Team:Aachen/lab-notebook
Notebook
Week 1
25.04.2019
Prepared six cultures of 3 mL LB medium with 10 µL 1000x kanamycin (from prior teams, one triplet old from 26.03.2019 and one triplet new from 11.04.2019) and inoculated with one of the following strains:
E. coli BL21 (DE3) peT28a(+)::eGFP-17x-TEV-LCI
E. coli BL21 (DE3) peT28a(+)::eGFP-17x-TEV-TA2
E. coli BL21 (DE3) peT28a(+)::eGFP-17x-TEV (control)
Cultures were incubated at 37°C, 250 rpm overnight.
26.04.2019
The overnight cultures showed good growth and a plasmid prep was performed according to the instructions of the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel.
Resuspended plasmids were stored at -20°C.
Week 4
14.05.2019
Prepared 1L FSM medium supplemented with EDTA-chelated trace element mixture and Vitamin solution according to their respective recipes. The FSM medium was prepared with Sodiumlactate instead of Potassiumlactate according to the modified recipe.
Let bacterial cultures (M. gryphiswaldense and M. magnetotacticum) of the DSMZ warm up to room temperature slowly.
M. gryphiswaldense
Preparation of one 100 mL Erlenmeyer flask with 100 mL FSM.
Inoculated flask with 10 mL DSMZ bacterial strain culture.
M. magnetotacticum
Preparation of one 100 mL Erlenmeyer flask with 100 mL FSM.
Preparation of one 50 mL Erlenmeyer flask with 50 mL FSM.
Both flasks were inoculated using 6 mL of DSMZ bacterial strain culture.
All cultures were incubated in a shaker at 28°C at 100 rpm.
17.05.2019
Both flasks inoculated with M. magnetotacticum on the 14.05.2019 show no visible growth/opaqueness.
The flask inoculated with M. gryphiswaldense was opened on the 16.05.19 under the clean bench for oxygenation. The medium looks slightly opaque, OD = 0,232.
Microscopic evaluation showed no contamination with other bacteria.
Two new 100 mL Erlenmeyer flasks were prepared, each containing 50 mL fresh FSM medium and 50 mL of the old culture. Both flasks are kept at 28°C and 100 rpm, but one is sealed tightly to allow for anaerobic conditions, while in the other, oxygen can pass freely.
Week 5
20.05.2019
Microscopic evaluation of both M. magnetotacticum flasks from the 14.05.2019 revealed only dead cells without significant increase in cell density, so both were autoclaved.
Microscopic evaluation of the M. gryphiswaldense culture from the 14.05.2019 showed significant growth in the aerobic culture (OD = 0,998) and less growth in the anaerobic culture (OD = 0,245), but with both flasks showing high death rates with many immobile, dead cells, higher in the anaerobic culture.
Three new cultures were prepared from the aerobic culture:
Two 250 mL flasks with each 50 mL FSM and 50 mL of the old culture and one 100 mL flask with 30 mL FSM and 20 mL of the old culture.
Two new cultures were prepared from the anaerobic culture:
Two 250 mL flasks with each 50 mL FSM and 50 mL old culture.
All five new cultures were kept at 28°C, 100 rpm under aerobe conditions to allow for faster growth.
22.05.2019
Microscopic evaluation of the M. gryphiswaldense culture from the 20.05.2019 showed mostly dead or immobile cells with a contamination of coccobacilli in one of them.
1 mL of a sterile filtrated 2.7M KCl solution was added to one 100ml flask (aerobic culture #1) in order to check for a potential lack of potassium due to the modified recipe from the 14.05.2019.
23.05.2019
Microscopy and macroscopic comparison of M. gryphiswaldense flasks with and without KCl supplementation showed no significant difference in growth, so the use of sodium lactate instead of potassium lactate does not seem problematic.
The cells were not mobile with most of them probably dead.
Another flask was contaminated with coccobacilli, so they were all autoclaved and wasted.
Inoculation of new flasks with M. gryphiswaldense.
One 500ml flask with 100ml Medium and 500μl culture taken from aerobic culture #2.
One 500ml flask with 50ml Medium and 500μl culture taken from aerobic culture #2.
Both flasks were kept at 28°C, 100 rpm under aerobic conditions.
Preparation of activated charcoal (AC) agar plates according to recipe.
AC agar plates were supplemented with different antibiotics (Ampicillin, Kanamycin, Streptomycin, Rifampicin, Chloramphenicol) to check for natural resistances of M. gryphiswaldense.
1 mL of uncontaminated culture each was centrifuged at 3500 rpm for 2 min, the supernatant was decanted and the remaining about 100 µl were plated onto the different antibiotic AC agar plates.
Week 6
29.05.2019
Ordered IDT-gBlocks arrived (OmpA-eGFP-17xHelix-TEV-LCI and OmpA-eGFP-17xHelix-TEV-TA2) and were stored at 4°C.
Two cryostocks of E. coli BL21 (DE3) were made according to protocol and stored at -80°C.
Chemically competent E. coli BL21 (DE3) were made according to protocol and stored at -80°C.
100 mL of TE-buffer was prepared according to recipe.
31.05.2019
Polyurethane (PU), polystyrene (PS), polyethylene (PE) and polypropylene (PP) samples were shredded in a coffee grinder (PP: pipet tips, PS: microtiter plates, PE: cables, PU: hose).
Ground samples were put in reaction tubes together with 0,75 g glass beads and 1 mL H2O and put into the pebble mill for 9 min.
PP did not grind up.
Samples (all but PP) and Impranil (industrial PU microparticles) were evaluated microscopically.
Week 7
04.06.2019
We recieved new cultures from Prof. Schüler from Bayreuth:
Magnetospirillum gryphiswaldense wildtype MSR-1
Magnetospirillum magneticum AMB-1
Rhodospirillum rubrum “magneticum” ABG6x_feoAB
Escherichia coli BW29427
Stocks of each new culture were created.
2 mL of each culture were centrifuged at 900 rpm for 5 min.
1 mL supernatant was taken off and the pellet was resuspended in the remaining 1 mL.
500 μL of resuspended pellet were mixed with 500 μL 50% Glycerole, frozen in liquid nitrogen and stored at -80 °C.
Escherichia coli BW29427 was stored at 4 °C.
1 L of 10X Sistrom's minimal medium (SMM) was prepared according to recipe, but it was impossible to reach pH 7, so it was discarded.
100 mL of trace element solution for SMM was prepared according to recipe and stored at 25°C.
100 mL of vitamin solution for SMM was prepared according to recipe and stored at 4°C in the dark.
Two separate cultures of each strain received were made with 1.2 mL of each culture and 10.8 mL FSM medium in closed 15 mL falcon tubes. All were stored at room temperature, one each with and without day light.
05.06.2019
Macroscopic evaluation of AC agar plates from the 23.05.2019 revealed no growth on rifampicin and kanamycin, but round, white colonies on every other plate.
Microscopic evaluation of one colony of each plate showed coccobacilli but no bacteria with the characteristic spiral shape of one of our cultures, so the contaminated plates were all discarded.
1 L of 10x SMM was prepared according to recipe.
pH was adjusted after dilution to working concentration in two steps. First 100 mL 10x SMM were diluted by adding 400 mL ddH2O and pH was adjusted with 46 platelets solid KOH to pH 6,9.
Then another 500 mL of ddH2O were added, shifting the pH to 6,99. The diluted medium was then autoclaved for sterilisation, before adding of the vitamin and trace element solutions.
The trace element solution showed insoluble particles. After sterile filtration the green solution turned blue, while the filter turned yellow. Because of that a new 1 L dilution was made with 100 μL of unsterile trace element solution added before autoclaving.
The vitamin solution was added after autoclaving as described before, because it is very heat sensitive.
The final SMM was stored at 4 °C, encased in aluminium foil for light protection and marked with "ATES" (AutoclavedTraceElementSolution).
Newly prepared sterile filtered trace element solution and vitamin solution were aliquoted in 2 mL reaction tubes and stored at 4° C with aluminium foil for light protection.
100 μL of each sterile filtered solution was given to another 1 L sterile SMM at working concentration, which was stored at 4° C with aluminium foil for light protection.
A growth experiment to determine the favourable media conditions for the growth of magnetotactic bacteria was prepared as follows, with duplicates of each strain being kept at 30°C under light conditions:
| Strain | Preculture [mL] | SMM [mL] | Colour of tube |
| M. gryphiswaldense | 1.2 | 10.8 | red |
| M. magneticum | 1.2 | 10.8 | red |
| R. rubrum magneticum | 1.2 | 10.8 | red |
| M. gryphiswaldense | 1.2 | 10.8 | yellow |
| M. magneticum | 1.2 | 10.8 | yellow |
| R. rubrum magneticum | 1.2 | 10.8 | yellow |
06.06.2019
The E. coli BW29427 we received together with other cultures by Prof. Schüler were in two 1,5 mL reaction tubes with two Agar-blocks each.
One tube was filled with 1 mL LB medium and it was stored for 3 h at 37 °C.
After the three hours the bacteria showed no growth and the experiment was cancelled due to a lack of diaminopimelic acid (DAP), since E. coli BW29427 has a DAP auxotrophy.
The tubes were stored at 4 °C.
GBlocks (OmpA-eGFP-17xHelix-TEV-LCI and OmpA-eGFP-17xHelix-TEV-TA2) arrived at the 27.05.2019 were resuspended in 100 µl TE-buffer according to protocol and their concentration was measured using nanodrop.
| Gene fragment | Concetration [ng/µL] |
| OmpA-eGFP-17xHelix-TEV-LCI | 4.5 |
| OmpA-eGFP-17xHelix-TEV-TA2 | 5.0 |
Restriction of OmpA-eGFP-17xHelix-TEV-LCI and OmpA-eGFP-17xHelix-TEV-TA2 as inserts and pET28a_pT7_eGFP-TA2 as backbone with XbaI and BlpI in Cutsmart buffer was performed according to the protocols for slow restriction digest for plasmids and inserts, respectively.
Ligation of pET28a_pT7_eGFP-TA2 with one of the inserts OmpA-eGFP-17xHelix-TEV-LCI or OmpA-eGFP-17xHelix-TEV-TA2 each was performed according to the slow ligation protocol.
07.06.2019
Both Magnetospirillum gryphiswaldense and Magentospirillum magneticum showed the best growth in FSM medium, but no growth in SMM.
Rhodospirillum rubrum magneticum showed the best growth in SMM.
New 50 mL cultures of Magnetospirillum gryphiswaldense, Magentospirillum magneticum, Rhodospirillum rubrum magneticum were made.
All 12 mL of the 15 mL falcon tube cultures from 04./05.06.2019 were transferred into 50 mL falcon tubes and filled up to 45 mL with new medium.
FSM was used for Magnetospirillum gryphiswaldense and Magnetospirillum magneticum.
SMM and SMM-ATES were used for Rhodospirillum rubrum magneticum.
One R. rubrum magneticum SMM-ATES falcon tube was only filled up to 40 mL to test different medium/headspace ratios.
All falcon tubes were stored at 30 °C without shaking, but Magnetospirillum gryphiswaldense and Magentospirillum magneticum were kept in the dark, while R. rubrum magneticum was kept under light conditions.
Heatshock transformation of E. coli DH5α with one of the new plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 made on 07.06.2019 was performed according to the protocol and plated to LB +kanamycin.
Week 8
10.06.2019
No growth was observed on LB +kanamycin plates with E. coli DH5α with pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 made on 07.06.2019.
All plates were discarded.
11.06.2019
200 mL LB agar were made according to protocol and supplemented with 200 µL Kanamycin to make LB +Kanamycin, which were stored at 4°C.
Restriction of OmpA-eGFP-17xHelix-TEV-LCI and OmpA-eGFP-17xHelix-TEV-TA2 as inserts and pET28a_pT7_eGFP-TA2 as backbone with XbaI and BlpI in Cutsmart buffer was performed according to the protocols for slow restriction digest for plasmids and inserts, respectively.
Ligation of pET28a_pT7_eGFP-TA2 with one of the inserts OmpA-eGFP-17xHelix-TEV-LCI or OmpA-eGFP-17xHelix-TEV-TA2 each was performed according to the slow ligation protocol.
Heatshock transformation of E. coli DH5α with one of the new plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 made on 07.06.2019 was performed according to the protocol and plated to LB +kanamycin.
100 mL LB medium, supplemented with DAP (1mM) was prepared according to the recipe.
1 mL medium from the reaction tube (06.06.2019) was transferred to 4 mL LB-DAP in a test tube.
1 mL LB-DAP was added to both reaction tubes containing agar blocks with E. coli BW29427.
All tubes were stored at 37 °C and 250 rpm.
Best growth of R. rubrum magneticum was observed in SMM, where sterile filtered trace element solution was added after autoclaving.
Only little growth of M. gryphiswaldense and M. magneticum was observed in FSM.
1 L of SMM working concentration was prepared as described before. Sterile filtered vitamine solution and trace element solution (100 μL each) were added after autoclaving.
Two 500 mL cultures of R. rubrum magneticum were made by transferring the 45 mL cultures from 07.06.2019 to 455 mL SMM in a 500 mL schott flask.
Note: The total volume is greater than 500 mL to generate the favourable 4:1 medium:headspace ratio.
Schott flasks were stored at 30 °C in light conditions.
One 500 mL culture of M. gryphiswaldense and on of M. magneticum was made by transferring one of the 45 mL cultures from 07.06.2019 with 455 mL fresh FSM into a 500 mL schott flask.
The schott flasks were stored at 30 °C in the dark.
R. rubrum magneticum was tested for natural antibiotic resistances by creating new 15mL falcon tube cultures (12 mL culture), where 1 mL of R. rubrum magneticum culture from 07.06.2019 in SMM ATES was added to 11 mL fresh SMM.
Different falcon tubes where supplemented with each 12 µl of 1000x antibiotic stock solutions of one of the following antibiotics: kanamycin, ampicillin, chloramphenicol, streptomycin.
Two cultures were grown without antibiotics as positive control.
All tubes were stored at 30 °C under light conditions. One positive control without antibiotics was stored at 24°C under 16/8 h light/dark conditions in a phytochamber to check for the influence of different growth temperatures.
All 15 mL falcon tube cultures of M. gryphiswaldense and M. magneticum in SMM from 07.06.2019 were discarded due to lack of growth.
12.06.2019
Two cryostocks from the overnight culture of E. coli BW29427 from 11.06.2019 were made according to protocol and stored at -80 °C.
1 L of fresh LB-medium was prepared according to recipe.
200 mL of that LB-medium were supplemented with 1mM DAP to generate 200 mL LB-DAP.
A new overnight culture of E. coli BW29427 was prepared in a 1 L Erlenmeyer flask with 100 mL LB-DAP to generate material for chemically competent cells. The flask was stored at 37°C, 250 rpm.
50 mL SOC medium was prepared according to recipe and supplemented with 1mM DAP, then aliquoted for transformation of BW29427 in 1 mL aliquots in 1.5 mL reaction tubes and were stored at -20°C.
No growth was observed on LB +kanamycin plates with E. coli DH5α with pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 made on 11.06.2019.
All plates were discarded.
Heatshock transformation of E. coli DH5α with one of the new plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 made on 11.06.2019 was performed according to the protocol and plated to LB +kanamycin.
Transformation of pET28a_pT7_eGFP-TA2 was included as positive control.
13.06.2019
Overnight culture of E. coli BW29427 was diluted to OD(600) = 0,54.
Chemically competent cells of E. coli BW29427 were prepared according to protocol and stored at -80 °C.
6 colonies each were picked from the heatshock transformation of E. coli DH5α with pBam_PmamDC_mamC-eGFP from 12.06.2019 and transferred to test tubes with 4 mL LB +kanamycin, which were incubated overnight at 37 °C, 250 rpm.
14.06.2019
Microscopic evaluation of the 500 mL R. rubrum magneticum cultures from 11.06.2019 revealed no contamination with other bacteria, yet the cells were not motile.
Heatshock transformation of the pET28a_pT7_eGFP-TA2 plasmid into E. coli BW29427 was done according to protocol as a test.
Cells were plated on LB-kanamycin plates without DAP and stored overnight at 37°C.
Heatshock transformation of E. coli DH5α with the plasmid pET28a_pT7_eGFP-TA2 was performed according to the protocol and plated to LB +kanamycin to test their competence.
None of the colonies picked on 13.06.2019 grew in liquid culture, so they were discarded.
15.06.2019
E. coli BW29427 pET28a_pT7_eGFP-TA2 grew on LB +kanamycin plates and were stored at 4°C.
(Note: E. coli BW29427 should NOT be able to grow without DAP -> first hint that there was something wrong with those cells.)
E. coli BL21 pET28a_pT7_eGFP-TA2 grew on LB +kanamycin plates, which proved their competence.
16.06.2019
6 colonies each were picked from the heatshock transformation of E. coli DH5α with the plasmid pET28a_pT7_eGFP-TA2 from 14.06.2019 and transferred to test tubes with 4 mL LB +kanamycin, which were incubated overnight at 37 °C, 250 rpm.
Week 9
17.06.2019
Heatshock transformation of the pBam_PmamDC_mamC-eGFP plasmid in E. coli DH5α was performed according to protocol.
Heatshock transformation of the pET28a-pT7-eGFP_TA2 plasmid into R. rubrum magneticum was performed according to protocol, with different variations to check for the possibility of transforming R. rubrum magneticum without biparental conjugation.
The variations were all combinations two different DNA-concentrations (0,5 μL, 5 μL), duration of the heatshock (00:30 min, 01:00 min, 01:30 min) and duration of resting phase after heatshock (1 h, 3 h, 6 h).
150 mL of LB-agar was prepared according to recipe and supplemented with 15 µL of ampicillin 1000x stock to make LB-DAP-Amp plates, which were stored at 4 °C.
Three cultures each were generated for M. magneticum in FSM and R. rubrum magneticum in SMM, with 3 mL old culture (all from 50 mL falcon tube cultures from 11.06.2019) and 9 mL fresh medium.
All cultures were stored at 30°C without shaking, M. magneticum in the dark and R. rubrum magneticum under light conditions.
These cultures were made as permanent cultures for experiments to come.
Culture PCR from overnight cultures made on 16.06.2019 of E. coli DH5α pET28a_pT7_eGFP-TA2 was performed according to protocol with “prä pT7 F” and “post tT7 R” as primers.
Agarose-gel electrophoresis was made according to protocol.
The gel was not polymerized correctly and dissolved during the run.
18.06.2019
Culture PCR from overnight cultures made on 16.06.2019 of E. coli DH5α pET28a_pT7_eGFP-TA2 was performed according to protocol with “prä pT7 F” and “post tT7 R” as primers.
Agarose-gel electrophoresis was made according to protocol.
The gel was clumpy and no sharp bands could be observed.
19.06.2019
All SMM-kanamycin plates with the heatshock transformed cells from 17.06.2019 showed colonies, which were picked and put in 5 mL SMM medium, supplemented with kanamycin in test tubes with parafilm and were stored at 30°C under light conditions.
One 500 mL R. rubrum magneticum culture was harvested according to the cell harvest protocol and the resuspended pellet was aliquoted to 1,5 mL in 2 mL reaction tubes for further testing.
| Designation | Method of cell lysis |
| F | Freeze-thaw cycles |
| U1 | Ultrasonication 5x 1 min |
| U2 | Ultrasonication 50% pulse for 3 min |
| U1+F | Ultrasonication 5x 1 min and subsequent freeze-thaw cycles |
| U2+F | Ultrasonication 50 % pulse for 3 min and subsequent freeze-thaw cycles |
U1 and U2 were centrifuged at 650 g for 5 min to remove cellular debris in a pellet, as the supernatant should contain the magnetosomes.
10 μL U1 were diluted with 490 μL ddH2O in a reaction tube with a magnet fixed to one side to pull magnetosomes to said side.
Three times 200 µL of each U1 and U2 were put into a 96-well microtiter plate with 6 magnets underneath each well to pellet magnetosomes at the bottom.
F, U1+F and U2+F were stored at -20 °C.
R. rubrum magneticum showed growth in positive control without antibiotics and in SMM+kanamycin, but no growth could be observed in SMM + chloramphenicol, streptomycin or ampicillin.
R. rubrum magneticum is naturally resistant against kanamycin.
The second positive control at 24°C and 16/8 light/dark cycle showed less growth than the positive control at 30°C and constant light conditions.
Culture PCR from overnight cultures made on 16.06.2019 of E. coli DH5α pET28a_pT7_eGFP-TA2 was performed according to protocol with “prä pT7 F” and “post tT7 R” as primers.
Agarose-gel electrophoresis was made according to protocol.
As all bands were smaller than 250 bp, we concluded that the plasmid closed without insertion of the fragment.
Week 10
24.06.2019
Three new perma-cultures of R. rubrum magneticum were made by transferring 3 mL of the old cultures from 17.06.2019 into new 15 mL falcon tubes and adding 9 mL fresh SMM, which were then stored at 30 °C in light conditions.
1 L FSM medium was prepared according to modified recipe with 27 mM potassium lactate instead of sodium lactate (6,92 g/L of 50 % potassium lactate).
A new Erlenmeyer flask culture of M. magenticum was prepared by transferring 25 mL of old culture (from 11.06.2019) into a 250 mL Erlenmeyer flask and add 225 mL fresh FSM, which was then stored at 30°C in the dark.
3 colonies were picked from heatshock transformation of E. coli DH5α with pBam_PmamDC_mamC-eGFP from 17.06.2019 and transferred to test tubes with 4 mL LB medium, which were incubated overnight at 37 °C, 250 rpm.
To check for magnetosome expression, magnets were fixed to the sides of the 500 mL schott flask cultures of M. gryphiswaldense and M. magneticum from 11.06.2019.
25.06.2019
6 plasmid preps of E. coli DH5α pBam_PmamDC_mamC-eGFP overnight culture (24.06.2019) were performed according to the QIAprep Spin Miniprep Kit.
The concentrations of the plasmid preps were measured using nanodrop.
| Designation | Concentration [ng/µL] |
| A1 | 18.2 |
| A2 | 18.4 |
| B1 | 14.8 |
| B2 | 13.8 |
| C1 | 20.9 |
| C2 | 19.7 |
Ordered gBlock fragments from IDT (EcoRI_mCherry-LCI_BamHI and EcoRI_eCFP-TA2_BamHI) arrived and were resuspended in each 50 μL TE buffer (pH = 8,0) according to protocol.
Restriction of OmpA-eGFP-17xHelix-TEV-LCI and OmpA-eGFP-17xHelix-TEV-TA2 as inserts and pET28a_pT7_eGFP-TA2 as backbone with XbaI and BlpI in Cutsmart buffer was performed according to the protocols for slow restriction digest for plasmids and inserts, respectively.
Restriction of EcoRI_mCherry-LCI_BamHI and EcoRI_eCFP-TA2_BamHI as inserts and pBam_pmamDC_mamC-eGFP as backbone with EcoRI-HF and BamHI-HF in Cutsmart buffer was performed according to the protocols for slow restriction digest for plasmids and inserts, respectively.
Ligation of pET28a_pT7_eGFP-TA2 with one of the inserts OmpA-eGFP-17xHelix-TEV-LCI or OmpA-eGFP-17xHelix-TEV-TA2 each was performed according to the slow ligation protocol.
Ligation of pBam_pmamDC_mamC-eGFP with one of the inserts EcoRI_mCherry-LCI_BamHI and EcoRI_eCFP-TA2_BamHI each was performed according to the slow ligation protocol.
Heatshock transformation of R. rubrum magneticum with pBam_pmamDC_mamC-eGFP was performed according to the protocol, with the variation of 1 min heatshock and 6 h resting phase after the heatshock. The transformation was plated onto SMM +Kanamycin + Ampicillin and stored at 30 °C under light conditions.
26.06.2019
50 mM HEPES buffer with 4 mM EDTA was prepared according to recipe for the French press.
The second 500 mL R. rubrum magneticum culture from 11.06.2019 was harvested according to the cell harvest protocol, pellets after the first centrifugation step were resuspended in 2 mL HEPES+EDTA buffer each, combined and directly used for further processing.
Solution was processed using ultrasonication with 5 min sonification at pulse 10 and an amplitude of 70%, before transferring the sample to the French press with 5 runs with 1000 bar pressure. The lysed sample was stored at -20°C.
Heatshock transformation of E. coli DH5α with one of the new pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI, pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2, pBam_pmamDC_mamC-mCherry-LCI or pBam_pmamDC_mamC-eCFP-TA2 made on 25.06.2019 was performed according to the protocol and plated to LB +kanamycin.
150 mL LB agar were prepared according to recipe and supplemented with 150µL 1000x ampicillin to make LB +amp plates, which were stored at 4°C.
27.06.2019
Fungal growth was observed in 10x SMM concentrate solution, so new 10x SMM was prepared according to recipe.
Two new 500 mL schott flask cultures were prepared by transferring old 50 mL falcon tube cultures from 19.06.2019 to sterile 500 mL schott flasks and adding 500mL SMM, while the third 50 mL falcon tube from 19.06.2019 was discarded.
3 new perma-cultures of R. rubrum magneticum were prepared by transferring 3 mL old culture from each 15 mL falcon tube (24.06.2019) and add 9 mL fresh SMM.
Both old and new falcon perma-cultures at 30°C under light conditions.
The 500 mL schott flask cultures, the cells of M.gryphiswaldense and M. magneticum showed obvious migratorial behaviour towards the source of a strong magnetic field, which implies the formation of functional magnetosomes in these cells.
The magnet was removed afterwards to not restrict the availability of nutrients and oxygen to the cells fixed to this portion of the flask.
Three new 50 mL falcon tube cultures of M. gryphiswaldense were prepared by transferring 10mL culture out of 500mL flasks and adding 40mL fresh FSM medium. Falcon tubes were stored at 30°C in the dark.
The LB +Kan plates with heatshock transformations made 26.06.2019 showed no growth for E. coli DH5α pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2, and only small colonies for E. coli DH5α pBam_pmamDC_mamC-mCherry-LCI or pBam_pmamDC_mamC-eCFP-TA2.
Heatshock transformation of E. coli DH5α with one of the plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI, pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2, made on 25.06.2019 was performed according to the protocol and plated to LB +kanamycin.
28.06.2019
Two colonies from heatshock transformation of R. rubrum magneticum from 25.06.19 were picked according to protocol and transferred into SMM + kanamycin + ampicillin.
The LB +Kan plates with heatshock transformations made 26.06.2019 showed two colonies each for E. coli DH5α pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI and pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2, and extensive growth with multiple defined colonies for E. coli DH5α pBam_pmamDC_mamC-mCherry-LCI and pBam_pmamDC_mamC-eCFP-TA2.
Week 11
01.07.2019
Three colonies each were picked from the heatshock transformation of for E. coli DH5α pBam_pmamDC_mamC-mCherry-LCI and pBam_pmamDC_mamC-eCFP-TA2 and two colonies each for E. coli DH5α pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI and pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 made at 28.06.2019 and transferred to test tubes with 4 mL LB +kanamycin, which were incubated overnight at 37 °C, 250 rpm.
02.07.2019
Picked colonies from 01.07.2019 showed no growth in LB +Kan.
Three colonies each for E. coli DH5α pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI and pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 were picked and transferred to test tubes with 4 mL LB +ampicillin and incubated at 37°C, 250 rpm overnight.
Heatshock transformation of E. coli DH5α with one of the plasmids pBam_pmamDC_mamC-mCherry-LCI or pBam_pmamDC_mamC-eCFP-TA2 was performed according to the protocol and plated to LB +kanamycin.
03.07.2019
Picked R. rubrum magneticum colonies from 28.06.2019 showed some growth, but no red colour. Microscopic evaluation revealed contamination with coccobacilli and rod cells, while some Rhodospirillum cells were moving.
Two 50 mL falcon cultures were prepared with 10 mL culture and 35mL SMM + Amp + Kan. Both cultures were stored at 30°C under light conditions.
Two times 500 mL LB medium was made according to recipe.
3 colonies were picked from LB +Kan plates with E. coli DH5α pBam_pmamDC_mamC-eCFP-TA2 made at 02.07.2019 and transferred to test tubes with 4 mL LB +Kan and incubated at 37°C, 250 rpm.
04.07.2019
200 mL LB agar was prepared according to recipe and supplemented with 200 µL 1000x kanamycin to generate LB +Kan plates, which were stored at 4°C.
CulturePCR on one picked colony with visible growth from 03.07.2019 was performed according to protocol with the following primer combinations (Position 5 was the positive control with original plasmid pBam_pmamDC_mamC-eGFP):
| Position | Forward primer | Reverse Primer | Band with Insert | Band without Insert |
| 1 | oriR6K F | KanR R | >2500bp | 1518bp |
| 2 | oriR6K F | Glylink R | 623bp | - |
| 3 | mCherry F | KanR R | - | - |
| 4 | 17xHelix F | KanR R | 373bp | - |
| 5 | oriR6K F | KanR R | >2500bp | 1518bp |
Agarose gel electrophoresis was performed according to protocol, but not successful. Initial denaturation time of CulturePCR was insufficient and the protocol was reworked to have 10 min initial denaturation time at 98°C. Positive control was successfully detected at 1518bp, which means the original plasmid does not contain our TA2-Insert.
R. rubrum magneticum cells were prepared for electron microscopy according to protocol.
Integrity of cells was confirmed by microscopic evaluation before suspension in HEPES buffer and after final suspension in 100% ethanol.
Heatshock transformation of E. coli DH5α with one of the plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 was performed according to the protocol and plated to LB +kanamycin.
Heatshock transformation of E. coli DH5α with the plasmid pBam_pmamDC_mamC-eGFP was performed according to the protocol and plated to LB +kanamycin.
05.07.2019
1 L FSM was prepared according to recipe with the variation of the use of 100μl trace element solution we received from Professor Schüler.
New Erlenmeyer flask cultures were prepared. 100 mL FSM with 50 mL culture in a 250 mL Erlenmeyer flask for M. gryphiswaldense and 400 mL FSM with 50 mL culture in a 1 L Erlenmeyer flask for M. magneticum culture from 24.06.2019. Both flasks were incubated at 28°C, 100 rpm under closed, anoxic conditions in the dark.
A new 50 mL falcon tube culture was prepared with 35 mL FSM and 10 mL M. gryphiswaldense culture from 24.06.2019.
Two new 50 mL falcon tube cultures were prepared with 35 mL FSM and 10 mL M. magneticum culture from 24.06.2019.
All cultures were incubated at 30°C in the dark.
CulturePCR on one picked colony with visible growth from 03.07.2019 was performed according to protocol with the following primer combinations:
| Position | Forward primer | Reverse Primer | Band with Insert | Band without Insert |
| 1 | oriR6K F | KanR R | >2500bp | 1518bp |
| 2 | oriR6K F | Glylink R | 623bp | - |
| 3 | mCherry F | KanR R | - | - |
| 4 | 17xHelix F | KanR R | 373bp | - |
Agarose gel electrophoresis was performed according to protocol, but not successful. Initial denaturation time of CulturePCR was insufficient and the protocol was reworked to have 10 min initial denaturation time at 98°C. Positive control was successfully detected at 1518bp, which means the original plasmid does not contain our TA2-Insert.
E. coli DH5α with one of the plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2, made on 04.07.2019 showed growth on LB +kanamycin and five colonies were transferred into test tubes with 4 mL LB, without antibiotics and incubated at 37°C, 250 rpm.
E. coli DH5α pBam_pmamDC_mamC-eGFP made on 04.07.2019 showed growth on LB +kanamycin and five colonies were transferred into test tubes with 4 mL LB, without antibiotics and incubated at 37°C, 250 rpm.
E. coli DH5α with one of the plasmids pBam_pmamDC_mamC-mCherry-LCI or pBam_pmamDC_mamC-eCFP-TA2 showed no growth and were autoclaved.
The remaining schott flask culture of R. rubrum magneticum was autoclaved due to old age and build-up of bacterial aggregates.
All remaining schott flask cultures of M. gryphiswaldense were autoclaved due to contamination with coccobacilli.
06.05.2019
E. coli test tube cultures were taken out of the incubator and stored at 4°C.
Week 12
08.07.2019
Plasmid prep of E. coli DH5α with one of the plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI or pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 and E. coli DH5α pBam_pmamDC_mamC-eGFP made on 04.07.2019, was done according to the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel, but Nanodrop concentration measurement showed negative DNA concentrations.
E. coli DH5α pBam_pmamDC_mamC-eGFP culture from 05.07.19 was transferred into two new tubes WITHOUT Ampicillin, so that a new culture can be used after 24 h of growth.
E. coli DH5α pBam_pmamDC_mamC-eGFP made 02.07.19 were picked and transferred it into 25ml LB +amp in a 250 mL Erlenmeyer flask.
Two colonies each for E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-TA2 and E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-LCI from 05.07.2019 were picked and transferred it into 25ml LB +amp in an 250 mL Erlenmeyer flask.
PCR Amplification of the inserts was done according to protocol to generate more insert sequence using a LongTaq Polymerase MasterMix and 35 cycles with the following primer combinations:
pT7_OmpA-eGFP-17xHelix-TEV-TA2 and pT7_OmpA-eGFP-17xHelix-TEV-LCI
E. coli Insert F and E. coli Insert R
mamC-mCherry-LCI and mamC-eCFP-TA2
Magneto Insert F and Magneto Insert R
PCRs were purified using the NEB PCR & DNA CleanUp Kit (20 µL elution volume) and concentrations were measured via Nanodrop:
| Insert | Concentration [ng/µL] | 260/280 |
| pT7_OmpA-eGFP-17xHelix-TEV-LCI | 734.8 | 1.87 |
| pT7_OmpA-eGFP-17xHelix-TEV-TA2 | 802.0 | 1.86 |
| mamC-mCherry-LCI | 359.5 | 1.86 |
| mamC-eCFP-TA2 | 690.4 | 1.87 |
Original gBlocks and PCR-amplified inserts were sent for sequencing to check for errors occurring during PCR:
| gBlock/Insert | ID of sequencing run |
| PCR pT7_OmpA-eGFP-17xHelix-TEV-LCI | EF30365293 |
| PCR pT7_OmpA-eGFP-17xHelix-TEV-TA2 | EF30365292 |
| PCR mamC-mCherry-LCI | EF30365291 |
| PCR mamC-eCFP-TA2 | EF30365290 |
| gBlock pT7_OmpA-eGFP-17xHelix-TEV-LCI | EF30365289 |
| gBlock pT7_OmpA-eGFP-17xHelix-TEV-TA2 | EF30365288 |
| gBlock mamC-mCherry-LCI | EF30365287 |
| gBlock mamC-eCFP-TA2 | EF30365286 |
Two 1L flasks of FSM medium were prepared according to recipe with the variation of using 27mM potassium lactate.
Two 100 mL Erlenmeyer flask cultures of M. gryphiswaldense were prepared and stored at 28°C, 100 rpm.
The M. gryphiswaldense cultures from 27.06.2019 were added new FSM until 45 mL was reached.
The light bulb for the R. rubrum magneticum culture burned out, so it was replaced by an 8.7 W, 806 lm, 2700 K bulb.
09.07.2019
200ml LB Agar were prepared according to recipe and supplemented with 200 µL 1000x Ampicillin and 0.038 g DAP to make LB +amp +DAP plates
The E. coli DH5α pBam_pmamDC_mamC-eGFP culture made 08.07.2019 did not grow in LB +amp, which is a strong indicator that no plasmid was present in this culture, so it was autoclaved.
The 25 mL E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-TA2 and E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-LCI cultures made 08.07.2019 did grow and they were concentrated 4-fold by centrifugation and resuspension to increase plasmid concentracions. Plasmid prep was performed using the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel, and concentration measurements showed concentrations between 56.5 ng/µL and 69.4 ng/µL with 260/280 ratios of 1.79 to 1.85. Preps were stored at -20°C.
100 µL of the 25 mL E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-TA2 culture #4 were plated onto LB +kan and stored at 37°C.
Cryostocks of the four 25 mL E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-TA2 and E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-LCI cultures, made 08.07.2019 were made according to protocol.
All remaining cultures were autoclaved.
Four colonies each from plates with E. coli DH5α pET28a_pT7_eCFP-17xHelix-TEV-TA2, E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-LCI and E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-TA2 from 12.06.19 were picked, plated on LB +kan and inoculated in 4 mL LB-Kan in test tubes. They were stored at 37°C, test tubes at 250 rpm.
Heatshock transformation of two of each E. coli DH5α and E. coli BW29427 with the plasmid pBam_pmamDC_mamC-eGFP was performed according to protocol and plated to LB +amp for E. coli DH5α and LB +amp +DAP for E. coli BW29427, which were stored at 37°C.
50 mL SOC medium was prepared according to recipe and 1 mL aliquots were stored at -20°C.
Both M. gryphiswaldense cultures in 100 mL Erlenmeyer flasks from 08.07.2019 were contaminated with coccobacilli and were autoclaved.
Due to slower growth of R. rubrum magneticum the lightbulb was changed to an Osram 25 W, 230 V bulb.
10.07.2019
Two new M. gryphiswaldense cultures were made with 50 mL FSM in 100 mL Erlenmeyer flasks, with bacterial material from cryostock and incubated at 28 °C, 100 rpm.
Sequencing results for the gBlocks arrived; 6 of them did not read past 20 bp and the remaining two PCR E. coli TA2 and PCR Magneto TA2 showed 100% sequence identity for the first 800 bp.
Further samples were prepared for sequencing:
| PCR DNA | ID of sequencing run | read direction |
| E. coli LCI | EF30365285 | Reverse |
| E. coli LCI | EF30365284 | Forward |
| E. coli TA2 | EF30365283 | Reverse |
| E. coli TA2 | EF30365282 | Forward |
| Magneto LCI | EF30365294 | Reverse |
| Magneto LCI | EF30365295 | Forward |
| Magneto TA2 | EF30365296 | Reverse |
| Magneto TA2 | EF30365297 | Forward |
To test for efficiency of CulturePCR vs ColonyPCR, both were performed according to protocols on the E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-LCI and E. coli DH5α pET28a_pT7_OmpA-eCFP-17xHelix-TEV-TA2 made on 09.07.2019.
Agarose gel electrophoresis was performed using 1.2% Agarose, 1kb+ ladder and 120V for 20 min.
PCR did not seem to work, yet at least DNA was present in the slots of ColonyPCR samples.
PCR amplification was performed according to protocol for the isolated pET28a_pT7_OmpA-eCFP-17xHelix-TEV-TA2 and pET28a_pT7_OmpA-eCFP-17xHelix-TEV-LCI made on 09.07.2019 with the primers E. coli Insert F and E. coli Insert R.
New LB +kan masterplates were made from the originally received plates from 25.03.2019 and incubated at 37°C.
E. coli BL21 (DE 3) pET28a(t)::his-eGFP-17x-TEV (negative control)
E. coli BL21 (DE 3) pET28a(t)::his-eGFP-17x-TEV-LCI
E. coli BL21 (DE 3) pET28a(t)::his-eGFP-17x-TEV-TA2
Agarose gel electrophoresis was performed on the amplified inserts from 09.07.2019 with 1.2% Agarose, 1 kb+ ladder and 120V for 20 min, which showed clear indication of the E. coli Insert LCI and both Magneto Inserts at 1500 bp.
11.07.2019
Sequencing results for the mamC-mCherry-LCI insert showed the insert to be correctly synthesized. Eurofins stops sequencing after 1 kb!!
Three new 15 mL perma-cultures for R. rubrum magneticum were made by transferring 1 mL of old culture each into new falcon tubes and filling it up with 11 mL fresh SMM, which were then stored at 30 °C in light conditions. The three oldest cultures were then discarded.
ColonyPCR was performed according to protocol to test for pBam_pmamDC_mamC-eGFP (primers: oriR6K F and KanR R) in 16 colonies of E. coli BW29427, which were picked from LB +amp +DAP plates. A masterplate for those 16 colonies was made and stored at 37°C.
Agarose gel electrophoresis was performed on the ColonyPCR 1-16 and the PCR amplification made 10.07.2019 with 1.2 % agarose, 50V for 22 min.
Colonies 3, 4, 7, 9, 13 and 16 were picked from the masterplate and put into 4 mL LB +DAP and 4 mL LB +DAP +Amp and incubated at 37°C, 250 rpm.
Amplification of Magneto LCI and Magneto TA2 was performed like on 08.07.2019, with a final concentration after CleanUp of:
Magneto LCI: 277,4 ng/μL, 260/280: 1.83
Magneto TA2: 284,8 ng/μL, 260/280: 1.81
Week 13
15.07.2019
Heatshock transformation of E. coli BW29427 with the plasmids pBam_pmamDC_mamC-mCherry-LCI and pBam_pmamDC_mamC-eCFP-TA2 was performed according to protocol, plated to LB +amp +DAP and stored at 37°C.
The M. gryphiswaldense cultures showed no growth over the weekend, so two new cultures were inoculated from cryostock in a test tube with 3 mL FSM and incubated at 30 °C, 250 rpm.
A new 500 mL culture of R. rubrum magneticum was made by transferring 50 mL of old 500 mL culture into a new schott flask with 400 mL SMM, which was stored at 30 °C in light conditions.
The old 500 mL culture was autoclaved.
Samples of M. magneticum culture were prepared for TEM microscopy according to protocol and stored at -20°C.
A new culture was made from the M. magneticum culture made 05.07.019 with 100 mL old culture in 400 mL FSM in a 1 L Erlenmeyer flask and incubated at 28°C, 100 rpm.
The M. magneticum culture made 05.07.019 was harvested according to protocol and stored at -20°C.
Heatshock transformation of E. coli BL21 with the plasmids pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI and pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 was performed according to protocol, plated to LB +kan and stored at 37°C.
200 mL LB Agar were prepared according to protocol and supplemented with 200 μL Kanamycin to make LB +kan plates.
16.07.2019
The M. gryphiswaldense 3 mL cultures showed no growth.
The 1 L Erlenmeyer flask culture of M. magneticum showed growth.
The prepared TEM samples of M. magneticum in ethanol showed only cell debris under the microscope, so they were discarded.
E. coli BW29427 showed colonies on LB +DAP +amp, which were too small for ColonyPCR yet.
ColonyPCR was performed on the heatshock transformation on E. coli BL21 done 15.07.2019 according to protocol with the primer combinations präpT7 F + OmpA R and 17xHelixF/mCherryF (depending on plate) and posttT7 R.
Agarose gel electrophoresis was performed with 3% agarose, 50bp ladder and 120V for 20 min.
Restriction and ligation seemed to work, as the bands are as expected O: 415 bp and I: 602 / 606 bp.
Notes: in colony 1 was a mistake during PCR preparation, as in colony 6, which had the double amount of primers added.
17.07.2019
ColonyPCR was performed on 8 colonies each of E. coli BW29427 pBam_pmamDC_mamC-mCherry-LCI and E. coli BW29427 pBam_pmamDC_mamC-eCFP-TA2 (made 15.07.2019), with a masterplate made for both, which was incubated at 37°C.
PCR was done with two primer combinations per colony:
For LCI: ori6K + Glylink R (623 bp), mCherry F (LCI) + KanR R (918 bp)
For TA2: ori6K + Glylink R (623 bp), 17xHelix (TA2) + KanR R (373 bp)
Agarose gel electrophoresis was done with 1.2 % Agarose gel, 120 V for 20 min.
Unexpected secondary bands were found.
In the 500 mL culture and 500 mL FSM in 1000 mL Flask culture of M. magneticum, only coccobacilli could be detected, so both of them were autoclaved.
A new 1 L 1x SMM was prepared by dilution from 10x SMM, added vitamin and trace element solution after autoclaving.
Three new R. rubrum magneticum cultures were made in 15 mL falcon tubes and the oldest three were discarded.
100 mL of 0,1 M DAP-solution were prepared and stored at 4°C.
200 mL LB agar were made according to recipe and supplemented with 1 mM DAP and 200 µL 1000x ampicillin to generate LB +amp +DAP plates, which were stored at 4°C.
Two cultures of E. coli BL21 from LCI-colony 3 and TA2-colony 5 were made in 10 mL LB +kan in an 100 mL Erlenmeyer flask and incubated at 37 °C, 250 rpm.
Masterplates from ColonyPCR were stored at 4°C.
18.07.2019
Two Cryostocks each of E. coli BL21 pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI and E. coli BL21 pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 were made according to protocol.
A ColonyPCR was performed on 8 colonies each of E. coli BW29427 pBam_pmamDC_mamC-mCherry-LCI and E. coli BW29427 pBam_pmamDC_mamC-eCFP-TA2 like on 16.07.2019.
In addition, a PCR with only the respective vectors and the same primers was made to check on the outcome of ligation.
Agarose gel electrophoresis was made with 140V for 45 min, which turned out negative, because the time was far too long and the samples were lost to the buffer.
100mM IPTG solution was made according to recipe, sterile filtrated and stored at -20°C.
200 µL each E. coli BL21 culture (17.07.2019) were transferred to 10 mL LB+kan in an 100 mL Erlenmeyer flask and inoculated with 10 µL IPTG, incubated at 37°C, 250 rpm.
Week 14
22.07.2019
1 L LB medium and 1 L LB agar were made according to recipe.
One colony each of masterplates from 16.07.2019 were transferred into test tubes with 4 mL LB+kan and incubated at 37°C, 250 rpm.
The E. coli BL21 pET28a_pT7_OmpA-eGFP-17xHelix-TEV-LCI and E. coli BL21 pET28a_pT7_OmpA-eGFP-17xHelix-TEV-TA2 cultures were induced with 90 μL IPTG.
Microscopic evaluation after 4,5 h showed only low fluorescence and binding to plastic particles (impranil), while microscopy of impranil alone showed no fluorescence.
1.2% agarose gel was made according to recipe.
23.07.2019
The M. magneticum cultures made from cryostocks showed no growth, so they were discarded.
Two new cultures were made in 250 mL Erlenmeyer flask with 25 mL FSM and 3 mL M. magneticum culture from 500 mL flask stored on shaker at 28 °C, 100 rpm.
Cultures for E. coli BW29427 from 22.07.2019 showed not enough growth for plasmidprep, so two 100 mL culture in 1000 mL Erlenmeyer flask were made with 4 mL culture (made on 22.07.2019) and 100 mL LB + Amp + DAP, stored at 37 °C, 250 rpm.
Three colonies each were picked from the new masterplates made 10.07.2019 and transferred into test tubes with 4 mL LB (for BL21 without inserts as negative control) and LB +kan for E. coli BL21 pET28a_pT7_eGFP-LCI and E. coli BL21 pET28a_pT7_eGFP-TA2 cultures, incubated at 37°C, 250 rpm.
24.07.2019
Plasmid prep of E. coli BW29427 cultures made 23.07.2019 was done according to the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel, concentration was measured using Nanodrop.
| Plasmid | Concentration batch 1 [ng/µL] | Concentration batch 2 [ng/µL] |
| pBam_pmamDC_mamC-eGFP | 407.6 | 473.6 |
| pBam_pmamDC_mamC-mCherry-LCI | 512.0 | 484.3 |
| pBam_pmamDC-mamC-eCFP-TA2 | 518.9 | 515.0 |
Agarose gel electrophoresis was done on pBam_pmamDC_mamC-mCherry-LCI batch 1 with 1.2% agarose, 1kb+ and 120V for 20 min
No bands were visible, the measured DNA was most likely genomic DNA.
A slow digest was performed on pBam_pmamDC_mamC-mCherry-LCI batch 1 and batch 2 according to protocol, using EcoRI-HF and BamHI-HF.
Agarose gel electrophoresis was done on the digest with 1.2% agarose, 1kb+ and 120V for 20 min.
E. coli BL21 cultures from 21.07.2019 were induced with 100 µL IPTG for microscopy and incubated at 37°C, 250 rpm.
26.07.2019
DNA-purification was performed on mamC plasmids from plasmidprep and restriction from 24.07.2019 using the NEB PCR & DNA CleanUp Kit.
Agarose gel electrophoresis was perfomed with 1.2% agarose, 1kb+, 120V for 20 min with no bands found under UV light.
Three additional colonies from masterplates with E. coli BW29427 pBam_pMamDC_mamC-mCherry-LCI and E. coli BW29427 pBam_pmamDC_mamC-eCFP-TA2 were picked and transferred into test tubes with 4mL LB +Amp +DAP each and incubated at 37°C, 250 rpm to eventually do a new plasmid prep.
Slow ligation of purified plasmid pBam_mamDC_mamC-eGFP and mamC-mCherry-LCI/mamC-eCFP-TA2 inserts was performed according to protocol and incubated overnight.
27.07.2019
Heatshock transformation of E. coli BW29427 with the ligation from 26.07.2019 was performed according to protocol and plated to LB +amp +DAP.
28.07.2019
8 colonies each were picked from the plated transformation done 27.07.2019 and transferred into test tubes with 4 mL LB +amp +DAP and incubated at 37°C, 250 rpm.
Week 15
29.07.2019
Plasmidprep of E. coli BW29427 pBam_mamDC_mamC-mCherry-LCI colonies 3 and 4 and E. coli BW29427 pBam_mamDC_mamC-eCFP-TA2 colony 8 was done using the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel.
DNA concentration was measured using Nanodrop:
| Colony | Batch# and concentration [ng/µL] | ||||
| 1 | 2 | 3 | 4 | 5 | |
| LCI 3 | 188.6 | 176.1 | 161.4 | 202.9 | 145.4 |
| LCI 4 | 155.5 | 219.0 | 171.8 | 138.7 | 149.2 |
| TA2 8 | 33.0 | 51.3 | 57.1 | 69.3 | 64.0 |
ColonyPCR was performed on E. coli BW29427 pBam_mamDC_mamC-mCherry-LCI colonies 3 and 4 + 5 additional colonies and E. coli BW29427 pBam_mamDC_mamC-eCFP-TA2 colony 8 + 4 additional colonies according to protocol, with the following primer combinations:
TA2: 17xHelix F and KanR R
LCI: mCherry F and KanR R
A masterplate was made with the colonies used on LB +DAP +Kan and stored at 37 °C.
The PCR thermocycler broke during PCR protocol, but agarose gel electrophoresis was performed nonetheless, since 27 cycles should have already been run.
Agarose gel electrophoresis was performed with 3% agarose, 50 bp, 120V for 20 min.
31.07.2019
Plasmid prep of E. coli BW29427 pBam_mamDC_mamC-mCherry-LCI (culture from 30.07.19) was done using the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel.
PCR of plasmidprep was performed according to protocol with the following primers:
O: OriR6K + Glylink; I: KanR + mCherry
Quick restriction digest of ligated plasmid from 26.07.19 with EcoR1 and Blp1 was performed according to protocol.
Agarose gel electrophoresis was performed using 1.2% agarose, 1kb+ and 50 bp ladders, 120V for 20 min.
Colony 1 was transferred to different media, because of the possibility of contamination, LB + DAP + Amp; LB + DAP + Kan; LB - DAP + Amp; and LB + DAP + Amp without colony as negative control and incubated at 37°C, 250 rpm.
Shredded plastic was suspended in ethanol and filtered through a 450 nm filter.
01.08.2019
All cultures made 31.07.2019 grew, except the negative control, which means that no contamination occurred.
Agar plates with E. coli S17-1 were received from iAMB (LB and LB +kan).
E. coli S17-1 was transferred into two test tubes with 6 mL LB and one tube with 8 mL LB without any antibiotics, which were incubated at 37°C, 250 rpm.
OD measurement after six hours showed an OD(600) = 2,6.
One 250 mL Erlenmeyer flask with 27 mL LB was inoculated with 3 mL of one culture above in a 250 mL flask and one 100 mL Erlenmeyer flask with 8 mL LB was inoculated with 2 mL of one culture above, which both were incubated at 25°C, 250 rpm over the weekend.
Week 16
05.08.2019
A 1 L Erlenmeyer flask with 200 mL LB was inoculated with 30 mL of culture from 01.08.2019 for competent cells and incubated at 37 °C, 250 rpm.
OD measurement after two hours showed an OD(600) = 0.642.
Chemically competent cells were made according to protocol.
Eight cryostocks of E. coli S17-1 were made according to protocol.
Three heatshock transformations of E. coli S17-1 was done according to protocol with pBam_pmamDC_mamC-eGFP, pBam_pmamDC_mamC-mCherry-LCI and pBam_pmamDC_mamC-eCFP-TA2, which were plated to LB +amp and incubated at 37°C.
06.08.2019
All transformations from 05.08.2019 showed colonies.
ColonyPCR was performed according to protocol with 8 colonies for E. coli S17-1 pBam_pmamDC_mamC-eGFP, 4 colonies for E. coli S17-1 pBam_pmamDC_mamC-mCherry-LCI and 4 colonies for E. coli S17-1 pBam_pmamDC_mamC-eCFP-TA2 with the primer combinations:
pBam_pmamDC_mamC-eGFP: oriR6K F and KanR R
pBam_pmamDC_mamC-mCherry-LCI: oriR6K F and Glylink R; mCherry F and KanR R
pBam_pmamDC_mamC-eCFP-TA2: oriR6K F and Glylink R; 17xHelix F and KanR R
A masterplate was made with those colonies on LB+amp.
Agarose gel electrophoresis was performed with 1.2% agarose, 1kb+, 120V for 20 min.
Inserts should show bands at ~1000 bp, ~600 bp, ~350 bp
Positive colonies were mamC #7; LCI #3 and TA2 ‘3 and #4.
07.08.2019
Positive colonies from 06.08.2019 were transferred to test tubes with 4 mL LB+amp and incubated at 37°C, 250 rpm overnight.
08.08.2019
Plasmid prep from the test tube cultures made 07.08.2019 was made using the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel.
Nanodrop measurement showed low concentration between 10 and 25 ng/µl.
Resuspended plasmids were stored at -20°C.
A 500 mL schott flask culture of R. rubrum magneticum was first sonicated on ice (time: 5:00 min, pulse 30, amplitude 60%), before using the french press in three cycles with 1000 bar pressure. The cell lysate was then cleaned up using Miltenyi Biotech magnetic LS columns in a QuadroMACS seperator.
50 mL of lysate were put onto the column. The column was then washed two times with 5 mL ethanol, before it was removed from the QuadroMACS separator and the samples were eluted two times with 500 μL ethanol.
09.08.2019
Cultures were made from the masterplate made 08.08.2019 (1x mamC-Schüler #7, 1x LCI #3, TA2 #3 and #4)
Since plasmids showed low concentrations in Nanodrop measurements, they were concentrated using a vacuum concentrator with the following program parameters: V-AL, 60 °C, first 15 Min, then 20 Min. again.
In the same run, the cleaned magnetosomes were concentrated.
Week 17
12.08.2019
ColonyPCR of plasmids from 09.08.19 was done according to protocol.
Agarose gel electrophoresis was done using 1.2% agarose, 1kb+, 120V for 20min.
Plasmids should show bands at ~1500 bp, ~300 bp, ~600 bp.
All plasmids were negative.
15.08.2019
New gBlock oripMB1 arrived from IDT and was dissolved in 10 μL nucleasefree water to achieve a final concentration of 50ng/μL.
PCR of oripMB1 with longTaq PCR Mastermix was performed according to protocol with the primers oripMB1 F and oripMB1 R.
Plasmid concentration was determined using Nanodrop.
| Concentration [ng/µL] | 260/280 |
| 183.9 | 1.87 |
| 119.1 | 1.86 |
Agarose gel electrophoresis was performed using 3% agarose, 50 bp ladder, 120V for 20 min.
oripMB1 should show bands at 700-730 bp.
Quick restriction of pBam_mamDC_mamC-eGFP and the gBlock oripMB1 was performed according to protocol using FseI as restriction enzyme.
Slow ligation of pBam_mamDC_mamC-eGFP and gBlock oripMB1 was performed according to protocol and incubated at 18°C overnight.
16.08.2019
Heatshock transformation of E. coli DH5α with the ligation from 15.08.2019 (pBam_pmamDC_mamC-eGFP + oripMB1) was performed according to protocol and plated to LB +kan plates, which were incubated at room temperature over the weekend.
A 1 L Erlenmeyer flask with 200 mL LB medium was inoculated with 100 μL E. coli DH5α and incubated 25°C, 250rpm over weekend.
Culture from masterplates (mamC-GFP, mamC-LCI, mamC-TA2) was transferred to new agarplates and incubated at 37°C over the weekend.
Week 18
19.08.2019
Quick restriction of copied oripMB1 gBlock with FseI was done according to protocol.
Clean-up of restricted gBlock was done with Monach PCR & DNA Cleanup Kit and the concentration was measured using Nanodrop: 41,3ng/μl at 260/280 = 1,88.
Slow ligation of restricted gBlock with the restricted plasmid pBam_pmamDC_mamC-eGFP from 15.08.19 was done according to protocol.
Chemically competent E. coli DH5α were made according to protocol (OD = 0,55).
A masterplate was made from agar plate made 15.08.2019 (colony 1-8) and parts of colonies were transferred to test tubes with 5 mL LB +kan and incubated at 37°C overnight, test tubes at 250 rpm.
Colony PCR from masterplate (colony 1-8) was performed according to protocol with primers oripMB1 F and Glylink R.
Three new perma cultures of R. rubrum magneticum were made in 15mL falcon tubes with 2mL old culture and 9mL fresh SMM. The three oldest cultures were discarded.
20.08.2019
Agarose gel electrophoresis of ColonyPCR (10.08.2019) was done with 1.2% agarose, 1kb+,120 V for 20 min.
oripMB1 should show bands at 1650 bp.
ColonyPCR of masterplate (colony 1-8) from 19.08.19 was performed according to protocol with primers oripMB1 F & oripMB1 R, which should show bands at 713bp.
Agarose gel electrophoresis of ColonyPCR was performed with 3% agarose, 50bp ladder, 120V for 25 min.
Heatshock transformation of E. coli DH5α with ligation from 19.08.19 was performed according to protocol and plated to LB +kan and incubated at 37°C overnight.
Plasmid prep of test tube cultures of colonies 6-8 made 19.08.19 was done using the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel.
Concentrations were measured using Nanodrop.
| Colony# | Concentration [ng/µL] | 260/280 |
| 6.1 | 169.9 | 1.84 |
| 6.2 | 268.7 | 1.70 |
| 7.1 | 144.5 | 1.88 |
| 7.2 | 279.2 | 1.75 |
| 8.1 | 326.9 | 1.80 |
| 8.2 | 408.9 | 1.78 |
21.08.2019
LB +kan plates made 19.08.19 showed no colonies, so a heatshock transformation was performed to check for transformation efficiency of competent E. coli DH5α with pET28a_pT7_eGFP-LCI with E. coli BL21 and E. coli S17.1 as positive control, according to protocol, plated to LB +kan and incubated at 37°C overnight.
New 1 L Erlenmeyer flask culture was prepared with 200 mL LB-medium and 8 mL E. coli DH5α culture from 16.08.19 and incubated at 37°C, 250rpm overnight.
PCR of plasmid prep 6.2, 7.2, 8.2 made 20.08.19 was done according to protocol with primers oripMB1 F & Glylink R, which should show bands at 1650bp.
Agarose gel electrophoresis was performed with 1.2% agarose, 1kb+, 120V for 20 min.
The gelelectrophoresis of remaining PCR 6.2, 7.2, 8.2 for purification purposes failed, because the machine broke.
New PCR of plasmid prep 6.2, 7.2, 8.2 was performed the same as above.
Fast restriction digest of plasmid prep 8.2 (408,9 ng/μL), mamC-mCherry-LCI and mamC-eCFP-TA2 with EcoRI-HF and BamHI-HF in Cutsmart was performed according to protocol.
CleanUp of the plasmid double digest was done with the NEB DNA & PCR CleanUp Kit and concentration was measured using Nanodrop.
Concentration: 30,8 ng/μl, 260/280 = 1,83
Fast ligation of restricted pBam_oripMB1_pmamDC_mamC-eGFP with either mamC-mCherry-LCI or mamC-eCFP-TA2 was performed according to protocol.
Heatshock transformation of the fast ligation into E. coli DH5α was performed according to protocol, plated to LB +kan plates and incubated at 37°C overnight.
22.08.2019
Agarose gel electrophoresis of second PCR of plasmid prep 6.2, 7.2, 8.2 from 21.08.19 was done with 1.2% agarose, 1kb+, 120 V for 20 min.
Purification from gel was done using the Monarch DNA Gel extraction Kit.
Concentration was measured using Nanodrop.
| Sample | Concentration [ng/µL] | 260/280 |
| 6.2 | 16.1 | 1.64 |
| 7.2 | 18.6 | 1.65 |
| 8.2 | 20.8 | 1.70 |
Samples were sent for sequencing.
| Sample | Primer | Sequencing ID |
| 6.2 | oripMB1 F | EF30365298 |
| 6.2 | Glylink R | EF30365299 |
| 7.2 | oripMB1 F | EF30365300 |
| 7.2 | Glylink R | EF30365301 |
| 8.2 | oripMB1 F | EF30365302 |
| 8.2 | Glylink R | EF30365303 |
Transformation efficiency of chemically competent E. coli DH5α was negative, so new chemically competent E. coli DH5α were made according to protocol (OD 1:10 = 0,436).
Old competent E. coli DH5α from 19.08.19 were wasted.
Heatshock transformation of E. coli DH5α with the ligations done 21.08.19 was performed according to protocol, plated to LB +kan and incubated at 37°C overnight.
23.08.2019
Fast ligation of restricted pBam_oripMB1_pmamDC_mamC-eGFP with either mamC-mCherry-LCI or mamC-eCFP-TA2 made 21.08.2019 was performed according to protocol.
Week 19
26.08.2019
Heatshock transformation of E. coli DH5α with the ligations done 22.08.19 was performed according to protocol, plated to LB +kan and incubated at 37°C overnight.
Slow ligation of restricted pBam_oripMB1_pmamDC_mamC-eGFP with either mamC-mCherry-LCI or mamC-eCFP-TA2 made 21.08.2019 was performed according to protocol.
27.08.2019
All transformations were negative, so the plates were wasted.
29.08.2019
Fast restriction digest of pBam_oripMB1_pmamDC_mamC-eGFP plasmid preps 6.1, 7.1, 8.1, made 20.08.2019 using BglII in NED3.1 buffer was performed according to protocol.
CleanUp of the plasmid digest was done with the NEB DNA & PCR CleanUp Kit and eluted in 10 µL.
Fast restriction digest of pBam_oripMB1_pmamDC_mamC-eGFP plasmid preps 6.1, 7.1, 8.1, 10 µL each, with EcoRI-HF in Cutsmart was done according to protocol.
Agarose gel electrophoresis was done with 1.2% agarose, 1kb+, 120 V for 23 min.
Bands were visible at 5435bp & 776bp, which means that oriT & oripMB1 are inserted in the same direction, which explains why prior ColonyPCRs with more than just the insert did not work correctly.
Chemically competent E. coli DH5α were made according to protocol and old DH5α from 22.08.19 were wasted.
Heatshock transformation of E. coli DH5α with the ligations done 26.08.19 was performed according to protocol, plated to LB +kan and incubated at 37°C overnight.
30.08.2019
Since plates with transformations made 29.08.19 showed growth, a masterplate (LB +kan) with 8 colonies each (pBam_oripMB1_mamDC_mamC-mCherry-LCI and pBam_oripMB1_mamDC_mamC-eCFP-TA2) was made and the same 16 colonies were put into test tubes with 5 mL LB +kan, which were stored at 4°C over the weekend.
ColonyPCR of masterplate colonies was done according to protocol with primers mCherry F and KanR R for pBam_oripMB1_mamDC_mamC-mCherry-LCI and 17xHelix F and KanR R for pBam_oripMB1_mamDC_mamC-eCFP-TA2.
Agarose gel electrophoresis was done with 1.2% agarose, 1kb+, 120 V for 20 min.
Bands should show at 918 bp (LCI) & 373 bp (TA2).
All E. coli DH5α pBam_pmamDC_mamC-mCherry-LCI colonies were tested positive for the plasmid containing the right insert and the E. coli DH5α pBam_pmamDC_mamC-eCFP-TA2 colonies 1,4,5 and 6 were also positively tested, while colonies 2,3,7 and 8 were negative.
Week 20
02.09.2019
The Masterplate and the precultures of E. coli DH5α pBam_pmamDC_mamC-mCherry-LCI colonies 1-4 and E. coli DH5α pBam_pmamDC_mamC-eCFP-TA2 colonies 1,4,5 and 6, made 30.08.19 were incubated at 37°C overnight.
03.09.2019
Plasmid prep of precultures of E. coli DH5α pBam_pmamDC_mamC-mCherry-LCI colonies 1-4 and E. coli DH5α pBam_pmamDC_mamC-eCFP-TA2 colonies 1,4,5 and 6, made 30.08.19 was performed with the Nucleospin Plasmid MiniPrep Kit from Macherey-Nagel and concentrations were measured using Nanodrop.
| Culture# | Concentration [ng/µL] | 260/280 |
| LCI 1.1 | 211.0 | 1.88 |
| LCI 1.2 | 211.2 | 1.88 |
| LCI 2.1 | 239.7 | 1.88 |
| LCI 2.2 | 236.6 | 1.88 |
| LCI 3.1 | 154.4 | 1.83 |
| LCI 3.2 | 248.9 | 1.83 |
| LCI 4.1 | 129.8 | 1.86 |
| LCI 4.2 | 140.0 | 1.86 |
| TA2 1.1 | 269.6 | 1.88 |
| TA2 1.2 | 182.6 | 1.88 |
| TA2 4.1 | 285.6 | 1.88 |
| TA2 4.2 | 265.2 | 1.89 |
| TA2 5.1 | 203.5 | 1.85 |
| TA2 5.2 | 269.0 | 1.85 |
| TA2 6.1 | 338.9 | 1.80 |
| TA2 6.2 | 272.4 | 1.89 |
Samples were sent to sequencing.
| Sample# | Primer | Sequencing ID |
| LCI 1.1 | SeqAmpR F | EF30365309 |
| LCI 1.1 | SeqmamC R | EF30365310 |
| LCI 1.1 | SeqmamC F | EF30365311 |
| LCI 1.1 | SeqKanR R | EF30365305 |
| LCI 2.1 | SeqAmpR F | EF30365313 |
| LCI 2.1 | SeqmamC R | EF30365314 |
| LCI 2.1 | SeqmamC F | EF30365315 |
| LCI 2.1 | SeqKanR R | EF30365316 |
| LCI 3.2 | SeqAmpR F | EF30365317 |
| LCI 3.2 | SeqmamC R | EF30365318 |
| LCI 3.2 | SeqmamC F | EF30365319 |
| LCI 3.2 | SeqKanR R | EF30365322 |
| LCI 4.2 | SeqAmpR F | EF30365323 |
| LCI 4.2 | SeqmamC R | EF30365324 |
| LCI 4.2 | SeqmamC F | EF30365325 |
| LCI 4.2 | SeqKanR R | EF30365326 |
| TA2 1.1 | SeqAmpR F | EF30365327 |
| TA2 1.1 | SeqmamC R | EF30365328 |
| TA2 1.1 | SeqmamC F | EF30365329 |
| TA2 1.1 | SeqKanR R | EF30365330 |
| TA2 4.1 | SeqAmpR F | EF30365331 |
| TA2 4.1 | SeqmamC R | EF30365332 |
| TA2 4.1 | SeqmamC F | EF30365333 |
| TA2 4.1 | SeqKanR R | EF30365334 |
| TA2 5.2 | SeqAmpR F | EF30365335 |
| TA2 5.2 | SeqmamC R | EF30365336 |
| TA2 5.2 | SeqmamC F | EF30365337 |
| TA2 5.2 | SeqKanR R | EF30365338 |
| TA2 6.1 | SeqAmpR F | EF30365339 |
| TA2 6.1 | SeqmamC R | EF30365340 |
| TA2 6.1 | SeqmamC F | EF30365341 |
| TA2 6.1 | SeqKanR R | EF30365342 |
Three new permacultures of R. rubrum magneticum were made in 15 mL falcon tubes by transferring 1 mL old culture into 12 mL fresh SMM. The oldest three tubes were discarded.
Two additional cultures in 50 mL falcon tubes were made by transferring 1 mL old culture from 15 mL falcon tube cultures into 45 mL fresh SMM.
05.09.2019
Samples of the original pBam_pmamDC_mamC-eGFP and pBam_oripMB1_pmamDC_mamC-eGFP, made 20.08.2019 were sent for sequencing:
| Plasmid | Primer | Sequencing ID |
| pBam_pmamDC_mamC-eGFP | SeqAmpR F | EF30365312 |
| pBam_pmamDC_mamC-eGFP | SeqmamC R | EF30365343 |
| pBam_oripMB1_pmamDC_mamC-eGFP | SeqmamC F | EF30365344 |
| pBam_oripMB1_pmamDC_mamC-eGFP | SeqKanR R | EF30365345 |
| pBam_oripMB1_pmamDC_mamC-eGFP | SeqAmpR F | EF30365346 |
| pBam_oripMB1_pmamDC_mamC-eGFP | SeqmamC R | EF30365347 |
Week 21
09.09.2019
Heatshock transformation of pBam_oripMB1_pmamDC_mamC-mCherry-LCI 1.1 and 1.2 and pBam_oripMB1_pmamDC_mamC-eCFP-TA2 1.1 and 1.2, made 03.09.2019 into E. coli BW29427 was done according to protocol and plated to LB +DAP +Kan and LB +DAP +Amp plates.
1 L LB-Medium was made according to recipes.
200 mL LB agar was made according to recipe and supplemented with 0.1mM DAP and 200 µL 1000x kanamycin to make LB +DAP +Kan plates.
10.09.2019
Plates of transformation from 09.09.19 showed colonies, so a masterplate (LB +DAP +Kan) was made from LB +DAP +Amp plates with 3 colonies each of LCI & TA2.
The same colonies were transferred into test tubes with 5 mL LB +DAP +Kan and incubated at 37°C, 250 rpm overnight.
ColonyPCR of masterplate was performed according to protocol with primers oripMB1 R and oripMB1 F.
Agarose gel electrophoresis was performed with 3% agarose, 50 bp, 120V for 25 min.
11.09.2019
Conjugation was performed according to protocol with both E. coli BW29427 pBam_oripMB1_pmamDC_mamC-mCherry-LCI 1.1 and E. coli BW29427 pBam_oripMB1_pmamDC_mamC-eCFP-TA2 1.1, made 09.09.2019.
100 mL YPS agar was made according to recipe and supplemented with 100 µL 1000x ampicillin to yield YPS +amp plates, which were stored at 4°C.
100 mL YPS agar was made according to recipe and supplemented with 100 µL 1000x kanamycin to yield YPS +amp plates, which were stored at 4°C.
12.09.2019
Both conjugation plates made 11.09.19 showed little contamination outside the droplet of mixed conjugation cells, which were carefully regained with 1ml YPS without washing off the contamination.
Regained cells were transferred into test tube with 4ml YPS without antibiotics and incubated for 2h at 30°C, 250 rpm, before being centrifuged at 4000 rpm for 10 min and resuspended in 1 mL YPS medium for smaller volume, then being replated to YPS +kan and YPS +amp plates and incubated at 30°C according to protocol.
Week 22
16.09.2019
Plates made 12.09.19 showed excessive growth of bacteria without discernible colonies, so they were plated to new YPS-Kan-Amp plates, closed with parafilm, and further incubated at 30°C under light conditions.
17.09.2019
500 mL YPS agar were made according to recipe. Some were supplemented with 1000x kanamycin and ampicillin to yield YPS plates and YPS +Kan +Amp plates
10 mL CaCl2- & MgSO4- solution were made and sterile filtrated.
New overnight cultures of E. coli BW29427 and R. rubrum magneticum were made according to conjugation protocol, 1 mL in 2 mL reaction tubes and incubated spinning at 30°C under light conditions.
Chemically competent E. coli BW29427 were thawed and transferred to test tubes with 5 mL of either LB or LB +DAP and incubated at 37°C, 250 rpm overnight, to check for the possibility of DAP-auxotrophy lost.
18.09.2019
A new E. coli BW29427 culture was revived from original agar stocks, received at 04.06.2019, stored since 06.06.2019 at 4°C and transferred to test tubes with 5 mL either LB with 50μl DAP and control without DAP and incubated at 37°C, 250 rpm.
New E. coli BW29427 pBam_oripMB1_pmamDC_mamC-mCherry-LCI and E. coli BW29427 pBam_oripMB1_pmamDC_mamC-eCFP-TA2 were transferred into test tubes with 5 mL LB +DAP without antibiotics and incubated at 37°C, 250 rpm.
19.09.2019
Cultures of E. coli BW29427, made 18.09.2019 showed growth in LB +DAP medium, but not in medium without DAP, which means that the auxotrophy still persists.
The new E. coli BW29427 culture with DAP made 18.09.19 (5 mL) were transferred into 1 L Erlenmeyer flasks with 200 mL LB + 2ml 1mM DAP and incubated at 37°C, 250 rpm for a few hours.
OD measurement showed OD(600) = 0,573.
Competent cells were made according to protocol.
Chemically competent E. coli BW29427, made 19.09.2019 were transferred to test tubes with 5 mL of either LB or LB +DAP and incubated at 37°C, 250 rpm overnight, to check for the possibility of DAP-auxotrophy lost after E. coli BW29427 were made competent.
20.09.2019
Chemically competent E. coli BW29427 test tube cultures, made 19.09.2019, exhibited growth in both LB with and without DAP, which means that these cells somehow loose their DAP auxotrophy during the process of making them chemically competent.
Week 23
25.09.2019
New R. rubrum magneticum perma cultures were made by transferring 2 mL old culture into new falcon tubes with 10 mL fresh SMM.
26.09.2019
Magnetic Dynabeads were received (2x 50 μL of suspension in 1,5ml reaction tubes), which were put next to a magnet to take off the supernatant.
700μl purified 6xHis-eGFP-LCI or 6xHis-eGFP-TA2 were added and filled up with ddH2O to 1,5 mL.
Control time for separation with neodymium magnet 1-2 sec, with electromagnet 06:30 min.
Microscopic evaluation of coated beads showed fluorescence.
After addition of Impranil (polyurethane) no significant binding between beads and plastic could be observed, as no plastic was seen; the beads seemed to cluster together.
27.09.2019
New primers were received to generate micro-Inserts to either delete (deleteKanR F and deleteKanR R) or destroy (destroyKanR R and destroyKanR R) the kanamycin resistance in the plasmids pBam_oripMB1_pmamDC_mamC-mCherry-LCI and pBam_oripMB1_pmamDC_mamC-eCFP-TA2 to circumvent the impossibility of selection after conjugation for R. rubrum magneticum and against E. coli BW29427 without DAP auxotrophy by using the natural kanamycin resistance of R. rubrum magneticum (proven 19.06.2019).
Complementary primers (deleteKanR F + deleteKanR R or destroyKanR R + destroyKanR R) were equimolarly mixed and incubated at room temperature for 45 min to generate dsDNA fragments.
Quick restriction digest of pBam_oripMB1_pmamDC_mamC-mCherry-LCI, pBam_oripMB1_pmamDC_mamC-eCFP-TA2 and dsdeleteKanR with AatII and BamHI was performed according to protocol.
Quick restriction digest of pBam_oripMB1_pmamDC_mamC-mCherry-LCI, pBam_oripMB1_pmamDC_mamC-eCFP-TA2 and dsdestroyKanR with NsiI was performed according to protocol.
Slow ligation of pBam_oripMB1_pmamDC_mamC-mCherry-LCI, pBam_oripMB1_pmamDC_mamC-eCFP-TA2 and dsdeleteKanR was performed according to protocol and incubated at 37°C over the weekend at room temperature.
Slow ligation of pBam_oripMB1_pmamDC_mamC-mCherry-LCI, pBam_oripMB1_pmamDC_mamC-eCFP-TA2 and dsdestroyKanR was performed according to protocol and incubated at 37°C over the weekend at room temperature.
Microscopic evaluation of magnetic beads was performed as on 26.09.19.
The beads again showed fluorescence under the microscope.
No fluorescence was observed in uncoated beads.
Polystyrene (PS) showed no fluorescence without beads.
After addition of cryoshredded polystyrene (PS), the fluorescence of the beads greatly decreased, possibly due to the interaction of the His-tag to the beads being far weaker than the interaction of LCI or TA2 to the polymer and thus spreading the protein over a greater surface, seen in the slight fluorescence of the plastic particle in the picture.
Week 24
30.09.2019
Heatshock transformation of E. coli BW29427 with the ligation done 27.09.19 was performed according to protocol, plated to LB +Amp plates and incubated at 37°C until evening.
Masterplates were made of each transformation, with the same colonies being transferred to LB +Amp for storage and LB +Kan as negative control.
01.10.2019
New colonies were picked of the transformation-plates (30.09.2019), because the picked colonies all showed growth on kanamycin.
02.10.2019
Following colonies showed growth on ampicillin but not on kanamycin:
BW+TA2 destroyKanR 1:7, colony 7
BW+TA2 destroy 1:50, colony 5
BW+TA2 deleteKanR 1:50, colony 7
BW+LCI deleteKanR 1:7, colony 7
BW+LCI destroyKanR 1:7, colony 7
Each colony was transferred to two test tubes each, one with 5 mL LB +DAP +Kan and the other with 5 mL LB +DAP +Amp and incubated at 37 °C, 250 rpm.
03.10.2019
Cultures made 02.10.2019 were checked for growth in ampicillin and kanamycin.
The following colonies showed growth in ampicillin, but not in kanamycin:
A BW+TA2 destroyKanR 1:7, colony 7
B BW+TA2 destroyKanR 1:50, colony5
C BW+TA2 deleteKanR 1:50, colony 7
D BW+LCI deleteKanR 1:7, colony 7
The following colonies showed growth in both antibiotics and was autoclaved:
E BW+LCI destroyKanR 1:7, colony 7
1 mL of each culture (A-D) was centrifuged at 5000 rpm for 1 min and the pellet resuspended in 1 mL LB without antibiotics as to not interfere with the conjugation.
Cultures were made in 100 mL Erlenmeyer flasks by transferring the cultures (A-D) and adding 9 mL LB, then incubated for 2 h at 37 °C, 250 rpm.
Conjugation of R. rubrum magneticum with the E. coli BW29427 (A-D) was performed according to protocol.
Plasmid prep of cultures A – D was performed according to protocol.
| Designation of culture | Concentration [ng/µL] | 260/280 |
| TA2 destroyKanR 1:50 (B) | 51.0 | 1.61 |
| LCI deleteKanR 1:7 (D) | 58.3 | 1.83 |
| TA2 deleteKanR 1:50 (C) | 38.3 | 1.82 |
| TA2 destroyKanR 1:7 (A) | 78.2 | 1.61 |
04.10.2019
Conjugation of R. rubrum magneticum with the E. coli BW29427 (A-D) was continued according to protocol.
Week 25
07.10.2019
One red colony could be seen on TA2 destroyKanR 1:50 plate, no colonies on the other plates.
08.10.2019
No new colonies were seen on agar plates.
ColonyPCR was performed on the TA2 destroyKanR 1:50 colony according to protocol with the primers 17xHelix F and KanR R.
Agarose gel electrophoresis was done with 1.2% agarose, 1kb+, 120V for 24 min.
PO = original plasmid with KanR
Pd = plasmid pBam_oripMB1_pmamDC_mamC-eCFP-TA2_woKanR
Rr = R. rubrum magneticum colony, conjugated with TA2 destroyKanR
ColonyPCR showed a clear band in the plasmid still harbouring the kanamycin resistance, but neither in the purified plasmid, nor in the R. rubrum magneticum colony.
The R. rubrum magneticum pBam_oripMB1_pmamDC_mamC-eCFP-TA2_woKanR
(TA2 1:50 destroy) was transferred into a new 2 mL reaction tube with 1 mL SMM and incubated at 30 °C under light conditions.
09.10.2019
CulturePCR was performed according to protocol with the R. rubrum magneticum pBam_oripMB1_pmamDC_mamC-eCFP-TA2_woKanR culture and the primers oriR6K F and Glylink R. Bands should be shown at around 630 bp.
Agarose gel electrophoresis was done with 1.2% agarose, 1kb+, 120V for 25 min.
No plasmid was detected in R. rubrum magneticum, so the culture was discarded new E. coli BW29427 were transferred to new test tubes for new conjugations with 5 mL LB +amp and incubated at 37 °C, 250 rpm.
11.10.2019
E. coli BW29427 TA2 deleteKanR 1:50 showed no growth and was autoclaved.
Conjugation with TA2 plasmids was performed according to protocol.
E. coli BW29427 with LCI plasmid were autoclaved since the flask broke during shaking.
Three new R. rubrum magneticum permacultures were made.
Rest of old cultures from 03.10.2019 was used for antibiotic-resistance check, by transferring 1 mL of each into new falcon tubes with 12 mL SMM with and without kanamycin.
Week 26
15.10.2019
TA2 destroyKanR 1:50 on old plate (04.10.2019) showed small colonies.
TA2 destroy KanR 1:7 showed 1 colony.
TA2 destroyKanR 1:50 on plate from 11.10.2019 showed growth.
ColonyPCR was done according to protocol with different colonies from different dates, primers oriR6K F and Glylink R.
Agarose gel electrophoresis was done with 1.2% agarose, 1kb+, 120 V for 24 min.
1: R. rubrum magneticum culture from 08.10.2019
2: pBam_oripMB1_pmamDC_mamC-eCFP-TA2
3: TA2 destroyKanR 1:7 (11.10.2019)
4: TA2 destroyKanR 1:50 (04.10.2019)
5: TA2 destryKanR 1:7 (11.10.2019)
Every ColonyPCR was negative, but the positive control for the purified pBam_oripMB1_pmamDC_mamC-eCFP-TA2 plasmid.
Cultures of Rhodospirillum rubrum magneticum showed growth on kanamycin so the resistance is still present.
16.10.2019
For spectrometry the following was put in a MTP:
→ E. coli BL21, without plasmid, treated like E. coli BL21 pET28a(+)::his-eGFP-17xhelix-TEV-LCI
→ purified his-eGFP-17xhelix-TEV-LCI
→ eGFP
→ magnetic beads (Dynabeads from thermo fisher)
→ magnetic beads coated with his-eGFP-17xhelix-TEV-LCI, different dilutions
→ pure PP
→ pure PS
→ pure PU
each polymer mixed with magnetic beads coated with his-eGFP-17xhelix-TEV-LCI with the same amount of coated beads and different amounts of plastics
Spectrometry was done in a Tecan M1000 reader with an excitation wavelength of 548 nm and emission wavelength from 500 nm - 540 nm in 5 nm steps.
18.10.2019
No growth could be observed in SMM-Amp, inoculated at 15.10.2019, but the lamp broke most likely two days ago, which could be the reason why there is no growth.
A PCR of conjugated R. rubrum magneticum from 04.10.2019 (with LCI fusion protein) was executed like described in the protocol. Positive control PCR (plasmid) was done like described before. Two different primer combinations were used as a double control: GlylinkR and oriR6KF (GO), pmB1F and pmB1R(pmB).
For positive control the ligated plasmid was used.
For negative control R. rubrum magneticum prior to conjugation was used.
Besides a PCR made like described in the special protocol a normal culture PCR was made.
The plasmid insertion could be validated.
1-2: positive control with different primer combinations, expected bands at ~600 bp (No. 1,GO ) and ~700 bp (No.2, pmB); 3-6 Rhodospirillum rubrum magneticum prior to conjugation (negative control) Number 3 shows bands because liquid was sloshed over by number 2.
1-6: PCR of Rhodospirillum rubrum magneticum after conjugation with different primer combinations and different amount of template; 1-4 PCR after special PCR protocol (1 = 5 µL template, GO; 2 = 5 µL template, pmB; 3 = 10 µL template, GO; 4 = 10 µL template, pmB) ; 5-6 culture PCR with 5 µL culture (5 = GO, 6 = pmB)