Notebook

Follow all of our steps we took during this exciting journey in the lab!

Deletion of BsaI and SapI Restriction Recognition Side
  • 02.06.
    • Mutation of pET22b(+)-Ferritin for deletion of BsaI or SapI restriction recognition sites (RRS)
      • two samples each for BsaI mutation step and SapI mutation step
  • 05.06.
    • PCR to check mutation of BsaI mutant and SapI mutant
    • Agarose electrophoresis
      • 120 V for 40 min
      • if PCR was successful, presence of intense bands at approximately 6,000 base pairs should be obtained
      Agarose gel (1 %) with PCR results to check mutation to remove BsaI or SapI restriction recognition sites. Two samples for each mutation step were used. Bands which show positive results are marked with a red box.
  • 06.06.
    • PCR purification
  • 21.06.
    • Ligation (KLD, kinase, ligase, DpnI)
    • Transformation of SapI Mut and BsaI Mut samples in DH5α onto LB-agar plates containing 100 µg/mL ampicillin
  • 26.06.
    • pre-culture of transformed colony
      • in 5 mL LB medium containing 100 µg/mL ampicillin
      • incubated at 37 °C and 200 rpm
  • 27.06.
    • Miniprep of pre-culture
    • Double digest
      • enzymes used for all mutation samples:
        • BsaI
        • SapI
    • Agarose gel to check digest results
      • 120 V for 35 min
      • expected result: just one digest which results in one band in agarose gel
      Agarose gel (1 %) with products after the double digest. Wild type ferritin (pET22b(+)) without restriticion enzymes used as negative control (-); pET22b(+) with enzyme BsaI used as a positive control for BsaI digest (BsaI (+)); pET22b(+) with enzyme SapI used as a positive control for SapI digest (SapI (+)); Positive results of Plasmid with suspected deleted BsaI RRS (Sample BsaI 1-3) or SapI RRS (SapI Mut 1 and 2) marked by a red box.
      • problem:
        • period for the digest was too short which is why not everything got cleaved, resulting in to bands (digested product and undigested plasmid)
  • 28.06.
    • Second PCR mutagenesis of SapI mutant and BsaI mutant to eliminate second RRS
      • mutation to remove SapI RRS in BsaI Mut
      • mutation to remove BsaI RRS in SapI Mut
  • 29.06.
    • Agarose gel to check second PCR mutagenesis from 28.06.
      • if PCR mutation was successful, presence of intense bands at approximately 6,000 base pairs should be obtained
      Agarose gel (1 %) with PCR results to check mutation to eliminate BsaI RRS in SapI Mut or SapI RRS in BsaI Mut. Two samples for each mutation were used. Bands which show positive results marked with a red box.
    • PCR Purification
    • Ligation
  • 30.06.
    • Transformation of mutated pET22b(+)_WT-Ferritin (BBa_K4669027) in NEB 10-beta onto LB-agar plates containing 100 µg/mL ampicillin following the protocol of DH5α transformation, using NEB 10-beta/Stable outgrowth medium instead of SOC medium
  • 02.07.
    • pre-culture of transformed clones
      • in 5 mL LB media containing 100 µg/mL ampicillin
      • incubated at 37 °C and 200 rpm
  • 03.07.
    • Miniprep of pre-culture
    • Double digest
      • enzymes used:
        • BsaI for SapI Mut 1
        • SapI for BsaI Mut 1 and BsaI Mut 2
        • both enzymes used for SapI (BsaI) Mut 1 and BsaI (SapI) Mut 1
      • pET22b(+)_WT-Ferritin without enzymes used as negative control
      • pET22b(+)_WT-Ferritin with both enzymes used as positive control
    • Agarose electrophoresis to check digest results
      • run at 120 V for 35 min
      • expected results: after second mutagenesis plasmids without RRS should not show any digest
      Agarose gel (1 %) with products after the double digest. Wild type ferritin (pET22b(+)) without restriticion enzymes BsaI and SapI used as positive control (+); pET22b(+) with both enzymes used as negative control; Sample with eliminated SapI RRS (SapI Mut 1 1-5) showed one digest. Plasmids with suspected eliminated RRS had no digest (SapI (BsaI) Mit 1 1-5) and are marked by a red box.
      Agarose gel (1 %) with products after the double digest. Sample with eliminated BsaI RRS 1 (BsaI Mut 1 1-5 and BsaI Mut 2 1-5) showed one digest. Plasmids BsaI (SapI) Mut sample 1 2,3 and 5 with suspected eliminated RRS BsaI were not digested whereas sample 4 got digested. Positive results are marked by a red box.
    • DNA of one BsaI (SapI) Mut 1 and SapI (BsaI) Mut 1 sample was sent to whole plasmid sequencing
      • both samples had positive mutation results

Mutation with CPPs
  • 08.07.
    • PCR mutation of pET22b(+)_WT-Ferritin (without BsaI and SapI RRS) with CPPs TAT (BBa_K1202006), R9 (BBa_K4669001), and R12 (BBa_K4669002).
      • Program used:
        1. Primary denaturation 98 °C 30 sec
        2. Denaturation 98 °C 10 sec
        3. Annealing 62.2 °C 10 sec
        4. DNA-synthesis 72 °C 2 min
        5. Go to 2, repeat 29x - -
        6. Final DNA-synthesis 72 °C 5 min
        7. Hold 4 °C -
    • Agarose gelelectrophoresis to check success of PCR
      • expected results: intense bands at approximately 6,000 base pairs
      1% agarose gel of pET22b(+) after PCR mutagenesis with CPPs TAT, R9 and R12. Result should be around 6,000 bp
  • 10.07.
    • Gradient PCR (Gradient of 5 °C) for PCR mutation of pET22b(+)_WT-Ferritin to check optimal annealing temperature
      • Program used:
        1. Primary denaturation 98°C 30 s
        2. Denaturation 98°C 10 s
        3. Annealing 60°C 10
        G = 5 °C
        4. DNA-synthesis 72°C 2 min
        5. Go to 2, repeat 34x - -
        6. Final DNA-synthesis 72°C 5 min
        7. Hold 4°C -
    • Agarose gelelectrophoresis to check success of PCR
      • run at 120 V for 35 min
      • expected results: intense bands at approximately 6,000 base pairs
      Agarose gel (1 %) with gradient PCR results (gradient of 5 °C) to check optimal primer annealing temperature. Faint bands with approximately 2,000 base pairs in size at 55.7 °C, 56.6 °C and 57.8 °C.
      • Problems:
        • faint bands with not enough base pairs in size
        • DNA-synthesis time too short
  • 11.07.
    • PCR Mutation of pET22b(+)_WT-Ferritin (without BsaI and SapI restriction recognition sites) with CPP TAT using different template concentrations
      • program used:
        1. Primary denaturation 98 °C 30 s
        2. Denaturation 98 °C 10 s
        3. Annealing 62 °C 10 s
        4. DNA-synthesis 72 °C 3.5 min
        5. Go to step 2, repeat 34x - -
        6. Final DNA-synthesis 72 °C 5 min
        7. Hold 4 °C -
    • Agarose Gelelectrophoresis to check success of PCR
      • run at 120 V for 35 min
      • expected results: intense bands at approximately 6,000 base pairs
      Agarose gel (1 %) of TAT mutagenesis results using different template concentrations. Plasmid template amounts used in reaction ranged from 10-70 ng in steps of 10 ng. Except with the lowest template concentration of 10 ng, all lanes displayed positive results with a faint band at approximately 6,000 bp. The area with the suspected bands in the agarose gel is marked by the red box.
  • 12.07.
    • PCR purification of WT (60 ng), TAT-Ferritin (30 ng*), TAT-Ferritin (40 ng) and TAT-Ferritin (50 ng)
    • Ligation
    • Transformation of WT (60 ng), TAT-Ferritin (30 ng*), TAT-Ferritin (40 ng) and TAT-Ferritin (50 ng) in NEB 10-beta onto LB-agar plates containing 100 µg/mL ampicillin
      Sample NameOriginal Sample
      WTWT Ferritin (60 ng)
      TAT-Ferritin 30TAT-Ferritin (30 ng*)
      TAT-Ferritin 40TAT-Ferritin (40 ng)
      TAT-Ferritin 50TAT-Ferritin (50 ng)
  • 13.07.
    • Colony PCR of transformation colonies from 12.07.
      • program used:
        Denaturation94 °C5 min
        94 °C10 sec
        60,5 °C10 sec
        go to step two, repeat 34X72 °C3:30 min
        Final extension72 °C5 min
        Hold4 °C
      • negative control without template
    • Agarose gelelectrophoresis to check results of Colony PCR
      • run at 120 V for 35 min
      • expected result: intense bands with approximately 600 bp in size
      Agarose gel (1 %) with results of the colony PCR. Negative control without template had no band. In lanes of WT Ferritin after mutation (WT) and TAT-Ferritin (30, 40, and 50) are intense bands present at approximately 600 base pairs in size which are marked by a red box.
  • 15.07.
    • Pre-culture of clones with positive Colony-PCR results from 13.07
      • 5 mL LB media containing 100 µg/mL ampicillin
      • incubated at 37°C and 200 rpm
  • 16.07
    • Miniprep of pre-culture of colonies with positive Colony PCR results
    • PCR Mutation with CPPs TAT, R9 or R12
      • used program:
        1.Primary denaturation98 °C30 s
        2.Denaturation98 °C10 s
        3.Annealing62 °C10 s
        4.DNA-synthesis72 °C3.5 min
        5.Go to 2, repeat 34x--
        6.Final DNA-synthesis72 °C5 min
        7.Hold4 °C-
    • Agarose gelelectrophoresis to check success of the PCR mutations
      • run at 120 V for 35 min
      • expected result: bands at approximately 6,000 bp
      Agarose gel (1 %) after PCR mutagenesis with CPPs TAT, R9 or R12. Two samples used for each CPP mutation. All samples showed bands at approximately 6,000 bp. Bands of interest are marked by a red box.

    • PCR purification
    • Ligation
    • Transformation in DH5α onto LB-agar plates containing 100 µg/mL ampicillin
  • 17.07.
    • Colony PCR of transformed colonies from 14.07.
      • used program:
        Denaturation94 °C5 min
        go to step 2, repeat 34X94 °C10 sec
        60,5 °C10 sec
        72 °C3:30 min
        Final extension72 °C5 min
        Hold4 °C
    • Agarose gelelectrophoresis to check Colony PCR
      • run at 120 V for 40 min
      • expected results: bands at approximately 600 bp
      Agarose gel (1 %) with Colony PCR results of R9-Ferritin 1 and 2, WT Ferritin (pET22b(+)) as well as a positive control (+) with WT Ferritin Colony PCR sample from the 13.07. and a negative control (-) without template. R9-Ferritin 1 sample 1, 2, 4, 5, 6, 7, 8, 9, 10 and R9-Ferritin 2 sample 1, 2, 3, 4, 7, 8, 10 showed the expected band at approximately 600 bp. The area with the bands for a positive results is marked by a red box.
      Agarose gel (1 %) with Colony PCR results of R12-Ferritin 1 and 2. R12-Ferritin 1 sample 1, 3, 4, 7, 9, 10 and R12-Ferritin 2 sample 3, 4, 5, 6 showed the expected band at approximately 600 bp. The area with the bands for a positive results is marked by a red box.
      Agarose gel (1 %) with Colony PCR results of TAT-Ferritin 1. TAT-Ferritin 1 sample 7, 8, 9, 10 showed the expected band at approximately 600 bp. The area with the bands for a positive results is marked by a red box.
      Agarose gel (1 %) with Colony PCR results of TAT-Ferritin 2, WT Ferritin (pET22b(+)) as well as a positive control (+) with WT Ferritin Colony PCR sample from the 13.07 and negative control (-) without template. TAT-Ferritin 2 sample 2, 4, 6, 7, 9 showed the expected band at approximately 600 bp. The area with the bands for a positive results is marked by a red box.
  • 20.07.
    • Pre-culture of colonies with positive colony PCR results of 17.07.
      • in 5 mL LB-media containing 100 µg/mL ampicillin
      • incubated at 37 °C and 200 rpm
  • 21.07.
    • Miniprep of pre-culture from 20.07
    • sent 2 samples of each CPP mutation for sequencing
      • One sample of TAT-Ferritin mutation and R12-Ferritin mutation had positive mutation results
  • 25.07.
    • Pre-culture of R9-Ferritin
      • in 5 mL LB-media containing 100 µg/mL ampicillin
      • incubated at 37 °C and 200 rpm
  • 26.07.
    • Miniprep of pre-culture R9-Ferritin
    • sent sample to sequencing
      • positive sequencing result
  • 31.07.
    • Pre-culture of R9-Ferritin with positive sequencing results
      • in 5 mL LB-media containing 100 µg/mL Ampicillin
      • incubated at 37 °C and 200 rpm
  • 01.08.
    • Miniprep of R9-Ferritin culture with positive sequencing results

Protein Expression and Purification
  • 17.04.
    • Transformation of WT-Ferritin (pET22b(+)) in BL21 (DE3) Star
      • 100 ng/µL plasmid added to the aliquot of 50 µL of competent cells
  • 18.04.
    • pre-culture for expression analysis of WT-Ferritin BL21 (DE3) Star
      • clone cultured in 50 mL LB media in a 500 mL Erlenmeyer flask + 1:1000 (1 mM) ampicillin
  • 19.04.
    • IPTG induction of expression
      • pre-culture from 18.04. transferred into TB media with 150 µg/mL of ampicillin and incubated at 37 °C and 180 rpm
      • 0.25 mM IPTG used for induction when OD600 0.6 was reached
      • incubation at 18 °C with 180 rpm for 48 h
  • 21.04.
    • Cell harvest of WT Ferritin BL21 (DE3) Star
  • 25.04.
    • pre-culture for expression analysis of WT Ferritin BL21 (DE3) Star
      • Three BL21 Star (DE3) pET22b(+) WT-Ferritin colonies were used to inoculate 25 mL TB-Medium (150 µg/mL ampicillin) in a 250 mL flask each
  • 26.04.
    • IPTG induction of expression
      • 10 mL pre-culture from 25.04. transferred into 1 L TB media in 5 L Erlenmeyer flasks with 150 µg/mL ampicillin and incubated at 37 °C and 180 rpm
      • 0.25 mM IPTG used for induction when OD600 0.6 was reached
      • incubation at 18 °C with 180 rpm for 48 h
  • 28.04.
    • Cell harvest of WT Ferritin BL21 (DE3) Star
  • 16.06.
    • Heat precipitation and ammoniumsulfate precipiation of WT Ferritin BL21 (DE3) Star to check the protocol
      • centrifuged 40 min at 3300 g and 4 °C
      • resuspend pellet in 50 mL lysis buffer
      • protein concentration very low
    • concentration of WT-Ferritin by using Amicron Ultra centrifugal units 3k MWCO from 50 mL to 1 mL final volume
    • protein concentration measured with NanoDrop:
      Ferritin WT SampleC_Heat PrecipiationC_after Concentration
      K10.039 mg/mL3.380 mg/mL
      K20.051 mg/mL2.76 mg/mL
  • 19.06.
    • SDS-PAGE of heat precipitation and ammoniumsulfate precipitation results
      • 12 % SDS-Gel
      • run at 160 V for 70 min
      12 % SDS-PAGE of WT-Ferritin K1 after heat precipitation and ammonium sulfate precipitation with different distinct masses (30 µg, 20 µg, 10 µg, 1 µg, 0.039 µg, 3.380 µL). Protein ferritin expected at 21.14 kDa (area marked in red)
      • Problem:
        • heat precipitation and ammonium precipitation leads to residual impurities which compromised its purity
  • 24.07.
    • Heat precipitation and ammoniumsulfate precipiation of WT-Ferritin BL21 (DE3) Star
      WT Ferritin SampleConcentration
      K1 #115.307 mg/mL
      K1 #F1.277 mg/mL
      Sample numbersample after following steps in the protocol
      11. (resuspended pellet)
      22. (after sonification)
      33. (supernatant)
      43. (pellet)
      55. (supernatant)
      65. (pellet)
      77. (supernatant)
      88. (resuspended pellet)
      910. (supernatant)
      1011. (before filtration)
      • sample 8 was inadvertently omitted from both sets of probes
    • SDS-gelelectrophoresis
      • 12% SDS-gel
      • run for 140 V for 70 min
      • F 10 were not applied on the SDS-PAGE
      12 % SDS-PAGE of heat precipitation and ammonium sulfate precipitation with in-between samples 1 (#11 1, resuspended pellet), 2 (#11 2, after sonification), 3 (#11 3, supernatant after centrifugation), 4 (#11 4, pellet after centrifugation, diluted 1:200), 5 (#11 5, supernatant after centrifugation) and 6 (#11 6, pellet after centrifugation, diluted 1:200) of WT-Ferritin #11 to check purification.
      12 % SDS-PAGE of heat precipitation and ammonium sulfate precipitation with in-between samples 7 (#11 7, supernatant after centrifugation), 9 (#11 9, supernatant after centrifugation) and 10 (#11 10, before filtation), of WT-Ferritin #11 to check purification.
      12 % SDS-PAGE with WT-Ferritin #11 after heat precipitation and ammonium sulfate precipitation with 10 µg, 20 µg, 30 µg protein and undiluted (UD) with 15 µL of 5.307 mg/mL protein solution . Protein ferritin, expected at 21.14 kDa, could not be detected.
      12 % SDS-PAGE of heat precipitation and ammonium sulfate precipitation with in-between samples 1 (F 1, resuspended pellet), 2 (F 2, after sonification), 3 (F 3, supernatant after centrifugation), 4 (F 4, pellet after centrifugation, diluted 1:200), 5 (F 5, supernatant after centrifugation) and 6 (F 6, pellet after centrifugation, diluted 1:200) of WT-Ferritin #F to check purification.
      12 % SDS-PAGE of heat precipitation and ammonium sulfate precipitation with in-between samples 7 (F 7, supernatant after centrifugation) and 9 (F 9, supernatant after centrifugation) of WT-Ferritin #F to check purification.
      12 % SDS-PAGE of WT-Ferritin #11 after heat precipitation and ammonium sulfate precipitation undiluted (UD) with 15 µL of 5.307 mg/mL protein solution and with 30 µg, 20 µg and 10 µg protein . Protein ferritin, expected at 21.14 kDa, could not be detected.
      • Problem:
        • SDS gelelectrophoresis was stopped too early which resulted in an incomplete seperation of proteins. Ferritin could not be detected
    • Transformation of TAT-Ferritin and R12-Ferritin in BL21 (DE3) Star
  • 26.07.
    • Colony PCR of colonies of TAT-Ferritin BL21 and R12-Ferritin BL21 (DE3) star
  • 28.07
    • Agarose gelelectrophoresis of Colony PCR Results
      • run at 120 V for 40 min
      • expected result: bands at approximately 600 bp
      Agarose gel (1 %) with Colony PCR results of TAT-Ferritin BL21 (DE3) Star (1-3) and R12-Ferritin (1-3) BL21 (DE3) Star. Results of Colony PCR of TAT-Ferritin DH5α (+) and R12-Ferritin DH5α (+) used as a positive control. The area with the bands for a positive result is marked by a red box.
  • 02.08.
    • Transformation of R9-Ferritin in BL21 (DE3) Star
  • 07.08.
    • Colony PCR of colonies of R9-Ferritin BL21 (DE3) Star
    • Agarose gelelectrophoresis of Colony PCR Results
      • expected results: bands at approximately 600 bp
      Agarose gel (1 %) with Colony PCR results of R9-Ferritin BL21 (DE3) Star (1-3) Results of Colony PCR of TAT-Ferritin DH5α (+) used as a positive control.
    • pre-culutre of TAT-Ferritin BL21 (DE3) Star, R9-Ferritin BL21 (DE3) Star and R12-Ferritin BL21 (DE3) Star in TB-media for expression analysis
  • 08.08.
    • IPTG induction of expression of TAT-Ferritin, R9-Ferritin and R12-Ferritin
  • 11.08.
    • Cell harvest of TAT-Ferritin BL21 (DE3) Star, R9-Ferritin BL21 (DE3) Star and R12-Ferritin BL21 (DE3) Star
  • 14.08.
    • Heat precipitation and ammonium sulfate precipitation of TAT-Ferritin, R9-Ferritin and R12-Ferritin
      • protein concentration measured by Nano-Drop:
        SampleConcentration
        TAT-Ferritin2.185 mg/mL
        R9-Ferritin1.704 mg/mL
        R12-Ferritin2.137 mg/mL
  • 16.08.
    • SDS-PAGE with purified Proteins
      • 15 % SDS-gel
      • run at 160 V for 70 min
      • gel was stained after blotting
      15 % SDS-PAGE of R9-Ferritin after heat precipitation and ammonium sulfate precipitation with 30 µg, 20 µg, 10 µg, 1 µg, 1.704 µg of protein and 15 µL undiluted protein solution (UD) of 1.704 mg/mL. The area where the ferritin protein construct (23 kDa) is expected is marked by a red box.
    • Western-Blot of R9-Ferritin with Ferritin Antibody with conjugated horse raddish peroxidase
      • no signal was obtained
  • 18.08.
    • pre-culture of TAT-Ferritin BL21 (DE3) Star, R9-Ferritin BL21 (DE3) Star and R12-Ferritin BL21 (DE3) Star in TB media
  • 19.08.
    • IPTG induction of expression
  • 21.08.
    • Cell harvest of TAT-Ferritin BL21 (DE3) Star, R9-Ferritin BL21 (DE3) Star and R12-Ferritin BL21 (DE3) Star
  • 22.08.
    • SDS-PAGE of whole cell samples before and after IPTG expression induction
      • 15 % SDS-gel
      • run at 160 V for 70 min
      • expected results: intense bands at approximately 21 kDa for WT-Ferritin, 23 kDa for CPP-Ferritin constructs after induction,
      15 % SDS-PAGE with whole cell samples of WT-Ferritin (WT-Ftn) TAT-Ferritin, R9-Ferritin and R12-Ferritin before (N) and after IPTG expression induction (I). The area where the ferritin protein construct is expected is marked by a red box.
    • Heat precipiation and ammonium sulfate precipitation of CPP-Ferritin samples and WT-Ferritin BL21 (DE3) Star
  • 23.08.
    • Whole cell SDS-PAGE and SDS-PAGE with purified proteins
      • 15 % SDS-gel
      • run at 160 V for 70 min
      • gel was stained after blotting
      • expected results: intense bands at approximately 22 kDa or 23 kDa, and less unspecified bands

      • Whole cell:

      15 % SDS-PAGE of whole cell samples of WT-Ferritin BL21 (DE3) Star, TAT-Ferritin BL21 (DE3) Star, R9-Ferritin BL21 (DE3) Star and R12-Ferritin BL21 (DE3) Star with an OD600 of 2.0 and 1.0. The area where the ferritin protein construct is expected is marked by a red box.

      Purified proteins:

      15 % SDS-PAGE of protein samples after heat precipitation and ammonium sulfate precipitation with 20 µg and 10 µg of WT-Ferritin, TAT-Ferritin, R9-Ferritin and R12-Ferritin. The area where the ferritin protein construct is expected is marked by a red box.
    • Ponceau S staining to check if blotting was successful
      • membrane stained with Ponceau S for 5 min
      • washed until excess staining was removed
      • after imaging membrane got washed until whole staining was removed

      Whole cell:

      Ponceau S membrane staining of membrane with whole cell samples of WT-Ferritin BL21 (DE3) Star, TAT-Ferritin BL21 (DE3) Star, R9-Ferritin BL21 (DE3) Star and R12-Ferritin BL21 (DE3) Star with an OD600 of 2.0 and 1.0 after blotting. The area where the ferritin protein construct is expected is marked by a red box.

      Purified proteins:

      Ponceau S membrane staining of membrane with protein samples after heat precipitation and ammonium sulfate precipitation with 20 µg and 10 µg of WT-Ferritin, TAT-Ferritin, R9-Ferritin and R12-Ferritin. after blotting The area where the ferritin protein construct is expected is marked by a red box.
    • Western-Blot whole cell samples and purified protein solution with Ferritin Antibody with conjugated Peroxidase

      Whole cell:

      Membrane after western blot and antibody conjugation of whole cell samples of WT-Ferritin BL21 (DE3) Star, TAT-Ferritin BL21 (DE3) Star, R9-Ferritin BL21 (DE3) Star and R12-Ferritin BL21 (DE3) Star with an OD600 of 2.0 and 1.0 after blotting. The area with the antibody signal is marked by a red box.
      Purified proteins:
      • no signal was obtained
  • 29.08.
    • SDS-PAGE of WT Ferritin and TAT-Ferritin, R9-Ferritin and R12-Ferritin protein solution
      • 15 % SDS-gel
      • run at 160 V for 70 min
      • gel was stained after blotting
      • expected results: intense bands at approximately 22 kDa or 23 kDa, and less unspecified bands
      15 % SDS-PAGE with protein samples after heat precipitation and ammonium sulfate precipitation with 20 µg and 10 µg of WT-Ferritin, TAT-Ferritin, R9-Ferritin and R12-Ferritin. The area where the ferritin protein construct is expected is marked by a red box.
    • Ponceau S staining to check if blotting was successful
      • membrane stained with Ponceau S for 5 min
      • washed until excess staining was removed
      • after imaging membrane got washed until whole staining was removed
      Ponceau S membrane staining of membrane with protein samples after heat precipitation and ammonium sulfate precipitation with 20 µg and 10 µg of WT-Ferritin, TAT-Ferritin, R9-Ferritin and R12-Ferritin. after blotting The area where the ferritin protein construct is expected is marked by a red box.
    • Western Blot of WT-ferritin and TAT-Ferritin, R9-Ferritin and R12-Ferritin with Ferritin Antibody conjugated with horse raddisch peroxidase peroxidase
      • no signal was obtained
  • 31.08.
    • Heat precipitiation and ammonium sulfate precipitation of CPP-Ferritin samples and WT-Ferritin BL21 (DE3) Star
    • SEC run with ferritin standard protein
      • 1 mL standard protein solution (3 mg/mL) injected
      SEC result after ferritin standard protein injection.
  • 27.09 - 29.09.
    • Ion exchange chromatography (IEC)
      • expected elution of ferritin protein constructs at a conductivity of 35 mS/cm

      WT-Ferritin 1

      IEC results of WT-Ferritin 1 with a general overview (A) and view of the area with the possible peak (B). The software did not detect a peak.
      • although the software could not detect a peak, samples were taken from the D3 to F3 sections

      WT-Ferritin 2

      IEC results of WT-Ferritin 2 with a general overview (A) and view of the area with the possible peak (B). The peak with the conductivity of 27.89 mS/cm was identified as the ferritin protein.

      TAT-Ferritin 1

      IEC results of TAT-Ferritin 1 with a general overview (A) and view of the area with the possible peak (B). The peak with the conductivity of 31.69 mS/cm was identified as the TAT-Ferritin protein.

      TAT-Ferritin 2

      IEC results of TAT-Ferritin 2 with a general overview (A) and view of the area with the possible peak (B). The peak with the conductivity of 31.92 mS/cm was identified as the TAT-Ferritin protein.

      R9-Ferritin 1

      IEC results of R9-Ferritin 1 with a general overview (A) and view of the area with the possible peak (B). The peak with the conductivity of 35.13 mS/cm was identified as the R9-Ferritin protein.

      R9-Ferritin 2

      IEC results of R9-Ferritin 2 with a general overview (A) and view of the area with the possible peak (B). The peak with the conductivity of 29.43 mS/cm was identified as the R9-Ferritin protein.

      R12-Ferritin 1

      IEC results of R12-Ferritin 1 with a general overview (A) and view of the area with the possible peak (B). The peak with the conductivity of 32.24 mS/cm was identified as the R12-Ferritin protein.

      R12-Ferritin 2

      IEC results of R12-Ferritin 2 with a general overview (A) and view of the area with the possible peak (B). The peak with the conductivity of 33.86 mS/cm was identified as the R12-Ferritin protein.
    • protein solution after IEC concentrated by using 30,000 MW centrifugal filter
  • 29.09.
    • SDS-PAGE with purified samples after IEC using section samples of the start of the peak, highest point of the peak and end of the peak as well as the wash step sample of R9-Ferritin to check the purification efficency of IEC
      • 15 % SDS-gel
      • run at 160 V for 70 min
      • expected results: intense bands at approximately 22 kDa or 23 kDa, and less unspecified bands
      15 % SDS-PAGE of WT-Ferritin 1 and R9-Ferritin 1 after IEC with section samples of the start of the peak (Start) the highest point of the peak (HP) and the end of the peak (End) as well as the washing step (Wash) during IEC of R9-Ferritin 1. The area where the ferritin protein construct is expected is marked by a red box.
      • Problem:
        • PAGE ladder drifted into lanes
        • just faint bands of expected R9-Ferritin
    • SDS-PAGE with purified samples after IEC
      • 15 % SDS-gel
      • run at 160 V for 70 min
      • expected results: intense bands at approximately 22 kDa or 23 kDa, and less unspecified bands
      15 % SDS-PAGE of sample 1 and 2 of WT-Ferritin, TAT-Ferritin, R9-Ferritin and R12-Ferritin after IEC. The area where the ferritin protein construct is expected is marked by a red box.
      • Problem: CPP-Ferritin constructs could not be detected after IEC
    • Size exclusion chromatography (first run) with WT Ferritin
      SEC result of WT-Ferritin 1 after the first run. Second peak with retention after 11.194 mL identified as ferritin monomer. First peak with retention after 8.091 mL possibly wrong assembled ferritin.
  • 02.10.
    • second run of size exclusion chromatography with WT-Ferritin to discard larger proteins
      SEC result of WT-Ferritin 1 after the second run. Second peak with retention after 10.897 mL identified as ferritin monomer. First peak with retention after 8.102 mL possibly wrong assembled ferritin.
    • SDS-PAGE of purified WT-Ferritin 1 sample after SEC
      • 15 % SDS-gel
      • run at 160 V for 70 min
      • expected results: intense bands at approximately 22 kDa or 23 kDa, and less unspecified bands
      15 % SDS-PAGE of WT-Ferritin after the first SEC run (WT Run 1) and second SEC run (WT Run 2). The WT-Ferritin protein is marked by a red box.
    • ion exchange chromatography (IEC) with R9-Ferritin 3 and TAT-Ferritin 3
      • lysis buffer and elution buffer at pH 9 due to the changed PI of R9-Ferritin and TAT-Ferritin
      • no peaks could be detected
      IEC result of TAT-Ferritin 3 (A) and R9-Ferritin3 (B). Peaks with an elution at a conductivity of 35 mS/cm which indicates for ferritin could not be detected.

      No peak present which could be indicated as CPP-Ferritin construct

  • 04.10.
    • Negative Staining of WT Ferritin (0.026 mg/mL) at pH 7.5
      • Glow discharching of copper grid at 25 mA for 30 sec
      • Protein Absorption, Washing (ddH2O), Uranylacetate-staining
      • Microscope: Talos L120c

Implementation of Nanobodies
  • 06.09.
    • QuikChange mutagenesis of pET22b(+)_WT-Ferritin and TAT-Ferritin for implementing the Amber codon (Q5 polymerase, long synthesis (3.5 minutes))
    • Agarose gel for examination of the PCR
      • expected result: band at approximately 6,000 bp
      Agarose gel (1 %) of PCR results to check success of the reaction for implementation of the amber codon. Two samples of WT-Ferritin DNA and TAT-Ferritin DNA were used. Intense bands which show positive results were obtained at approximately 6,000 bp.
  • 07.09.
    • PCR purification
    • DpnI digest to eliminate unmutated DNA
    • Transformation of mixes in DH5α onto ampicillin plates
  • 08.09.
    • pre-culture of transformed clones
      • 5 mL LB-media containing 100 µg/mL ampicilllin
  • 09.09.
    • Miniprep of pre-culture
  • 13.09.
    • Samples sent to sequencing
      • WT-Ferritin 2 and TAT-Ferritin 3 successfully mutated
  • 14.09.
    • PCR of p15A backbone for linearization and to add BsaI recognition sites for GGA reaction with fragments for helper plasmid (Q5 polymerase, Annealing: 69 °C)
  • 15.09.
    • Agarose gel for examination of PCR
      Agarose gel of p15A_CmR backbone PCR to integrate BsaI recognition sites to use in GGA. The ladder was re-dyed after taking the bands out for PCR purification, the first lane contains first replica, second lane contains second replica and third lane contains both replicas pooled. Bands are expected at about 2,000 kb.
  • Gel extraction for purifying PCR products
  • First GGA reaction of linear p15A backbone + 3 fragments for helper plasmid
    ReagentsFor GGA reactionFor negative control
    p15A backbone (40 ng/µL)1.64 µL1.64 µL
    Fragment 1 (50 ng/µL)2.19 µL-
    Fragment 2 (50 ng/µL)2.10 µL-
    Fragment 3 (25 ng/µL)1.13 µL-
    T4 DNA ligase buffer (10X)2 µL2 µL
    NEB GGA Mix BsaI1 µL1 µL
    MiliQ waterad 20 µLad 20 µL
    Total20 µL20 µL
    37°C10 min
    10 cycles (step 1-2)16°C10 min
    37°C20 min
    60°C20 min
    hold4°C
  • 19.09
    • Transformation of GGA in DH5α onto chloramphenicol plates
  • 20.09.
    • Colony PCR of transformed clones
    • Agarose gelelektrophoresis of Colony PCR results
      • expected result: bands at approximately 900 bp
      Agarose gel (1 %) with colony PCR results. The lane with negative control lane without template displayed unexpected bands. Bands with a size of about 900 base pairs are present in the traces of GGA colony PCR 2, 3, 4 and 5. The area with the suspected bands in the agarose gel is marked by a red box.
    • Pre-culture of clones with positive colony PCR results (GGA 2-5)
      • 5 mL LB-media containing 100 µg/mL ampicillin
      • incubated at 37 °C and 200 rpm
  • 21.09.
    • Miniprep of pre-culture
    • GGA 2 and GGA 3 were sent to sequencing
      • result: negative
    • Plating out of glycerol stock of eGFP expression culture onto ampicillin plate
  • 22.09.
    • Co-tranformation of WT-Amber-Ferritin and TAT-Amber-Ferritin plasmids, each with helper plasmid GGA#3
    • Concentrations were equalized and 2.5 µL of each plasmid was used
    • Ampicillin/chloramphenicol plates were used
  • 23.09.
    • Repitition of GGA
      ReagentsFor GGA reactionFor negative control
      p15A backbone (40 ng/µL)1.64 µL1.64 µL
      Fragment 1 (50 ng/µL)2.19 µL-
      Fragment 2 (50 ng/µL)2.10 µL-
      Fragment 3 (25 ng/µL)1.13 µL-
      T4 DNA ligase buffer (10X)2 µL2 µL
      NEB GGA Mix BsaI1 µL1 µL
      MilliQ waterad 20 µLad 20 µL
      Total20 µL20 µL
      37 °C10 min
      10 cycles (step 1-2)16 °C10 min
      37 °C20 min
      60 °C20 min
      Hold4 °C
    • Subcloning: Each fragment was also cloned on its own into a LacZ vector to amplify the amount of available DNA per fragment
  • 24.09.
    • Transformation of GGA in DH5α onto chloramphenicol plates
    • Transformation of LacZ vectors for blue white screening
  • 25.09.
    • Colony PCR of GGA transformed colonies
      Denaturation94 °C5 min
      29 cycles94 °C15 sec
      58 °C30 sec
      69 °C1 min
      Final extension72 °C5min
      Hold4 °Chold
    • Agarose gelelektrophoresis
      • expected result: bands at approximately 900 bp.
      Agarose gel (1 %) with Colony PCR results. The lane with negative control lane displayed no bands. Two samples of each transformation (GGA 1, GGA 2) were used. Only in the lane of GGA 2 2 a faint band at 900 bp could be observed. The area with the suspected bands in the agarose gel is marked by a red box.
  • 27.09.
    • Repetition of GGA
      ReagentsFor GGA reactionFor negative control
      p15A backbone (40 ng/µL)1.64 µL1.64 µL
      Fragment 1 (50 ng/µL)2.19 µL-
      Fragment 2 (50 ng/µL)2.10 µL-
      Fragment 3 (25 ng/µL)1.13 µL-
      T4 DNA ligase buffer (10X)2 µL2 µL
      NEB GGA Mix BsaI1 µL1 µL
      MiliQ waterTo 20 µLTo 20 µL
      Total20 µL20 µL
      37 °C5 min
      16 °C5 min
      Go to 1, repeat 29x
      60 °C5 min
      4 °CHold
    • Transformation of GGA into DH5α cells onto Chloramphenicol plates
  • 28.09.
    • Transformation was not successful
  • 30.09.
    • Repetition of PCR of p15A plasmid for linear backbone with BsaI restriction recognition sites at the end
    • Agarose gel for examination of PCR
      • expected result: intense bands at approximately 2000 bp.
      Agarose gel (1 %) with results of PCR reaction of p15A for linaerizing backbone with BsaI restriciton recognition sites. Two products of the reaction (PCR 1, PCR 2) were used. Intense bands which show positive results were obtained at approximately 2,000 bp.
    • PCR purification
  • 01.10.
    • Repetition of GGA for helper plasmid
      • 3 different approaches
      • according to protocol
      Reagents1.2.3.Negative
      p15A (70 ng/µL)0.88 µL0.88 µL1.76 µL1.76 µL
      Fragment 1 (50 ng/µL)2.19 µL2.19 µL4.38 µL-
      Fragment 2 (50 ng/µL)2.10 µL2.10 µL4.20 µL-
      Fragment 3 (25 ng/µL)1.13 µL1.13 µL2.26 µL-
      T4 DNA ligase buffer (10 X)2 µL2 µL2 µL2 µL
      NEB GGA Mix BsaI2 µL-2 µL2 µL
      BsaI enzyme-1 µL--
      T4 DNA ligase-1 µL--
      ATP (10 mM)1 µL1 µL1 µL1 µL
      MiliQ water8.7 µL8.7 µL2.4 µL8.7 µL
      Total20 µL20 µL20 µL20 µL
      37 °C1 min
      16 °C1 min
      Go to 1, repeat 29x
      60 °C5 min
      4 °CHold
    • Transformation of GGA in DH5α cells onto chloramphenicol plates
      • three plates for each approach
      • 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3 (naming according to the principle “approach.plate”)
  • 02.10.
    • Colony PCR
      • 6 colonies of each GGA approach plate
      • growth on negative control detected and 4 colonies got picked
      • 58 samples in total
      • naming according to the principle “approach.plate.colonie”
    • Agarose Gelelectrophoresis
    • Pre-culture of positive results (2.2.2, 2.2.3, 2.3.2, 2.3.4, 3.1.1, 3.1.3, 3.1.4, 3.1.6, 3.2.1, 3.2.2, 3.2.4, 3.2.5, 3.3.2)
      • 5 mL LB-media containing 100 µg/mL chloramphenicol
  • 03.10.
    • Miniprep of pre-culture and sent them to sequencing
  • 04.10.
    • Co-transformation of all plasmids each with WT-Amber-Ferritin and TAT-Amber-Ferritin onto ampicillin/chloramphenicol plates
  • 05.10.
    • Sequencing results: 2.3.4. possibly positive
    • Double digest of positive plasmid for checking sequencing results
      • used enzymes:
        • EcoRI
        • Cfr9l
      1% agarose gel, run at 160 V for 1 h with positive GGA product 2.3.4 with EcoRI and Cfr9I digest or without as negative control ( - ). Bands are expected at around 3,000, 1500, 1000 and 400 bp for digested plasmid and 5,800 bp for undigested. Since plasmid and probably supercoiled plasmid runs fast through the gel, the control band is lower than the expectad band of 5,800 bp.

Testing the susceptibility of bacteria to antibiotic molecules
  • 20.07.
    • Agar-diffusion test with flavone on Escherichia coli
      • no zone of inhibition could be observed
  • 26.07.
    • Repetition of agar-diffusion test with flavone on E.coli
      • no zone of inhibition could be observed
  • 03.08.
    • Agar-diffusion test with flavone, quercetin, rutin and ethylparaben individually and in combination on E.coli
      • no zone of inhibition could be observed

Penetration Efficiency
  • 11.05.
    • overnight culture of pET-17b-eGFP-6xHis BL21 DE3 in 20 mL LB medium with ampicillin in preparation for Miniprep
  • 12.05.
    • overnight culture from 11.05. split into two, Miniprep of pET-17b-eGFP-6xHis BL21 DE3 using Thermoscientific GeneJET Plasmid Miniprep Kit
      • nucleic acid concentration measured with nanodrop: 551 ng/µL and 458 ng/µL
  • 23.05.
    • incoming material from Dr. Dirk Becker I: purified proteins eGFP, R9-eGFP, R12-eGFP, R12-cpf1-GFP, TAT2-eGFP
      • eGFP-sythesis started on 11.05. stopped until further notice (pET-17b-eGFP-6xHis BL21 DE3)
    • incoming material from Dr. Dirk Becker II: glycerol stocks
      • 619: eGFP in BL17
      • 620: R12-eGFP in BL21 DE3 RIPL*
      • 621: TAT-eGFP in BL21 DE3 RIPL*
      • 622: R9-eGFP in BL21 DE3 RIPL*
      • 623: TAT2-eGFP in BL21 DE3 RIPL*
      • 624: TAT3-eGFP in BL21 DE3 RIPL*
      • 625: R9-TAT-eGFP in BL21 DE3 RIPL*
      • * pET28a(+)
  • 01.06.
    • penetration with TAT2-eGFP following protocol “penetration of E. coli DH5α cells with GFP-CPPs” until fixation; incubation with protein for 1 hour
  • 05.06.
    • DNA staining, mounting with 2.5 µL mowiol, wide field microscopy
      • results: GFP signals, location of fusion proteins (cytoplasma/ plasma membrane) not clear (see Results)
      Figure 1: E. coli DH5α cells incubated with TAT2-eGFP for 1 hour. Leica Dmi8 Wide field microscope. GFP channel. Display range: 0-3000 (64-bit format).
  • 06.06.
    • subcultivation of E. coli DH5α overnight culture, plating of 100 µL OD600 0.640 and 100 µL OD600 0.0795 to agar plates and incubated overnight at 37 °C
  • 14.06.
    • overnight culture of E. coli of DH5α (20 mL LB medium, 37 °C, 120 rpm)
  • 15.06.
    • penetration with R9-eGFP and R12-eGFP following the protocol “penetration of E. coli DH5α cells with GFP-CPPs” until fixation; incubation with protein for 1 hour
  • 20.06.
    • DAPI staining, mounting, wide field microscopy
      • Results: R12 assay showed GFP signals, R9 assay did not show GFP signals. R12 presumably binds or penetrated the membrane, binding or penetration of R9 cannot be shown. (more details see Results)
      Figure: E. coli DH5α cells incubated with R12-eGFP for 1 hour. Leica Dmi8 Wide field microscope. GFP channel. Display range: 0-3000 (64-bit format).
      Figure: E. coli DH5α cells incubated with R9-eGFP for 1 hour. Leica Dmi8 Wide field microscope. GFP channel. Display range: 0-3000 (64-bit format).
  • 21.06.
    • plating of glycerol stocks 619-625 (see entry 01.06.) on LB agar plates containing 50 µg/mL kanamycin, incubated at 37 °C overnight
      • all plates showed colonies except of 619 (eGFP plasmid in BL17, does not have kanamycin resistance)
  • 22.06.
    • overnight culture of glycerol stocks (30 mL of LB medium containing 50 µg/mL kanamycin inoculated with one colony), incubated at 37 °C and 180 rpm overnight
  • 23.06.
    • Miniprep of overnight cultures from 22.06. with Thermoscientific GeneJET Plasmid Miniprep Kit
      • concentrations [ng/µL nucleic acid]: 620 - 61.0, 621 - 69.9, 622 - 80.5, 623 - 51.8, 624 - 48.1, 625 - 64.2
    • sending to sequencing
      • results: sequences confirmed
  • 05.07.
    • penetration with R9-eGFP, R12-eGFP and TAT2-eGFP following the protocol “penetration of E. coli DH5α cells with GFP-CPPs” until fixation; incubation with protein for 1.5 hours
      • results:
  • 22.07.
    • transformation of E. coli DH5α competent cells with Miniprepped pET28a(+)-plasmids (received from Dr. Dirk Becker): R9-eGFP, R12-eGFP, TAT-eGFP, TAT2-eGFP, TAT3-eGFP, R9-TAT-eGFP
      • transformed DH5α, but BL21 needed for expression; plasmid DNA 621 and 622 contaminated with DH5α cells
  • 23.07.
    • overnight culture of transformed with 621 and 622 DH5α colony (15 ml LB medium, 37 °C, 120 rpm shaking)
  • 24.07.
    • Miniprep of 621 and 622 overnight culture with Thermoscientific GeneJET Plasmid Miniprep Kit
      • concentration of 621: 93.5 ng/µL
      • concentration of 622: 105.7 ng/µL
  • 29.07.
    • transformation of E. coli BL21 competent cells with plasmid DNA from 23.06. (620, 623, 624, 625) or 24.07. (621, 622)
  • 01.08.
    • overnight culture of transformed BL21 colony from 29.07. in TB medium containing 50 µg/mL kanamycin; incubated overnight at 37 °C and 120 rpm shaking
  • 02.08.
    • 100 mL of LB medium with 50 µg/mL kanamycin inoculated with 2 mL of overnight culture each, incubated at 37 °C and 150 rpm shaking
      • OD600:
      time620621622623624625
      1:400.2160.1810.2280.2420.2010.197
      2:150.3840.3380.3980.4150.3760.330
      3:000.544-----
      3:100.6260.5820.6310.7530.7310.550
    • after 3:10 hrs, protein epxression was induced with 0.5 mM IPTG end concentration, incubation overnight at 24 °C and 150 rpm shaking
  • 03.08.
    • cell pelleting at 3234 g, 4 °C for 15 min, supernatant discarded and storage of the pellets at -80 °C
      • results: pellets showed green color, GFP fusion proteins were expressed
  • 07.08.
    • E. coli DH5α overnight culture (35 mL LB medium, incubated at 37 °C and 120 rpm)
  • 08.08.
    • preparation of calcium competent cells: E. coli DH5α treated and washed with 50 mM CaCl2 and stored in 30 % glycerol containing 50 mM CaCl2
  • 10.08.
    • penetration with R9-eGFP, R12-eGFP and TAT2-eGFP following the protocol “penetration of E. coli DH5α cells with GFP-CPPs“; difference: usage of calcium competent cells
      • results: no GFP signal in all assays, penetration with CaCl2 treated cells not succesfull
  • 14.08.
    • preparation of buffers for protein purification following the protocol “Purification of Polyhistidine-Tagged Proteins“ from Bioke (October 2010): NPI-10, NPI-20, NPI-250
  • 15.08.
    • Ni-NTA agarose batch purification of proteins 620 to 625 (see entry 01.06.) following the protocol “Purification of Polyhistidine-Tagged Proteins“ from Bioke (October 2010)
  • 30.08.
    • SDS-PAGE (12% polyacrylamid gel) of purified proteins from 03.08.; samples loaded: pellets before IPTG induction (t=0), pellets after protein expression (t=1), purified protein (P)
      • results: successful protein expression unclear, purification needs to be proceeded
SDS-PAGE (12%) of protein purification: t=0 before IPTG induction, t=1 after IPTG induction, P=purified protein.

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