the L-, FL-, W- and E 1-6-samples were analysed on a SDS-PAGE gel in order to  proof the purification in the elution fractions.</p>

     <p>The gel  shows a huge protein concentration in E1 and E2 so that the purification could  be continued with the next step – gel filtration. For that purpose the filtrate  out of the concentrator was transferred into a new 2 mL tube resuspending very  well without air bubbles. </p>

     <p>17 mL  of GeFi-buffer werd injected into the loop. After that the protein filtrate was  injected as well. </p>

     <li>First endtimer at 90 mL volumen before fractioning</li>

     <p>After  observing a peak on the screen 40 µL of different fractions (C7 , C9, C11, D1,  D3, D5, D7) covering the peak were taken for analysis via SDS-PAGE gel.</p>

     <p>The gel  shows that the elutions contain the protein concentrated. The Elutions C7-D3  were pooled and transferred to the concentrator until an end volume of 200 µL.</p>

     <p>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.</p>

     <p>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.</p>

     <legend><a name="exp_10">results of Sequencing  of cPCR samples from pMAD transformation from 08.07.14</a></legend>

     <td>Hag-Spe1 in B.s.  genome clone 1</td>

     <td>No SpeI insertion</td>

     <td>Hag-Spe1 in B.s.  genome clone 3</td>

     <td>No SpeI insertion</td>

     <td>Hag-Spe1-DARPin  in B.s. genome clone 1</td>

     <td>Hag-Spe1-DARPin  in B.s. genome clone 3</td>

<p>The sequencing will be repeated with the reverse Primer instead of the  forward primer. In case of the Hag-SpeI clones, different clones have to be  taken for sequencing.</p>

     <td>Hag-Spe1 in B.s. genome clone 7</td>

     <td>Hag-BamHI-rv</td>

     <td>Hag-Spe1-DARPin  in B.s. genome clone 1</td>

     <td>Hag-BamHI-rv</td>

     <td>Hag-Spe1-DARPin  in B.s. genome clone 3</td>

     <td>Hag-BamHI-rv</td>

     <p>The  pellets which were stored at -80°C were resuspended in 20 mL buffer A and  cracked with the microfluidizer. The cell parts were spinned down at 20000 rpm  for 20 min at 4°C. The supernatand was transferred to a new 50 mL falcon and  stored on ice (!!!).</p>

     <li>taking 40 µL supernatant  (load)+ 10 µL SDS-Buffer - L-Sample</li>

     <li>taking 40 µL of flow through + 10 µL SDS-buffer - FT-Sample</li>

     <li>taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Sample</li>

     <li>hanging pipe on column, 6x2ml Evolutions - E 1-6</li>

     <li>taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E-sample 1-6</li>

<p>The gel  shows a huge protein concentration in E1 and E2 so that the purification could  be continued with the next step – gel filtration. For that purpose the filtrate  out of the concentrator was transferred into a new 2 mL tube resuspending very  well without air bubbles. </p>

   <li>Flow rate: 2500-3000  µL / min</li>

   <li>max. pressure: 0,60 – 0,65 mPa</li>

   <li>First  endtimer at 80-90 mL volumen before fractioning </li>

     <p>After  observing a peak on the screen 40 µL of different fractions (C7 , C9, C11, D1,  D3, D5, D7) covering the peak were taken for analysis via SDS-PAGE gel.</p>

<p>The gel  shows that the elutions contain the protein concentrated. The Elutions  C7-D7were pooled and transferred to the concentrator until an end volume of 200 µL.</p>

     <p>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 9clones were proven for the right  integration of the insert although flipping out the pMAD backbone.</p>

     <p>The  plates were incubated at 42°C overnight.</p>

     <legend><a name="exp13.66">13.66 Digest of piGEM-002 NcoI/ SpeI</a></legend>

     <p>Aim: digest for isolation of piGEM-002 without GFP, replacing it with mCherry out of pMA17 from Daniel Schindler</p>

     <p>piGEM-002 was digested with NcoI/ SpeI in order to isolate the backbone without the GFP so that an mCherry could be integrated via Gibson assembly.</p> <table width="100%" border="1">

     <th scope="col">Volume (µl)</th>

     <th scope="row">piGEM-002 (73 ng/µL)</th>

     <th scope="row">Cutsmart 10x</th>

     <th scope="row">SpeI</th>

     <th scope="row">NcoI</th>

<p>The cut vector was isolated via gel extraction.</p>

     <p>Aim: digest for isolation of piGEM-002 without GFP, replacing it with mCherry (Part I and II)</p>

     <th scope="row">Template: pMA17 1:10 (25 ng/ µL)</th>

     <th scope="row">Phusion</th>

</table

    ><p>The PCR was ran as a gradient from 55-65 °C annealing temperature overnight.</p>

     </div>

     <td>Hag-Spe1 in B.s. genome clone 7</td>

     <td>Hag-Spe1-DARPin  in B.s. genome clone 1</td>

     <td>1 dot mutations in domain, deletion in HagII/ Frameshift</td>

     <td>Hag-Spe1-DARPin  in B.s. genome clone 3</td>

     <td>2 dot mutations in domain, deletion in HagII/ Frameshift</td>

     <p>Aim: finding out if the mutations have been in the constructs or made by bacillus</p>

     <td>piGEM-005 – Hag-SpeI</td>

     <td>piGEM-016 – Hag-Cup</td>

     <td>piGEM-018 – Hag-DARPin</td>

     <p>Gel analysis shows that the PCR was successful at every annealing temperature. MgCl2 and DMSO were important for the function of the polymerase. </p>

     <th scope="col">Volume (µl)</th>

     <th scope="row">Insert I (mCherry I 460 ng/µL)</th>

     <th scope="row">Insert II (mCherry II 460 ng/µL)</th>

     <th scope="row">piGEM-002 Nco/SpeI (5,5 ng/ µL)</th>

<p>The mix was incubated 1h at 50 °C  , transformed into E. Coli XL1-Blue  and  plated out on LB-Amp plates. Incubation overnight at 37°C. </p>

     <p>Aim: finding out if the mutations have been in the constructs or made by bacillus</p>

     <td>piGEM-005 – Hag-SpeI</td>

     <td>piGEM-016 – Hag-Cup</td>

     <td>piGEM-018 – Hag-DARPin</td>

     <p>The Flank was checked by sequencing and showed no deletion in the Flank.</p>

     <th scope="col">1:2 (µl)</th>

     <th scope="col">1:3 (µl)</th>

     <th scope="row">Insert I (mCherry  I 460 ng/µL)</th>

     <th scope="row">Insert II (mCherry  II 460 ng/µL)</th>

     <th scope="row">piGEM-002 Nco/SpeI (42 ng/ µL)</th>

<p>Gibson reactions were transformed into E.Coli XL1-Blue after 1h incubation at 50°C and plated out on LB-Amp. Incubation was  proceeded at 37°C overnight.</p>

     <p>Overnight  cultures of Bacillus Transformants (DARPin clone 3 &amp; 8, Cup1-1 clone 2  &amp; 3, Hag-SpeI clone 7), PY79 and WT3610 were incubated at 37°C.</p>

     <p>Aim: Check motility of Bacillus transformants</p>

     <p>The overnight cultures were set to an OD of 10. 10 µL of each culture were spotted on a swarming and a swimming plate. The distance of the front line was measured after periods of time.</p>

     <th colspan="2" scope="col">Hag-SpeI cl. 7</th>

     <p>Aim: Check motility of Bacillus transformants</p>

     <p>In  order to repeat the swarming assay new overnight cultures of  Bacillus Subtilis WT3610, the mutants DARPin  clone 3 &amp; 8, Cup1-1 clone 2 , Hag-SpeI clone 7 in 10 mL LB and incubated at  37°C.</p>

     <p>The  overnight cultures were set to an OD of 10. 10 µL of each culture were spotted  on a swarming and a swimming plate. The plates with the drops were dried for  half an hour on the bench. The distance of the front line was measured after  periods of time. The measurement at t=0 is the radius of the start spot. The  following measurements refer to the difference between the start spot and the  new front line.</p>

     <th scope="row">Hag-SpeI cl. 7</th>

     <legend><a name="exp13.70">13.70 screening clones of Gibson plates</a></legend>

     <legend><a name="exp_15">Sequencing cPCR Products from B.s. mutants with Flank fw/rv</a></legend>

     <td>B.s.  – Hag-SpeI Cl.7</td>

     <td>B.s. – Hag-SpeI Cl.7</td>

     <td>B.s. – Hag-Cup1-1 Cl.2</td>

     <td>B.s. – Hag-Cup1-1 Cl.2</td>

     <legend><a name="exp19.9">19.9 Protein overexpression with transformed E.Coli BL21 (DE3) (piGEM-021)</a></legend>

     <p>Aim: Overexpression of flagellin modifications for a second crystallization try</p>

     <p>After newly packed GeFi Columns we decided to purify the Fla-Cup again for crystallization.</p>

     <p>For that purpose 2 x 1 L LB were inoculated with a solution of LB and  every clone on the cotransformation plate. All clones from the piGEM-021 +  FlaiS cotransformation plates were picked and resuspended in 1 mL LB. 500 µL were added to the 1 L culture flasks. Each flask was additionally mixed with 1  mL Ampicillin (100 mg/ mL) and 1 mL Canamycin (50 mg/ mL).</p>

     <p>The cultures were incubated overnight at 30°C.</p>

     <p>In order to check the expression of  the flagellum in the bacillus mutants cultures  in 100 mL LB medium were grown until an OD of 0,8 at 37°C. 10 mL culture were centrifuged  at 4000 rpm for 10 min to get the pellet. The pellet was resuspended in 60 µL water and 40 µ SDS-loading buffer. After that the resuspension was analysed via  SDS-PAGE. As control samples were overproduced E.Coli flagellin modifications  used. The analysed culture samples should run on the same level as the Coli  produced samples in case of a positive expression.</p>

     <p>The gel showed that the Hag-SpeI mutant generates a band on the same level as the WT3610 which is a positive result because the modified flagellin with speI site has no remarkable change of molecule mass. The Bacillus transformation was successful though.</p>

     <p>The mutants with the inserted Cup and DARPin  showed no significant expression of the flagellin. Either the export of the flagellin  is not possible for Bacillus because of the size/ folding of our constructs and  ends in degradation of our modifications or the expression is too low to be  analysed via SDS-PAGE. In that case it would be   not efficient enough for usage. The results of the SDS-PAGE fits to the  previously made Swarming assays. </p>

     <p>A new strategy has to be planned for further  work.</p>

     <legend><a name="exp_16">Sequencing cPCR Products from B.s. mutants with Flank fw/rv</a></legend>

     <td>B.s. – Hag-SpeI Cl.7</td>

     <td>B.s. – Hag-SpeI Cl.7</td>