iGEM 2023 — Team hamburg

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{% block lead %}Follow all of our steps we took during this exciting journey in the lab!{% endblock %}
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## 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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure1.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure2.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure3.png)

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)](http://parts.igem.org/Part%3ABBa_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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure4.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure5.png)

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)](http://parts.igem.org/Part%3ABBa_K1202006), R9 [(BBa\_K4669001)](http://parts.igem.org/Part%3ABBa_K4669001), and R12 [(BBa\_K4669002)](http://parts.igem.org/Part%3ABBa_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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure6.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure7.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure8.png)

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 Name | Original Sample |
    | --- | --- |
    | WT | WT Ferritin (60 ng) |
    | TAT-Ferritin 30 | TAT-Ferritin (30 ng\*) |
    | TAT-Ferritin 40 | TAT-Ferritin (40 ng) |
    | TAT-Ferritin 50 | TAT-Ferritin (50 ng) |

* **13.07.**
  + Colony PCR of transformation colonies from 12.07.
    - program used:

      |  |  |  |
      | --- | --- | --- |
      | Denaturation | 94 °C | 5 min |
      |  | 94 °C | 10 sec |
      |  | 60,5 °C | 10 sec |
      | go to step two, repeat 34X | 72 °C | 3:30 min |
      | Final extension | 72 °C | 5 min |
      | Hold | 4 °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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure9.png)

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 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 2, repeat 34x | - | - |
      | 6. | Final DNA-synthesis | 72 °C | 5 min |
      | 7. | Hold | 4 °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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure10.png)

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:

      |  |  |  |
      | --- | --- | --- |
      | Denaturation | 94 °C | 5 min |
      | go to step 2, repeat 34X | 94 °C | 10 sec |
      |  | 60,5 °C | 10 sec |
      |  | 72 °C | 3:30 min |
      | Final extension | 72 °C | 5 min |
      | Hold | 4 °C |  |
  + Agarose gelelectrophoresis to check Colony PCR
    - run at 120 V for 40 min
    - expected results: bands at approximately 600 bp

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure11.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure12.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure13.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure14.png)

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 Sample | C\_Heat Precipiation | C\_after Concentration |
    | --- | --- | --- |
    | K1 | 0.039 mg/mL | 3.380 mg/mL |
    | K2 | 0.051 mg/mL | 2.76 mg/mL |

* **19.06.**
  + SDS-PAGE of heat precipitation and ammoniumsulfate precipitation results
    - 12 % SDS-Gel
    - run at 160 V for 70 min

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure15.png)

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 Sample | Concentration |
    | --- | --- |
    | K1 #11 | 5.307 mg/mL |
    | K1 #F | 1.277 mg/mL |

    | Sample number | sample after following steps in the protocol |
    | --- | --- |
    | 1 | 1. (resuspended pellet) |
    | 2 | 2. (after sonification) |
    | 3 | 3. (supernatant) |
    | 4 | 3. (pellet) |
    | 5 | 5. (supernatant) |
    | 6 | 5. (pellet) |
    | 7 | 7. (supernatant) |
    | 8 | 8. (resuspended pellet) |
    | 9 | 10. (supernatant) |
    | 10 | 11. (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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure16.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure17.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure18.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure19.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure20.png)

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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure21.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure22.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure23.png)

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:

      | Sample | Concentration |
      | --- | --- |
      | TAT-Ferritin | 2.185 mg/mL |
      | R9-Ferritin | 1.704 mg/mL |
      | R12-Ferritin | 2.137 mg/mL |

* **16.08.**
  + SDS-PAGE with purified Proteins
    - 15 % SDS-gel
    - run at 160 V for 70 min
    - gel was stained after blotting

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure24.png)

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,

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure25.png)

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:

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure26.png)

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:

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure27.png)

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:

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure28.png)

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:

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure29.png)

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:

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure30.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure31.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure32.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure33.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure34.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure35.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure36.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure37.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure38.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure39.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure40.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure41.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure42.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure43.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure44.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure45.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure46.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure47.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure48.png)

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

![](https://static.igem.wiki/teams/4669/wiki/notebook/gelbild-jonas2.png)

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

  | Reagents | For GGA reaction | For negative control |
  | --- | --- | --- |
  | p15A backbone (40 ng/µL) | 1.64 µL | 1.64 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 µL | 2 µL |
  | NEB GGA Mix BsaI | 1 µL | 1 µL |
  | MiliQ water | ad 20 µL | ad 20 µL |
  | Total | 20 µL | 20 µL |

  |  |  |  |
  | --- | --- | --- |
  |  | 37°C | 10 min |
  | 10 cycles (step 1-2) | 16°C | 10 min |
  |  | 37°C | 20 min |
  |  | 60°C | 20 min |
  | hold | 4°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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure49.png)

+ 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

    | Reagents | For GGA reaction | For negative control |
    | --- | --- | --- |
    | p15A backbone (40 ng/µL) | 1.64 µL | 1.64 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 µL | 2 µL |
    | NEB GGA Mix BsaI | 1 µL | 1 µL |
    | MilliQ water | ad 20 µL | ad 20 µL |
    | Total | 20 µL | 20 µL |

    |  |  |  |
    | --- | --- | --- |
    |  | 37 °C | 10 min |
    | 10 cycles (step 1-2) | 16 °C | 10 min |
    |  | 37 °C | 20 min |
    |  | 60 °C | 20 min |
    | Hold | 4 °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

    |  |  |  |
    | --- | --- | --- |
    | Denaturation | 94 °C | 5 min |
    | 29 cycles | 94 °C | 15 sec |
    |  | 58 °C | 30 sec |
    |  | 69 °C | 1 min |
    | Final extension | 72 °C | 5min |
    | Hold | 4 °C | hold |
  + Agarose gelelektrophoresis
    - expected result: bands at approximately 900 bp.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure50.png)

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

    | Reagents | For GGA reaction | For negative control |
    | --- | --- | --- |
    | p15A backbone (40 ng/µL) | 1.64 µL | 1.64 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 µL | 2 µL |
    | NEB GGA Mix BsaI | 1 µL | 1 µL |
    | MiliQ water | To 20 µL | To 20 µL |
    | Total | 20 µL | 20 µL |

    |  |  |
    | --- | --- |
    | 37 °C | 5 min |
    | 16 °C | 5 min |
    | Go to 1, repeat 29x |  |
    | 60 °C | 5 min |
    | 4 °C | Hold |
  + 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.

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure51.png)

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

    | Reagents | 1. | 2. | 3. | Negative |
    | --- | --- | --- | --- | --- |
    | p15A (70 ng/µL) | 0.88 µL | 0.88 µL | 1.76 µL | 1.76 µL |
    | Fragment 1 (50 ng/µL) | 2.19 µL | 2.19 µL | 4.38 µL | - |
    | Fragment 2 (50 ng/µL) | 2.10 µL | 2.10 µL | 4.20 µL | - |
    | Fragment 3 (25 ng/µL) | 1.13 µL | 1.13 µL | 2.26 µL | - |
    | T4 DNA ligase buffer (10 X) | 2 µL | 2 µL | 2 µL | 2 µL |
    | NEB GGA Mix BsaI | 2 µL | - | 2 µL | 2 µL |
    | BsaI enzyme | - | 1 µL | - | - |
    | T4 DNA ligase | - | 1 µL | - | - |
    | ATP (10 mM) | 1 µL | 1 µL | 1 µL | 1 µL |
    | MiliQ water | 8.7 µL | 8.7 µL | 2.4 µL | 8.7 µL |
    | Total | 20 µL | 20 µL | 20 µL | 20 µL |

    |  |  |
    | --- | --- |
    | 37 °C | 1 min |
    | 16 °C | 1 min |
    | Go to 1, repeat 29x |  |
    | 60 °C | 5 min |
    | 4 °C | Hold |
  + 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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure52.png)

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)

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure53.png)

**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)

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure54.png)

**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).

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure55.png)

**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.)](#d6038583-7640-49fd-95e8-5a1fbfc6968e) 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.](#69b2fd2c-8c3b-48c8-8f5b-48033284c965) 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.](#69b2fd2c-8c3b-48c8-8f5b-48033284c965) (620, 623, 624, 625) or  [24.07.](#ef663cbe-fa67-4234-b48c-1bf0b7e7996c) (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:

    |  |  |  |  |  |  |  |
    | --- | --- | --- | --- | --- | --- | --- |
    | time | 620 | 621 | 622 | 623 | 624 | 625 |
    | 1:40 | 0.216 | 0.181 | 0.228 | 0.242 | 0.201 | 0.197 |
    | 2:15 | 0.384 | 0.338 | 0.398 | 0.415 | 0.376 | 0.330 |
    | 3:00 | 0.544 | - | - | - | - | - |
    | 3:10 | 0.626 | 0.582 | 0.631 | 0.753 | 0.731 | 0.550 |
  + 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.)](#d6038583-7640-49fd-95e8-5a1fbfc6968e) 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.](#3b10b459-4486-426e-a61f-ed7a93a66c53); 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

![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure56.jpeg)
![](https://static.igem.wiki/teams/4669/wiki/notebook/notebook-figure57.jpeg)

SDS-PAGE (12%) of protein purification: t=0 before IPTG induction, t=1 after IPTG induction, P=purified protein.

![](https://static.igem.wiki/teams/4669/wiki/design-elements/up.svg)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Over the past year, we have overcome many challenges in the lab. Here, we present you three of them.{%
endblock %}
{% block page\_content %}

{{ self.title() }}

![failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/01-engineering-cycle.svg)

## Engineering The Mutagenesis Of The Ferritin Plasmid For TAT-Implementation

#### Cycle 1

###### **Design**

In order to fuse the cell penetrating peptides (CPPs) to our ferritin, we designed primers that have
an overlapping part complementary to the ferritin. The CPPs and the glycine-serine linker were
included by a non-overlapping region for introducing them during the synthesis of the PCR (fig.
1).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/02-cpp-primer.png)

Fig. 1: Binding site of the primers for introducing the CPPs. The non-overlapping parts
of the primers included a start codon, one CPP and a glycine-serine linker for enabling correct
folding each. We fused the glycine-serine linker directly to the ferritin.

###### **Build**

We ordered the primers and promptly started the mutagenesis following the protocol that we used for
mutating the BsaI and SapI restriction sites beforehand. The annealing temperature of 62.2 °C was
calculated using the [Thermofisher Tm-calculator](https://www.thermofisher.com/de/de/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html).

###### **Test**

Afterwards, we examined the outcome by applying the samples to an agarose gel. 6,000 bp bands were
expected, however our first mutagenesis failed (fig. 2).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/03-gel-first-mutagenesis.png)

Fig. 2: Agarose gel (1%) of the first CPP-mutagenesis of the ferritin-plasmid. Three
samples per CPP were applied. Expected were bands at approximately 6,000 bp. One faint band at
approximately 6,000 bp for one TAT-ferritin sample can be seen.

###### **Learn**

The protocol we used proved to be unsuitable for the implementation of the CPPs. We had to optimize
the protocol.
We assumed that we may have used an incorrect annealing temperature, hence we had to find a way of
testing that.

#### Cycle 2

###### **Design**

To save resources, we agreed to continue with TAT-CPP for the optimzation of our mutagenesis
protocols.
To test the impact of the annealing temperature on our results, we decided to conduct a gradient PCR
to see would if the results would validate or dismiss our assumption.

###### **Build**

We created a temperature gradient of 5 °C centered around 60 °C to broaden the range of annealing
temperatures tested.

###### **Test**

We examined the second mutagenesis’ success by running the samples on an agarose gel and as before,
we did not obtain the expected results of bands at 6,000 bp (fig. 3).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/04-gel-gradient-pcr.png)

Fig. 3: Agarose gel (1 %) with gradient PCR results (gradient of 5 °C) to check optimal
primer annealing temperature for the fusion of TAT to ferritin. Faint bands with approximately
2,000 base pairs in size at 55.7 °C, 56.6 °C and 57.8 °C.

###### **Learn**

The results using a range of annealing temperatures did not deliver optimal results. We concluded
that we would have to adjust another parameter for success.

#### Cycle 3

###### **Design**

Next, we altered the parameter concerning the duration of the synthesis step of our PCR program.
Additionally, we include varying amounts of DNA to understand the impact of the templates’
concentration in the reaction.

###### **Build**

The guideline for the synthesis according to [NEB](https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492) was to plan 20-30 seconds per kb. Our plasmid is about 6,000 bp
large, which is why we decided to adjust our synthesis duration to 3.5 minutes instead of 2 minutes.
We began with samples of 10 ng of DNA and ended with 70 ng. Concerning the annealing temperature, we
returned to the first one we used since it was calculated based on the annealing part of our
primers.

###### **Test**

As before, we used an agarose gel to confirm the success of the mutagenesis and, this time, bands
were visible at the expected height of 6,000 bp (fig.4.)!

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/05-gel-tat-ferritin-10-70-ng.png)

Fig. 4: 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.

###### **Learn**

The impact of the template concentration used in the reaction was minimal.
But we now learned that, having a larger plasmid, incorperating a longer synthesis duration is
required for optimal results. For further experiments, we would amplify plasmids for a duration of
3.5 minutes each to be on the safe side.

#### **Engineering success!**

Now that we had optimized our method, we repeated the CPP-implementation of all three CPPs as in our
first step. We adjusted the synthesis time, increasing it from 2 minutes to 3.5 minutes. Again, we
checked the results using an agarose gel and were delighted to see the results (fig. 5)!

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/06-gel-engineering-success.png)

Fig. 5: Agarose gel (1 %) after PCR mutagenesis of the ferritin plasmid with CPPs TAT,
R9 and R12. Two samples used for each CPP mutation. All samples showed bands at approximately
6,000 bp. Also bands smaller in size are shown. Bands of interest are marked by a red box.

Then, it was possible for us to continue with the transformation of *E. coli* with the PCR product,
followed by colony PCR and sequencing, and could then obtain the correctly mutated plasmid. This plasmid
we could then use for expressing our CPP-ferritin constructs!

## Engineering The Ion Exchange Chromatography of TAT-, R9- and R12-Ferritin

#### Cycle 1

###### **Design**

The protocol for the purification of ferritin contains three steps for the purification: First heat
precipitation, then ion exchange chromatography (IEC), followed by size exclusion chromatography
(SEC). We used the protocol that [Prof. Dr. Tobias Beck](https://2023.igem.wiki/hamburg/human-practices)
 provided to us.

###### **Build**

For the ion exchange chromatography, following the protocol provided by Beck, we used an anion
exchange column. The proteins were dissolved in the lysis buffer used for the heat precipitation
with a pH value of 7.5. We let two samples of each of the four constructs - TAT-Ferritin ([BBa\_K469005](http://parts.igem.org/Part%3ABBa_K4669005)), R9-Ferritin ([BBa\_K4669004](http://parts.igem.org/Part%3ABBa_K4669004)), R12-Ferritin ([BBa\_K4669006](http://parts.igem.org/Part%3ABBa_K4669006)), and WT-Ferritin ([BBa\_K4669000](http://parts.igem.org/Part%3ABBa_K4669000)) - run over the column.

###### **Test**

We saw peaks in the chromatograms for all samples (fig. 6-9), one sample of each construct is
displayed. These peaks corresponded to our expectations with an elution at around 35 mS/cm. We
continued with the fractions of these peaks, concentrated them and applied them on an SDS-PAGE.
Unfortunately, clear bands were only visible for WT-Ferritin (fig. 10). Our TAT-, R9-, and
R12-Ferritin constructs were evidently lost during IEC purification.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/07-iec-wt-ferritin.svg)

Fig. 6: 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
WT-Ferritin protein.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/08-iec-tat-ferritin.svg)

Fig. 7: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/09-r9-ferritin.svg)

Fig. 7: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/10-r12-ferritin.svg)

Fig. 7: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/11-nach-iec.png)

Fig. 10: 15 % SDS-PAGE of WT-Ferritin, TAT-Ferritin, R9-Ferritin and R12-Ferritin after
IEC; two samples each. The area where the ferritin protein construct is expected is marked by
the red box.

###### **Learn**

We discussed our method again and quickly found an error source to address. When purifying our TAT-,
R9- and R12-Ferritin samples, we did not consider the need to adjust the pH of our lysis and elution
buffer. This is due to the fact that the attached CPPs would change the isoelectric point (pI) of
our protein (tab. 1). Therefore our constructs might not have been as negatively charged as we
assumed, leading to an earlier elution.

Tab. 1: The isoelectric points of WT-Ferritin and all CPP-Ferritin.

| Construct | Isoelectric point |
| WT-Ferritin | 5.3 |
| TAT-Ferritin | 6.87 |
| R9-Ferritin | 6.59 |
| R12-Ferritin | 7.91 |

For changing pIs, the pH of the buffer that the proteins are dissolved in has to be adapted in order to
keep them negatively charged and to have a well functioning anion exchange.

#### Cycle 2

###### **Design**

For the optimization of the IEC purification step we adapted the lysis buffer by changing its pH
value. Since we had prepared a number of heat precipitated samples, we decided on repeating this
step for R9- and TAT-Ferritin. With R12-Ferritin’s pI being the highest, we concluded that we would
exclude R12-Ferritin, until our method was successfully optimized.

###### **Build**

For both R9- and TAT-Ferritin we now used a buffer with a pH value of 9.

###### **Test**

Unfortunately, despite that the protein concentrations of the samples were similar to the samples
used before, we could not detect any peak (fig. 11).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/12-iec-ph9.svg)

Fig. 11: IEC results of TAT-Ferritin 3 (A) and R9-Ferritin3 (B) when using bufers with
pH 9. Peaks with an elution at a conductivity of around 35 mS/cm which would indicate for
ferritin could not be detected.

###### **Learn**

Somehow, we lost our protein again. Currently, we are still in the process of finding new approaches
for optimizing the protocol for protein purification. Due to the time constraints, we were only able
to purify WT-Ferritin and have not been successful in finding our lost protein yet.

## Engineering The Golden Gate Assembly Of The Helper Plasmid (aaRS/tRNA) For Nanobody Implementation

#### Cycle 1

###### **Design**

For expressing the ferritin with a non-canonical amino acid, we had to introduce the Amber-codon
into its backbone. Besides adding the non-canonical amino acid to the medium, we had to transform
the expression strain with a second plasmid: the helper plasmid containing the aminoacyl-tRNA
synthetase and the corrosponding tRNA. Both are essential when expressing ferritin.
 We designed
a plasmid with a chloramphenicol resistance, containing an origin of replication (ORI) that differs
from the one on the WT-Amber-Ferritin ([BBa\_K4669007](http://parts.igem.org/Part%3ABBa_K4669007)) and the TAT-Amber-Ferritin plasmids ([BBa\_K4669008](http://parts.igem.org/Part%3ABBa_K4669008)) (fig. 12-14).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/13-wt-ferritin-amber-map.svg)

Fig. 12: Map of the plasmid of WT-Ferritin containing the Amber-codon.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/14-tat-ferritin-amber-map.svg)

Fig. 12: Map of the plasmid of WT-Ferritin containing the Amber-codon.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/15-helper-plasmid-map.svg)

Fig. 12: Map of the plasmid of WT-Ferritin containing the Amber-codon.

The backbone was available to us in the lab. We only had to add the BsaI restriction recognition sites
to the end of it by PCR. The insert was divided into three fragments that we ordered. These were
designed for a Golden Gate Assembly (GGA) (fig. 15).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/16-helper-plasmid-history.svg)

Fig. 15: Planned GGA of the helper plasmid. Three inserts
(Fragment 1-3) are supposed to assemble into the vector, resulting in the approximately 6,000 bp
big plasmid.

###### **Build**

We then started our first attempt of the GGA reaction, using the following protocol:

| Reagents | Volume |
| --- | --- |
| p15A backbone (40 ng/µL) | 1.64 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 µL |
| NEB GGA Mix BsaI | 1 µL |
| MiliQ water | To 20 µL |
| Total | 20 µL |

| Temperature | Time |
| --- | --- |
| 37 °C | 10 min |
| 16 °C | 10 min |
| Go to 1, repeat 9x |  |
| 37 °C | 20 min |
| 60 °C | 20 min |
| 4 °C | hold |

We then transformed the products into DH5α cells, followed by a colony PCR for evaluation of the
colonies.

###### **Test**

PCR fragments of about 900 bp were expected. In our experience, colony PCR are fairly pronounced.
However, our obtained fragments were rather slim (fig. 16). But, since we had clear products at the
expected height, we sent the plasmids of GGA #2-5 to sequencing. The results came back negative.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/17-gga-1.png)

Fig. 16: Agarose gel (1 %) of the colony PCR results. The negative control (second lane
from the left) displayed unexpected bands. Bands with a size of about 900 base pairs are present
in the lanes of GGA Colony PCR 2, 3, 4 and 5. The area with the suspected bands in the agarose
gel is marked by the red box.

###### **Learn**

When repeating the program, we obtained negative results. We decided to optimize the program.

#### Cycle 2

###### **Design**

We used the same volumes, but increased the number and duration of the cycles.

###### **Build**

We used the following program:

| Temperature | Time |
| --- | --- |
| 37 °C | 5 min |
| 16 °C | 5 min |
| Go to 1, repeat 29x |  |
| 60 °C | 5 min |
| 4 °C | hold |

###### **Test**

Unfortunately, the transformation of these GGA products into DH5α cells was unsuccessful,
suggesting that the GGA failed.

###### **Learn**

We concluded that it may not have been the time of the cycle that was of significance.

#### Cycle 3

###### **Design**

For the next iteration we attempted to change two parameters: the DNA concentration of backbone and
fragments and the enzymes in the reaction. Up to this moment we used the NEB GGA enzyme mix for
BsaI. For the following attempt, we tested the T4 DNA ligase and the BsaI enzyme separately.

###### **Build**

We now used the following protocol:

| Reagents | 1. | 2. | 3. | Negative |
| --- | --- | --- | --- | --- |
| p15A (70 ng/µL) | 0.88 µL | 0.88 µL | 1.76 µL | 1.76 µL |
| Fragment 1 (50 ng/µL) | 2.19 µL | 2.19 µL | 4.38 µL | - |
| Fragment 2 (50 ng/µL) | 2.10 µL | 2.10 µL | 4.20 µL | - |
| Fragment 3 (25 ng/µL) | 1.13 µL | 1.13 µL | 2.26 µL | - |
| T4 DNA ligase buffer (10 X) | 2 µL | 2 µL | 2 µL | 2 µL |
| NEB GGA Mix BsaI | 2 µL | - | 2 µL | 2 µL |
| BsaI enzyme | - | 1 µL | - | - |
| T4 DNA ligase | - | 1 µL | - | - |
| ATP (10 mM) | 1 µL | 1 µL | 1 µL | 1 µL |
| MiliQ water | 8.7 µL | 8.7 µL | 2.4 µL | 8.7 µL |
| Total | 20 µL | 20 µL | 20 µL | 20 µL |

| Temperature | Time |
| --- | --- |
| 37 °C | 1 min |
| 16 °C | 1 min |
| Go to 1, repeat 29x |  |
| 60 °C | 5 min |
| 4 °C | hold |

We transformed the GGA products into DH5α (three plates per approach). From each plate, we picked
6 colonies for a colony PCR.

###### **Test**

For the colony PCR, we expected bands of 900 bp. While some lanes were negative, we had multiple
fragments on other lanes (fig. 17-18).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/18-final-gga-gel-1.png)

Fig. 17: Agarose gel (1 %) of colony PCR results. The ladder was not stained, ladder for
reference copied to the left. Some lanes displayed unspecific binding.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/engineering/19-final-gga-gel-2.png)

Fig. 18: Agarose gel (1%) of colony PCR results. Some lanes displayed unspecific
binding. Additionally the negative control (-) was positive.

Among many fragments, some were at around 900 bp. We picked these for sequencing. For most of them, the
results were negative with exception of 2.3.4. We had positive sequencing results, which suggested a
successful GGA reaction. We are currently in the process of investigating this. Due to lack of time we
had to end our experiments here. We did not get to the stage of expressing the ferritin.

![](https://static.igem.wiki/teams/4669/wiki/design-elements/up.svg)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %} Dare to Research: Experimentation Unleashed! {% endblock %}
{% block page\_content %}

{{ self.title() }}

## Protocols

#### Standard Protocols

###### Miniprep

**Day 1:** Transformation in DH5α Escherichia coli
**Day 2:** Preparation of overnight culture

* Pick clones with pipette tip and inoculate culture tube with 5 mL LB medium + 100 µg/mL ampicillin.
* Incubate culture at 37 °C overnight.

**Day 3:** Miniprep

* Follow Miniprep Kit protocol ROTI®Prep Plasmid MINI-XL kit, Carl Roth.

###### Colony PCR

Protocol for 25 µL sample.

* Prepare mix with with the following substances:

| Substance | Volume |
| --- | --- |
| 10 mM FW Primer | 0,5 µL |
| 10 mM Rev Primer | 0,5 µL |
| OneTaq® 2X Master Mix | 12 µL |
| Nucelase free Water | 11,5 µl |

+ For information about primers used in this protocol see chapter primers.

* Pick one colony with a pipette tip and mix with sample solution
* Run reaction as following:

| Step | Temperature | Time |
| --- | --- | --- |
| initial denaturation | 95 °C | 5 min |
| 35 cycles | 95 °C | 10 sec |
| 60,5 °C | 10 sec |
| 72 °C | 1 min |
| final extension | 72 °C | 5 min |
| 4 °C | hold |

###### PCR Purification

We purified DNA by centrifugation by using the Wizard® SV Gel and PCR Clean-Up System kit, Promega.

###### Transformation

* Thaw 100 µL of competent cells on ice for 20 min.
* Add 100 ng/µL plasmid to aliquot and resuspend carefully.
* Incubate the cell-plasmid suspension on ice for 30 min.
* Incubate solution at 42 °C for exactly 45 seconds.
* After incubation, transfer cells instantly on ice and incubate for 5 min.
* Add 400 µL of SOC medium at room temperature to the cells.
* Incubate cells at 37 °C and 300 rpm for 1 hour.
* Plate the sample on LB-Agar containing 100 µg/mL ampicillin.
* Incubate at 37 °C overnight.

#### Mutation to eliminate restriction sites and to add CPPs

###### PCR Mutagenesis to Remove Restriction Recognition Sites

| Substance | Volume | Final concentration |
| --- | --- | --- |
| Phusion MM (2X) | 25 µL | 1X |
| Template | variable | 8 ng/µL |
| Fw-Primer (with mutation) (10 µM) | 2.5 µL | 0.5 µM |
| Rev-Primer (10 µM) | 2.5 µL | 0.5 µM |
| H2O | 19 µL |  |
| Total | 50 µL |  |

|  |  |  |  |
| --- | --- | --- | --- |
| 1. | Primary denaturation | 98 °C | 30 s |
|  |  |  |  |
| --- | --- | --- | --- |
| 2. | Denaturation | 98 °C | 10 s |
| 3. | Annealing | 69 °C | 10 |
| 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 | - |

###### PCR Mutagenesis for Fusing CPPs

| Substance | Volume | Final concentration |
| --- | --- | --- |
| Phusion MM | 25 µL | 1X |
| Template KLD BsaI 1 6 #4 | 1 µL | 8 ng/µL |
| Fw-Primer (with mutation) (10 µM) | 2.5 µL | 0.5 µM |
| Rev-Primer (10 µM) | 2.5 µL | 0.5 µM |
| H2O | 19 µL |  |
| Total | 50 µL | - |

|  |  |  |  |
| --- | --- | --- | --- |
| 1. | Primary denaturation | 98 °C | 30 s |
| 2. | Denaturation | 98 °C | 10 s |
| 3. | Annealing | 62.2 °C | 10 |
| 4. | DNA synthesis | 72 °C | 3.5 min |
| 5. | Go to 2, repeat 29x | - | - |
| 6. | Final DNA synthesis | 72 °C | 5 min |
| 7. | Hold | 4 °C | - |

###### Double Digest

* Components:

| Substance | Volume |
| --- | --- |
| DNA | 0,3 ng |
| SapI | 1 µL |
| BsaI-HF-v2 | 1 µL |
| rCutSmart buffer | 5 µL |
| H20 | up to 50 µL |

* Incubate at 37 °C for 1 hour

###### Ligation (KLD)

Kinase, Ligase and DpnI digest to circularize PCR products.

1. Prepare buffer (for 5 reactions):

* 5 µL T4 DNA Ligase Buffer 10x
* 2.5 µL ddH20

2. Reaction mixture:

   | Substance | Volume |
   | --- | --- |
   | Buffer | 1.5 µL |
   | DNA (PCR Product) | 5.5 µL |
   | T4 Polynucleotidkinase (PNK) | 1.0 µL |
   | DpnI | 1.0 µL |
   | Total | 10 µL |
3. Incubate for 2 hours at room temperature (20 °C).
4. Store DNA at -20 °C.

For further information see  [KLD Enzyme Mix Reaction Protocol](https://www.neb.com/en/protocols/2017/03/31/kld-enzyme-mix-reaction-protocol-m0554).

Use template before PCR reaction as control.

#### Expression and Protein Purification

###### (CPP-)Ferritin Expression

Protocol adapted from group Prof. Dr. Tobias Beck.
 Used bacterial strain: BL21 (DE3) Star

**Day 1:** Transformation via heat stock method

* Thaw 100 µL of competent BL21 (DE3) Star cells on ice for 20 minutes.
* Transfer 50 µL of the cells in eppi tubes.
* Add 5 µL of DNA.
* Incubate samples on ice for 30 minutes.
* Heat cells at 42 °C for exactly 45 seconds.
* Place samples directly on ice and incubate them for another 5 minutes.
* Add 450 µL of preheated SOC-medium to the sample.
* Incubate cells for 1 h at 37 °C and 300 rpm.
* Centrifuge cells for 1 min at room temperature and 16,000 g.
* Discard 400 µL of the supernatant and resuspend the cells in the remaining medium.
* Plate cells out on LB Agar with ampicillin (100 µg/mL) plates and store overnight at 37 °C.

**Day 2:** Pre-culture preparation

* Pick colonies and add them into TB-Media containing 100 µg/mL ampicillin.
* Incubate cells at 37 °C and 180 rpm.

**Day 3:** Induction of the main culture

* Prepare a flask with a size of at least 5 times of the required volume of main culture.
* Prepare TB medium containing 100 µg/mL ampicillin.
* Add 2 mL of pre-culture into the TB medium.
* Incubate at 37 °C and 200 rpm shaking until OD600 0.6-0.7 is reached.
* Induce with 0.25 mM IPTG and incubate for 48 h at 18 °C and 200 rpm shaking.

**Day 4:** Harvest

* Spin down cells for 20 min and 5000 g at 4 °C.
* Discard supernatant and resuspend the pellet in 40 mL storage buffer (50 mM Tris, 0.30 M NaCl, pH 7.5).
* Transfer the suspension into a 50 mL falcon and spin down at 5000 g, 4 °C for 20 min.
* Discard supernatant and freeze pellet with liquid nitrogen.
* Store at -80 °C until lysis day.

###### Ferritin Purification

Protocols adapted from group Prof. Dr. Tobias Beck.

**Heat precipitation**

1. Resuspend pellet from 200 mL expression culture in 10 mL lysis buffer (50 mM Tris, 0.15 M NaCl pH 7.4).
2. Sonicate the cell suspension for 16 min (8 times 1 minute on, 1 minute off).
3. To discard cell debris centrifuge sample for 20 min at 14,000 g at 4 °C. Keep supernatant and discard pellet.
4. Heat supernatant to 65 °C in water bath for 10 minutes, slightly sway the sample solution.

* Solution is turning turbid due to the fall out of heat-instable proteins.

5. Centrifuge sample for 20 min at 14,000 g at 4 °C. Discard the pellet.
6. Add saturated (NH4)2SO4 solution to a final concentration of 70 % to the supernatant.

* Ferritin is salting out, solution gets turbid.

7. Spin down sample for 20 min at 14,000 g at 4 °C. Discard supernatant.
8. Resuspend pellet in 5 mL lysis buffer.
9. Add saturated (NH4)2SO4 solution to a final concentration of 70 % to the supernatant.
10. Spin down sample for 20 min at 14,000 g at 4 °C. Discard supernatant.
11. Resuspend pellet in 5 mL lysis buffer and filter with 0.22 µm pore size (syringe) filter.

**Ion exchange chromatography**

Used coloumn: HiTrap Q HP 5 mL

* Sample application:

+ 5 mL sample injected
+ Flow rate: 0.2 mL/min

* Washing step:

+ Flow rate: 2 mL/min
+ 6 CV

* Elution:

+ Flow rate: 3.5 mL/min
+ 60 % elution buffer
+ 20 CV
+ 1.5 mL sample collected for each fraction

* Column washing:

+ Flow rate: 2.0 mL/min
+ 5 CV

* Equilibration:

+ 5 CV with lysis buffer

* Concentrate the samples by using 30 MWCO.

**Size exclusion chromatography**

Used coloumn: Superdex 200 Increase 10/300 GL

* Flow rate: 0.5 mL/min
* Sample application:

+ 500 µL sample injected
+ Flow rate: 0.5 mL/min

* Elution:

+ Flow rate: 0.5 mL/min
+ 1 CV

* Equilibration:

+ Flow rate: 0.5 mL/min
+ 1.5 CV

2 SEC to discard larger proteins.
Due to high pressure of the column, the flow rate was about at 0.3 mL/min mostly.

###### SDS-PAGE

1. **Preparation of gels and SDS sample buffer**

1. Prepare seperation gels

* For each gel (12 %) aliquot (5 mL gel volume):

  | Substance | Volume |
  | --- | --- |
  | H20 | 1.5 mL |
  | 30 % acrylamide mix | 2.0 mL |
  | Tris -HCl (1.5 M, pH 8.8) | 1.3 mL |
  | SDS (10 %) | 50 µL |
  | 10 % ammonium persulfate | 50 µL |
  | TEMED | 2 µL |
* For each gel (12 %) aliquot (5 mL gel volume):

| Substance | Volume |
| --- | --- |
| H20 | 1.1 mL |
| 30 % acrylamide mix | 2.5 mL |
| Tris-HCl (1.5 M, pH 8.8) | 1.3 mL |
| SDS (10 %) | 50 µL |
| 10 % ammonium persulfate | 50 µL |
| TEMED | 2 µL |

2. Pour gel mix in gel casting form and leave about 2 centimeters below the top of the comb for stacking gel.
3. Pour a layer of isopropanol on top of the gel.
4. Leave gel at room temperature to polymerize.
5. Prepare stacking gel.

* For each (5 %) stacking gel (1 mL gel volume):

  | Substance | Volume |
  | --- | --- |
  | H20 | 0.68 mL |
  | 30 % acrylamide mix | 170 µL |
  | Tris-HCl (1.0 M, pH 6.8) | 130 µL |
  | SDS (10 %) | 10 µL |
  | 10 % ammonium persulfate | 10 µL |
  | TEMED | 1 µL |

6. Remove isopropanol.
7. Pour stacking gel in gel casting form on top the cast gels and insert comb.
8. Leave gel at room temperature to polymerize.
9. For storage, wrap the gel in moistened paper towel and store at 4 °C for one week maximum.
10. Prepare 4X SDS sample buffer stock.

* For 10 mL:

  | Substance | Volume |
  | --- | --- |
  | 100 % Glycerol | 4 mL |
  | Tris-HCl (1 M, pH 6.8) | 2.4 mL |
  | SDS | 0.8 g |
  | Bromphenol blue | 4 mg |
  | ß- Mercaptoethanol | 0.5 mL |
  | H2O | 3.1 mL |

2. **Sample preparation**

1. Dilute the protein sample to desired concentration.
2. Mix 4 X SDS-Samplebuffer with protein extract.
3. Incubate mix at 95 °C for 10 min.

3. **Running the gel**

1. Put gel into gel box and pour SDS-PAGE running buffer into the chamber.
2. Remove comb.
3. Wash gel pockets with SDS running buffer.
4. Pipette samples into the gel pockets.
5. Pipette molecular weight standards in one lane to determine the molecular weight of the sample.
6. Run the gel at 160 V until lowest molecular weight lane of the standard is down to 0.5 cm, stop electrophoresis.
7. Staining

* Remove gel from casting form and put gel in a staining box.
* Add Quick Coomassie Stain (by ProteinArk) to the gel.
* Incubate the gel at least for 15 minutes.
* Remove staining solution and wash the gel for 2 times with water.
* Incubate the gel for 2 h in water to remove background.

###### Western Blot

Used kit:  [Trans-Blot® Turbo™ Ready-to-Assemble (RTA) Kit](https://www.bioradiations.com/trans-blot-turbo-ready-to-assemble-rta-kit-enables-economical-rapid-and-efficient-western-blot-transfers/)

* Soak both stacks and membrane in transfer buffer.
* Place the first stack on blotting station, put the soaked membrane on top, carefully place SDS-Gel with protein on the membrane and place the last stack on top.

+ With a roller, carefully roll over each part to remove air.
+ Remove supernatant transfer buffer with a tissue.

* Start blocking program with 1.3 As and 25 V for 7 min.
* After blotting place membrane in a clean box.
* (If required, stain membrane with Ponceau S for 5 min to check if blotting have been successfull, after washing membrane with water to remove supernant staining, image the membrane.)
* Wash membrane 2 times for 5 min with TBS-T on a shaker.
* Place membrane in blocking buffer and incubate for 1 h on a shaker.
* Wash membrane 3 times for 10 min with TBS-T.
* Incubate membrane in antibody solution using a 1:12,000 dilution of 1µg/µL Ferritin Antibody with conjugated horseradish peroxidase for 1 hour at room temperature on a shaker or at 4 °C overnight.
* Wash membrane 3 times for 10 min with TBS-T on a shaker.
* Incubate membrane in substrate solution (1:1 HLP and luminol enhancer) for 1 min.
* Place membrane in foil to prevent it from drying out.

###### Negative Staining

**Sample preparation**

* Glow-discharge of a carbon-coated copper grid with GloQube(R) Plus air chamber for 30 s using negative polarity at 25 mA intensity.
* Negatively charged grid was incubated with protein solution (4 µL, 0.036 µL/mL) in buffer based by pH 7.5 for 30 s and washed 2x with MilliQ water (20 µL).
* Negative staining with uranyl acetate solution (4 µL, 2%).
* Liquid was carefully removed by side-blotting the grid.

**Talos L120C TEM**

Electron microscope from Thermo Fisher Scientific at cryoEM facility of Centre for Strucutral Systems Biology (CSSB) with 120 kV accelerating voltage, a CETA camera, with manual side entry, mainly for negative stain samples, but also capable of cryo-EM using holders from Gatan. Software options include SerialEM and Velox.

#### Work with Nanobodies

###### Golden Gate Assembly

* Reagents:

+ p15A backbone (40 ng/µL)
+ Fragment 1 (50 ng/µL)
+ Fragment 2 (50 ng/µL)
+ Fragment 3 (23 ng/µL)
+ T4 DNA ligase buffer (10x)
+ NEB GGA Mix BsaI
+ MiliQ water

* Mix reagents as shown and gently mix by pipetting up and down 4 times.
* Briefly microcentrifuge to bring material to the bottom of the tube.
* Transfer to thermocycler.
* Program:

  |  |  |  |
  | --- | --- | --- |
  |  | 37 °C | 5 min |
  | 29 cycles of step 1 and 2 | 16 °C | 5 min |
  |  | 60 °C | 5 min |
  | Hold | 4 °C |  |

For further informaton see  [Golden Gate Assembly Protocol for Using NEBridge ® Golden Gate Assembly Kit (BsaI-HF®v2) (E1601)](https://www.neb.com/en/protocols/2018/10/02/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1601).

###### QuikChange

| Substance | Volume | Final concentration |
| --- | --- | --- |
| Q5 Polymerase | 25 µL | 1X |
| Template KLD BsaI 1 6 #4 | variable | 8 ng/µL |
| Fw-Primer (with mutation) (10 µM) | 2.5 µL | 0.5 µM |
| Rev-Primer (10 µM) | 2.5 µL | 0.5 µM |
| H2O | 19 µL |  |
| Total | 50 µL |  |

|  |  |  |  |
| --- | --- | --- | --- |
| 1. | Primary denaturation | 98 °C | 30 s |
| 2. | Denaturation | 98 °C | 10 s |
| 3. | Annealing | 62.2 °C | 10 |
| 4. | DNA-synthesis | 72 °C | 3.5 min |
| 5. | Go to 2, repeat 29x | - | - |
| 6. | Final DNA synthesis | 72 °C | 5 min |
| 7. | Hold | 4 °C | - |

###### DpnI Digest

| Component | Volume |
| --- | --- |
| DNA | 1 µg |
| 10X rCutSmart NEB | 5 µL (1X) |
| DpnI (NEB) | 1 µL (20 units) |
| MiliQ water | To 50 µL |
|Total
50 µL

* Mix components.
* Incubate at 37 °C for 5-15 minutes.

#### Measuring the power of antibiotic molecules

###### Agar Diffusion Test

**Preparation for the filters:**

* Punch holes in filter paper.
* Prepare the concentrations of flavone, quercetin, rutin and ethylparaben individually and mixed in dishes with ethanol.

+ 100 µg/mL antibiotic substance
+ 50 µg/mL antibiotic substance
+ 10 µg/mL antibiotic substance
+ 1 µg/mL antibiotic substance
+ 100 µg/mL ampicillin (as positive control)
+ Ethanol (as negative control)

* Pipette 10 µL of the substance into filter paper circles (let it dry in between application).

**Preparation of the plates:**

* Take prepared LB-Agar plates.
* Plate 100 µL of BL21 (DE3) Star cell culture.
* Place the filter with antibiotic substances about 1.5 cm from plate border.
* Incubate plates at 37 °C for about 24 hours.

#### Penetration Efficiency

###### Penetration of Escherichia coli DH5α cells with eGFP-CPP fusion proteins

1. Preparation of solutions

* Fixation solution: 4 % formalin in 1X PBS
* DNA staining solution: 1:4000 DAPI in 1X PBS (DAPI stock solution: 1 mg/mL)

2. Overnight culture of *E. coli*  DH5α

* Pick colony from agar plate and inoculate 20 mL LB medium.
* Incubate overnight at 120 rpm and 37 °C.

3. Subculture and washing

* Inoculate 20 mL fresh LB medium with 1,000 µL overnight culture.
* Incubate at 120 rpm and 37°C until OD600 0.7-0.8 is reached.
* Harvest 1 mL culture for each assay in Eppendorf tubes.
* Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and resuspend in 500 µL PBS.

4. Penetration

* Add 10 µg of CPP-eGFP fusion protein or eGFP (negative control).
* Incubate for 1 hour at 37 °C and 150 rpm (mantle tubes in parafilm and fix horizontally in incubator).

5. Fixation

* Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and discard supernatant.
* Add 700 µL 4 % formaldehyde in PBS and incubate 20 min at room temperature.
* Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and discard supernatant.

6. DNA staining

* Add 200 µL of DNA staining solution per pellet and incubate for 10 min at 150 rpm shaking and room temperature in the dark (cover tubes in aluminium).
* Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and discard supernatant.
* Resuspend pellets in 10 µL PBS and pipette up and down several times.

7. Mounting

* Label glass slides for each assay.
* Load 4 µL mowiol mounting medium to glass slides.
* Add 2.5 µL bacterial suspension on mowiol droplet.
* Cover with cover slip, after 1 min press cover slip gently to the glass slide and let them dry at room temperature overnight in the dark.

## Primers

| Name | Use | Used in PCR of | Sequence |
| --- | --- | --- | --- |
| BsaI\_Fw | removal of BsaI recognition site in pET22b | Mutation restriction recognition site | GTGAGCGTGGGTCACGCGGTATCATTGC |
| BsaI\_Rev | removal of BsaI recognition site in pET22b | Mutation restriction recognition site | CGGCTCCAGATTTATCAGCAATAAACCAGCC |
| SapI\_Fw | removal of BsaI recognition site in pET22b | Mutation restriction recognition site | AGGAAGCGGAATAGCGCCTGATGCG |
| SapI\_Rev | removal of BsaI recognition site in pET22b | Mutation restriction recognition site | CGCTCACTGACTCGCTGCG |
| R9\_Fw | fuse R9 to pET22b WT ferritin | Mutation of CPP R9 | ATGcgtcgtcgccgtcgtcgtcgccgtcgtGGTGGTGGTGGCTCCATGACCACCGCGTCCACTAGC |
| R12\_Fw | fuse R12 to pET22b WT ferritin | Mutation of CPP R12 | ATGcgccgtcgtcgtcgccgtcgtcgccgccgtcgccgccgtcgtGGTGGTGGTGGCTCCATGACCACCGCGTCCACTAG |
| TAT\_Fw | fuse TAT to pET22b WT ferritin | Mutation of CPP TAT | ATGatgtacggtcgtaaaaaacgccgtcagcgtcgccgtGGTGGTGGTGGCTCCATGACCACCGCGTCCACTAGC |
| CPP\_Rev | fuse CPP to pET22b WT ferritin (universal) | Mutation of CPP | ATGTATATCTCCTTCTTAAAGTTAAACAAAATT |
| Colony\_R9\_Fw | Colony PCR to control R9 mutation | Colony PCR | TATACATATGcgtcgtcgccgtc |
| Colony\_R12\_Fw | Colony PCR to control R12 mutation | Colony PCR | TATACATATGcgccgtcgtcgtcTATACATATGcgccgtcgtcgtc |
| Colony\_TAT\_Fw | Colony PCR to control TAT mutation | Colony PCR | TATACATATGatgtacggtcgtaaaaaacgc |
| Colony\_CPP\_Rev | Sequencing to control CPP mutation | Colony PCR | GGTGGCAGCAGCCAACTCAG |
| Seq\_CPP\_Ftn\_Fw | Sequencing to control CPP mutation | Sequencing | ACGACTCACTATAGGGGAATTGTGAGC |
| Seq\_CPP\_Ftn\_Rev |  | Sequencing | GGTGGCAGCAGCCAACTCAG |
| Helper\_Fw | Helper Plasmid, linearising the vector plasmid p15A incl. BsaI cutting site for GGA | Linearising backbone | TATTATGGTCTCAtctattgagatcgttttggtctgcgcg |
| Helper\_Rev | Helper Plasmid, linearising the vector plasmid p15A incl. BsaI cutting site for GGA | Linearising backbone | TATTATGGTCTCAATAAatgatcatatcgtcaattattacctccacg |
| aaRS\_tRNA\_Col\_Fw | Colony PCR and sequencing to control GGA | Colony PCR and | gaaaggctcagtcgaaagactgggc |
| aaRS\_tRNA\_Col\_Rev\_Seq | Colony PCR and sequencing to control GGA | Colony PCR and sequencing | gaaccttcgaaaaaccgccctg |
| aaRS\_tRNA\_Seq\_Fw | Colony PCR and sequencing to control GGA | Sequencing | gcagtgtgaccgtgtgcttctc |

![](https://static.igem.wiki/teams/4669/wiki/design-elements/up.svg)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Putting our vision into practice{% endblock %}
{% block page\_content %}

{{ self.title() }}

## Summary

Our project transFERRITIN aimed to synthesize a modular drug delivery system,
a CPP-Ferritin-Container that allows the specific introduction of antibiotic molecules
into bacterial pathogens by using nanobodies bound to a modified codon in the L-loop
of the ferritin subunits using click-chemistry. During our journey we managed to accomplished
the following aspects:

* Deletion of SapI and BsaI restriction recognition sites to enable future iGEM teams to continue
  working with the ferritin construct
* Successful introduction of CPP TAT ([BBa\_K1202006](http://parts.igem.org/Part%3ABBa_K1202006)), R9 ([BBa\_K4669001](http://parts.igem.org/Part%3ABBa_K4669001)) or R12 ([BBa\_K4669002](http://parts.igem.org/Part%3ABBa_K4669002))
  on the N-terminus to ferritin ([BBa\_K4669027](http://parts.igem.org/Part%3ABBa_K4669027)).
* Optimization of the purification protocol and successful purification of WT-Ferritin
* Implementation of Amber codon into the L-loop of WT-Ferritin ([BBa\_K4669007](http://parts.igem.org/Part%3ABBa_K4669007)) and TAT-Ferritin ([BBa\_K4669008](http://parts.igem.org/Part%3ABBa_K4669008))
* Analysis of the penetration efficiency of CPP R9, R12 and TAT2 ([BBa\_K4669016](http://parts.igem.org/Part%3ABBa_K4669016))
* Visualizing the structural integrity of purified WT-Ferritin by negative staining EM

Click here for an overview of our workflow

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/workflow-komplettrichtigfuigem.png)

## Elimination of BsaI and SapI restriction recognition site

To commence with our project Prof. Dr. Tobias Beck provided us with the plasmid pET22b(+)
WT-ferritin (fig. 1) containing the conding reagion for human heavy chain ferritin.
In order to provide a plasmid for future iGEM teams that wish to proceed with our project or
want to develop their own ferritin project, we needed to eliminate the SapI and BsaI restriction
recognition sites (RRS) by using classic PCR mutagenesis.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/plasmid-pet22b-mitrrs.svg)

**Fig. 1: Plasmid pET22b(+) WT-Ferritin**  with SapI Restriction recognition site (4158 bp) and BsaI restriction recognition site (3087 bp).

Workflow:

* PCR mutation ro remove RRS
* PCR purification
* Ligation
* Transformation of the ligated product into DH5α
* Miniprep

+ double digest for control

* PCR mutation to remove the second RRS
* PCR purification
* Ligation
* Transformation of the ligated product into DH5α
* Miniprep

+ double digest for control

* Sequencing

In the initial phase of this process, we began with a PCR mutagenesis to excise the first RRS,
the success of which got subsequently confirmed by agarose gelelectrophoresis (fig. 2 A).
Following this confirmation, we purified the product, ligated the mutated DNA and transformed
via heat shock into DH5α for further mutation.
After a Miniprep of the transformed clones, we performed a double digest assay using
BsaI and SapI restriction enzymes. With an agarose gel, we checked if the elemination
of the first RRS was achieved. In theory, due to the absence of either the SapI or BsaI
RRS, the plasmid should be sliced at only one site, yielding a linear product with 6,000 bp
in size. Due to premature termination of the double digest assay after 15 min, not all plasmids
could undergo complete cleavage. As a consequence, the agarose gel analysis exhibited two visible
bands, as depicted in figure 2 B. The upper band showed the successfully digested plasmid,
while the lower band indicated the supercoiled uncut plasmid.
We used the plasmids with positive digestion results for the next PCR mutagenesis to delete the second RRS.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/rrs-part1.svg)

**Fig. 2: Agarose Gels of the first mutation step and double digest:**
 (A) Agarose gel of products of the PCR mutation to remove BsaI or SapI restriction recognition sites. Two samples for each mutation step were used. Bands which show positive results marked with a red box correspond to approximately 6,000 bp. (B) Agarose gel with products after 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.

After the second PCR mutagenesis we checked the outcome again through an agarose gel (fig. 3 A).
We repeated the methodology as in the previous mutation process and conducted again the double digest
to verify if both RRS got eliminated. However, this time we extended the time for the digest from 15 min
to 1 h in order to ensure complete enzymatic activity.
We used plasmid of the initial mutagenesis step for which one RRS is still intact to compare the result
with the double mutated pasmid. As shown in figure 3 B and C, the SapI Mut and BsaI Mut plasmids underwent
digestion which resulted in a band at approximately 6,000 bp. In contrast the double mutated plasmids (SapI
(BsaI) Mut and BsaI (SapI) Mut) did not exhibit any observable digestion, leading to a lower base pair indicative
of supercoiled plasmid.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/rrs-part2-new.svg)

**Fig. 3: Agarose gels of the first mutation step and double digest:**
 (A) Agarose gel with PCR results to check mutation to eliminate BsaI RRS in SapI Mut or SapI PRR in BsaI Mut. Two samples for each mutation were used. Bands which show positive results marked with a red box. (B) Agarose gel with products after the double digest. Wild type Ferritin (pET22b(+)) without restriction enzymes BsaI and SapI used as negative control (-); pET22b(+) with both enzymes used as positive 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. (C) 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.

As a final product we obtained the pET22b(+) WT-Ferritin plasmid without SapI and BsaI RRS ([BBa\_K4669027](http://parts.igem.org/Part%3ABBa_K4669027)) (fig. 4),
which was verified by sequencing and therefore could be used for further modifications.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/plasmid-pet22b.svg)

**Fig. 4: pET22b(+) WT-Ferritin plasmid with eliminated SapI and BsaI RRS**

## Insertion of CPPs into ferritin plasmid

We employed our previously mutated plasmid, pET22b(+) WT-ferritin without SapI | BsaI as a starting point to
establish a protocol for fusing cell-penetrating peptides (CPPs) TAT, R9, or R12
to the N-terminus of ferritin through site directed mutagenesis.

Workflow:

* PCR mutation for fusing CPPs with ferritin
* PCR purification
* Ligation
* Transformation of the mutated product into DH5α

+ Colony PCR

* Miniprep
* Sequencing

Following the PCR mutagenesis, agarose gelelectrophoresis was once again utilized to asses the
success of the reaction, as present in figure 5. Within the gel, the faint upper band corresponded
to the lineralized PCR product, representing the fusion of CPP with ferritin, measuring approximately
6,000 bp in size. Nevertheless, the presence of additional bands in the gel suggests deviations from the
expected outcome, indicating an abnormal reaction
Furthermore, it is important to note that the extended length of the forward primers for TAT (74 bp),
R9 (65 bp), and R12 (80 bp) may lead to the formation of secondary structures within the oligonucleotides
themselves. These secondary structures may have a negative impact on the PCR mutagenesis process.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/mutation-cpps.svg)

**Fig. 5: Agarose gel (1 %) after PCR mutagenesis with CPPs TAT, R9 or R12.**
 Two samples were used for each CPP mutation. All samples showed bands at approximately 6,000 bp. Bands of interest are marked by a red box

The DNA samples with positive agarose gel results and so exhibiting indications of CPP mutation underwent
purification and subsequent ligation. Following this, a transformation via heat shock was performed into
DH5α bacterial cells.
To have a verification for a successful mutation we checked the transformed colonies through colony PCR.
The primers employed in this analysis were designed to anneal upstream of the CPPs and downstream of the
ferritin DNA. So by checking the results via agarose gelelectrophoresis we expected intense bands at
approximately 600 bp signifying the success of the mutation. The successful sequence amplification
was achieved for each CPP mutation, although not for all of the individual samples used in the analysis (fig. 6).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/cpp-colonypcr.svg)

**Fig. 6: Agarose gel with colony PCR results**
(A) R9-Ferritin 1 and 2, WT-Ferritin (pET22b(+)) as well as positive control (+) with WT-Ferritin colony PCR sample and 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 bands at approximately 600 bp. The area with the bands for a positive results is marked by a red box. (B) 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 bands at approximately 600 bp. The area with the bands for a positive results is marked by a red box. (C) TAT-Ferritin 2, WT-Ferritin (pET22b(+)) as well as 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 bands at approximately 600 bp. The area with the bands for a positive results is marked by a red box. (D) TAT-Ferritin 1. TAT-Ferritin 1 sample 7, 8, 9, 10 showed the expected bands at approximately 600 bp. The area with the bands for a positive results is marked by a red box.

The colonies that resulted in a successful colony PCR amplification were used for Miniprep and subsequently
submitted for sequencing to final verify the insertion of the CPPs into our plasmid.
We managed to fuse each CPP with our plasmid (fig. 7). Following this, E.coli BL21 (DE3) star cultures
were transformed via heat shock to introduce the CPP-Ferritin clones for subsequent expression.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/cpp-plasmids.svg)

**Fig 7: Plasmids of (A) TAT-Ferritin ligation, (B) R9-Ferritin ligation and (C) R12-Ferritin ligation**

## Protein Expression

To continue with our work we needed to produce and extract our proteins.

Workflow:

* Transformation of plasmid into BL21 (DE3) star
* Preparation of expression culutre
* IPTG induction

After the successful transformation of the various CPP-Ferritin constructs into BL21 (DE3) star,
we initiated the preparation of expression cultures. These cultures were allowed to grow until
reaching an OD600 of 0.6 before inducing the expression. Since the T7 promoter is used in the
plasmid construct, 0.25 mM isopropyl β-D-1 thiogalactopyranoside (IPTG) was utilized as an
inducer for T7 RNA polymerase production followed by incubating the cultures at 18 °C and 180 rpm for 48 h
To check if our target protein got expressed after induction, we separated the proteins in a
15 % polyacrylamide gel with samples collected both prior to and 48 h after the induction with IPTG.
As shown in figure 8 after the induction (I), compared to the non induced samples (N), intense bands
were evident at the approximate weight of 21 kDa for WT-Ferritin (WT-Ftn), 23.6 kDa for TAT-Ferritin
and 23.1 kDa which corresponds to R9-Ferritin. Even though we did not obtain the expected band for
R12-Ferritin at the approximate weight of 23.6 kDa, we proceeded with our experiments.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/sds-inductionanalysis.svg)

**Fig. 8: Analysis of the expression before and after IPTG induction:**
 SDS-PAGE (15 % SDS gel) of whole cell samples of WT-Ferritin (WT-Ftn) TAT-Ferritin, R9-Ferritin and R12-Ferritin before (N) and after (I) IPTG expression induction. The area where the ferritin protein construct is expected is marked by a red box.

In order to validate the expression of our protein of interest, we performed SDS-PAGE and western
blot analysis with our cell culture normalized to OD600 values of 0.2 and 0.1. Notably,
bands corresponding to WT-Ferritin and the respective CPP-Ferritin construct could be detected in each lane,
as illustrated in figure 9 A.
After performing the transfer of the proteins to the membrane, the membrane got stained with Ponceau S
to assess the efficiency of the transfer process. Figure 9 B depicts successfully transferred proteins
to the membrane, however the band which represents R12-Ferritin at approximately 23.6 kDa is hardly recognizable.
Following the brief imaging process of the membrane stained with Ponceau S, the staining got removed during
two wash steps with water and we continued with the blotting protocol using the ferritin antibody with
conjugated peroxidase to detect the ferritin construct.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/wholecell-sds-ps.svg)

**Fig. 9: SDS-gel and PonceauS stainend membrane of whole cell analysis:**
 (A) SDS-PAGE 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. The area where the ferritin protein construct is expected is marked by a red box. (B) 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.

The application of the ferritin antibody revealed a protein in the lanes of TAT-Ferritin and R9-Ferritin
with a molecular weight of about 23 kDa which correlates with the molecular weight of TAT-Ferritin and R9-Ferritin
(fig. 10). However, it is noteworthy that no detectable signal was obtained for R12-Ferritin. Additionally,
in the lane containing WT-Ferritin with OD600 1.0, a faint signal was observed. The faint bands with higher molecular
weight in R9-Ferritin OD600 2.0 and in both R12-Ferritin samples could be an indicator for completely disassembled
ferritin protein, due to its heat-stability.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/wholecell-wb-new.svg)

**Fig. 10: Blotting result of whole cell samples with ferritin antibodies:**
 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.

We successfully expressed our CPP-Ferritin protein and validated this expression through the utilization of SDS-PAGE
and western blot. Furthermore, the ferritin antibody demonstrated the capability to recognize and bind our target protein,
despite its modification with CPPs.

## Protein Purification

In order to move forward with the subsequent analysis, it was essential that we purify our protein constructs.
For this purpose, we used a purification protocol for WT-Ferritin provided by Professor Dr. Tobias Beck,
which we adapted to our specific needs.

Workflow:

* Heat precipitation and ammonium sulfate precipitation
* Ion exchange chromatography
* Size exclusion chromatography run 1
* Size exclusion chromatography run 2

#### Heat Precipitation and Ammoniumsulfate Precipitation

Initially in the purification process, we began with heat precipitation at 65 °C and ammonium precipitation,
which can be used due to the great heat stability of ferritin.

After finishing the first part of the purification we checked the results via gelelectrophoresis using a
15 % SDS-gel and western blot just like for the expression analysis. Each lane was loaded with either 20 µg or
10 µg of total protein.
Since the gels were stained after blotting, it is difficult to detect the bands for small proteins like the
CPP-Ferritin constructs because they are already transfered on the membrane (fig. 11). The prominent
band observed in the WT-Ferritin lane with an approximate molecular weight of 21 kDa is indicative of the
presence of this specific protein.
However it can be noted that, after heat precipitation and ammoniumsulfate precipitation, a quite clear
protein solution was expected. In both SDS-PAGEs with the protein solution after the precipitation, a
lot of other proteins could be detected that indicated that the heat precipitation alone is not very
effective for high purity.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/sds-protein-1-und-2.svg)

**Fig. 11: 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 of experiment 1 (A) and experiment 2(B). The area where the ferritin protein construct is expected is marked by a red box.

After the blotting process the membrane got stained with Ponceau S to assess the success of the blotting
process (fig. 12). Using this staining, it is possible to detect WT-Ferritin with the predominant band
with approximately 21 kDa, as well as TAT-Ferritin, R9-Ferritin and R12-Ferritin with the bands of approximately
23 kDa. When the Ponceau S imaging was finished we continued with the antibody staining.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/sds-protein-1-und-2.svg)

**Fig. 12: 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 of experiment 1 (A) and experiment 2 (B). The area where the ferritin protein construct is expected is marked by a red box.

However, no signal was detected in both western blots.
Even if the heat precipitation and ammoniumsulfate precipitation did not work as well as expected and
the western blot experiment resulted with no signal, we assume that we were able to obtain WT-Ferritin
and CPP-Ferritin after the first purification step.

#### Ion Exchange Chromatography (IEC)

For ion exchange chromatography we used the HiTrap Q HP 5 mL column and injected 5 mL
of heat and ammonium precipitated protein solution.

For the elution we used 20 CV and 60 % of elution buffer. The following figures 13 to 21
show the chromatograms of the proteins. According to the protocol of Beck we expected to
detect WT-Ferritin and CPP-Ferritin constructs at nearly 35 mS/cm. Indeed, for nearly all
samples we could detect a peak around 30 mS/cm to 35/nbsp/cm except for sample WT-Ferritin 1,
where no peak could be detected indicating for WT-Ferritin protein

Click here for Chromatograms

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/wt1-iex.svg)

**Fig. 13: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/wt2-iex.svg)

**Fig. 14: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/z-stacks-tat2-egfp-1-5.png)

**Fig. 15: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/tat2-iex.svg)

**Fig. 16: IEC results of TAT-Ferritin 2**
 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/r91-iex.svg)

**Fig. 17: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/r92-iex.svg)

**Fig. 18: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/r92-iex.svg)

**Fig. 19: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/r121-iex.svg)

**Fig. 20: 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.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/r122-iex.svg)

**Fig. 21: 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.

After the chromatography process we collected the samples of the peak sections and concentrated the
solution by using 30 MWCO filter units. Subsequently SDS-PAGE was used to check if the WT-Ferritin,
TAT-Ferritin, R9-Ferritin and R12-Ferritin got purified, as shown in figure 19. Nonetheless, beside
the intense bands in the WT-Ferritin samples at approximately 20 / 21 kDa, that indicate the presence
of WT-Ferritin protein, no other bands in the CPP-Ferritin samples could be found. This implies that no
CPP-Ferritin construct could be purified. Probably, the proteins were already washed out of the column
during the washing process, but this would need further investigation. Besides that, it must be noted that
even after the purification step, a lot of unspecified proteins are still present.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/iex-samples.svg)

**Fig. 22: 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.

To improve the IEC for purification of R9-Ferritin and TAT-Ferritin, we adjusted the pH of the lysis buffer
(sample buffer) and elution buffer from pH 7.5 to pH 9 according to the isoelectric point (pI) to provide
enhanced binding affinity to the stationary phase of the column. Although we expected a better separation and
therefore a better signal with this adjustment, no peak indicating the presence of our proteins was detected in
the R9-Ferritin and TAT-Ferritin samples (fig. 23).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/iex-ph9.svg)

**Fig. 23: IEC results of TAT-Ferritin 3 (A) and R9-Ferritin 3 (B)**
 using lysis buffer and elution buffer at pH 9 . Peaks with an elution at a conductivity of 35 mS/cm which indicates for ferritin could not be detected.

We managed to successfully purify the WT-protein, however it was not possible to perform the purification method
with the CPP-Ferritin proteins, even though the pH was adjusted according to the pI.

#### Size Exclusion Chromatography (SEC)

Since it was not possible to purify the CPP-Ferritin proteins by IEC, SEC was performed only with WT-Ferritin.
As described in the original protocol, two SEC runs were performed in order to discard larger proteins. For this,
the Superdex 200 Increase 10/300 GL coloumn was used and 500 µL of post IEC concentrated protein solution was injected.
According to the standard protein injection, and the original protocol we expected the ferritin protein to
elute after 11 mL.
In the chromatogram of the first SEC run (fig. 24 A) two peaks can be identified. The first peak indicates wrong
assembled ferritin that must be discarded. The second peak represents the ferritin monomer.
Compared to the second run, the signal for both peaks is strongly reduced (fig. 24 B).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/wt1-sec.svg)

**Fig. 24: SEC result of WT-Ferritin 1**
 (A) first run. Second peak with retention after 11.194 mL identified as ferritin monomer. First peak with retention after 8.091 mL could be wrong assembled ferritin. (B) second run. Second peak with retention after 10.897 mL identified as ferritin monomer. First peak with retention after 8.102 mL could be wrong assembled ferritin.

To analyze if the WT-Ferritin is still present after the SEC runs, we performed a SDS-PAGE (fig. 25).
After both runs one band is visible at approximately 21 kDa. That indicates the presence of WT-Ferritin.
However, after the second run the signal in the gel is much less intense compared to the first run. Therefore,
it can be assumed that significant amounts of protein were lost between the runs.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/sds-sec.svg)

**Fig. 25: SDS-PAGE with 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.

Since no further bands can be detected after the first run, it can be concluded that WT-Ferritin was successfully purified in the first run.
The purified protein can now be used for further experiments.

## Negative Staining

To analyze the successful assembly of the ferritin complex after protein purification, we used the technique of negative
staining with uranyl acetate. This method plays a crucial role in transmission electron microscopy (TEM)
as it increases the contrast of biological samples that would otherwise be difficult to detect.
Negative staining involves depositing the staining agent around the specimen, leaving the specimen unstained. This makes the
sample appear bright against a dark background, which facilitates the identification and analysis of its structures.
We used the Talos TEM L120C to examine the sample. The microscope operated at an accelerating
voltage of 120 kV (HT 120 kV). A 70 µm aperture was employed to produce a magnification of 92000x by 4096 pixels.
These settings were carefully calibrated to ensure optimal representation of the sample's microstructure.
This method allowed us to capture high-resolution, clear and easily interpretable images of the specimen.
Figure 26 shows a structural representation of WT ferritin, annotated with a scale bar of 100 nm.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/negative-staining.png)

**Fig. 26: Negative stain image of WT-Ferritin, scale to 100 nm.**
 Taken with a 70 µm aperture to give a resolution of 92000x magnification with 4096 pixels and a pixel size of 154.2 pm.

Under the TEM, ferritin appears as a spherical protein whose 24 subunits
are arranged in a characteristic way to form a symmetrical hollow sphere. This cavity appears under the microscope
as a darker area in the centre of the molecule. At the current resolution, the surface of the ferritin molecule appears smooth.

## Implementation of Nanobodies

Workflow:

For the nanobody recognition to integrate the Amber Codon

+ PCR mutagenesis to intgerate the Amber Codon
+ PCR purification

- DpnI digestion as control

+ Miniprep

- Sequencing

For the concentration of the helper plasmid

+ PCR of the p15A backbone for linearization and addition of BsaI recognition site for the Golden Gate Assembly
+ Golden Gate Assembly of our three fragments
+ Transformation into DH5α and plating onto chloramphenicol agar plates
+ Colony PCR
+ Miniprep

- double digest as control

+ Sequencing

#### Amber Codon

As we settled for click chemistry to add the nanobodies onto our ferritin, we were in need of a
binding site. For that, we mutated the outwards facing lysine in the L-loop at position 88
Using QuikChange mutagenesis, we transformed our ferritin plasmid and successfully
introduced the mutation K88TAG, as proofed by the sequencing report.
After the mutagenesis, we performed a DpnI restriction and transformed DH5α competent
cells with the plasmid. Since the negative control did not grow any colonies when plated out on agar,
we can assume, that the DpnI restriction worked and we only introduced plasmid that was produced in our mutagenesis PCR (fig.27).
This result is backed up by the sequencing results.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/dpni-restriction.png)

**Fig. 27: Bacterial colonies after DpnI restriction of the PCR product from our mutagenesis of WT- and TAT-Ferritin and trafo into competent DH5α cells.**
 DpnI digests every methylated DNA which means every DNA that is not the product of our PCR. The negative control, where no colonies are growing, is bacterial plasmid and no PCR product.

#### GGA/Helper plasmid

To actually utilize the introduced Amber codon as binding site for the click-chemistry,
the bacteria needed a helper plasmid to produce the corresponding tRNA as well as the aminoacyl
tRNA synthetase to incorporate the ncAA that will be added later. To achieve that, we tried to assemble
the p15A plasmid via Golden Gate Assembly with three fragments. These fragments code for the synthetase
and two copies of the tRNA (fig. 28).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/helper-plasmid-15a.svg)

**Fig. 28: helper plasmid with p15A-backbone**
 with integrated chloramphenicol resistance (CmR) and added synthetase expression (ORF\_pyltRNA-aaRS) and tRNA (tRNA)

Afterwards, competent DH5α cells were transformed and plated out. From these plates, we picked colonies
for a colony PCR. With our primers designed to anneal in front of the second araBAD promotor
(4948-49726 bp) and inside the ori (62-84 bp) to flank our tRNA inserts.
The PCR product is expected to have a size of 900 bp. We used the colonies with positive results
to induce overnight cultures. From these overnight cultures, we extracted the DNA via Miniprep and
sent them in for sequencing. We managed to get only one colony so far with a positive result in both
the colony PCR as well as the sequencing report (fig. 29, red marked lane), but could not manage to
re-trafo the plasmid in time to use for further experiments.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/colonypcr-p15a.svg)

**Fig. 29: Results of the colony PCR for the integration of our three fragements into the p15A backbone,**
 run in an 1% agarose gel at 160V for 1h. Colonies 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 and 3.2.5 indicated positive results, with only 2.3.4 being also backed positive with the sequencing report. SYBRgreen was used to make bands visible and was forgotten to add to the marker lane, that’s why a ladder from another gel is added for reference. The expected product has a size of around 900 bp. The red box indicates the sample, which had a positive sequencing report for our inserts

We also attempted a double digest with two restriction sites (EcoRI and Cfr9I), that should
only be present in our GGA product. Sadly, this assay failed presumably cause of to little
amount of DNA, since in the negative control you can clearly see a band, but no band is visible
in the lane with our digested product (fig. 30).

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/dd-gga-new.svg)

**Fig. 30: Figure 26, 1% agarose gel run at 160 V for 1 h with double digested GGA product with EcoRI and Cfr9I.**
Plasmid without digestion enzymes as negative control (-). Bands are expected at around 3,000 bp, 1500 bp, 1000 bp and 400 bp for the digested plasmid and 5,800 bp for undigested plasmid. The control band is at 4,000 bp due to plasmid running faster through the gel. Additionally, it could be supercoiled which results in even faster running. Sample does not show any bands, presumably due to too little amounts of DNA.

#### Co-transformation

We also performed a co-trafo of our complete and finished WT- and TAT-Ferritin plasmid with our supposed
to be GGA transformed helper-plasmid, which resulted in only a handful of mini colonies to grow. Afterwards,
as we received the sequencing results, it showed that the helper-plasmid we used did not got mutated correctly
and therefore does not carry the tRNA and synthetase as wanted. Although, since we used two different resistances
in our two plasmids and both antibiotics on the agarplate, somehow the resistance must have been successfully implemented
into our helper-plasmid. Nontheless, since we had at least one successful introduction of all fragments into the
helper-plasmid as suggested by the sequencing report, we still have to validate this result and then retry the co-trafo
of our two plasmids.

The apparently successful GGA product needs to be re-transformed into bacteria culture. Afterwards, we can try the double
digestion again to double proof the correct insertion of our fragments. If this is succesful, we can continue with the co-trafo
of both of our plasmids and try if our nanobodies can bind to our ncAA on the Ferritin L-Loop. If they do so, all of our parts
are complete and we can undergo research on their selective target penetration and cargo release. If you want to know more in
detail, how we can proceed with our project after the iGEM competition, have a look at our [Outlook](https://2023.igem.wiki/hamburg/outlook)  page

## Penetration Efficiency

In order to test the capability of selected CPPs to penetrate the plasma membrane of gram-negative prokaryotes, CPP-eGFP
fusion proteins were incubated with *E. coli* DH5α cells and analyzed by fluorescence microscopy.
*E. coli* DH5α cells were cultured until OD600 0.7-0.8 and 1.5 · 108 cells in 500 µL PBS were incubated with 10 µg
of fusion protein - R9-eGFP, R12-eGFP, TAT2-eGFP, or eGFP without CPP as negative control - for 1 hour.
After incubation, cells were washed with 1X PBS, fixed with 4 % formalin and mounted with Mowiol mounting medium.
The samples were analyzed under the Leica Dmi8 Wide Field Microscope and Leica SP8 Confocal Microscope.
Two different thresholds were set to determine the efficiency of CPP penetration. By looking at the negative control,
the mimimum of threshold 1 was set to the value of 40, corresponding to the autofluorescence of the bacterial cells.
A second threshold was set to a mimimum value of 250 to include cells within the focal plane and exclude GFP signals
from outside the focal plane to determine the fluorescences intensity of the TAT2-eGFP incubated cells.
Cells incubated with TAT2-eGFP showed significantly increased GFP signals compared to the negative control of
cells incubated with eGFP. TAT2-eGFP incubated cells showed an approximately 10-fold increase in GFP signal with
a value of approximately 627 compared to the autofluorescence of approximately 67 (table 1).
Thus, TAT2-eGFP has either bound or penetrated the plasma membrane of *E.coli* DH5α.
In addition to fluorescence intensity measurements, the comparison of the DAPI and GFP channel of TAT2-eGFP
incubated cells show that all cells stained with DAPI have bound GFP and thus TAT2. The binding coverage
seems to be at approximately 100 %.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/e-coli-egfp-tat2.png)

**Fig. 31: *E. coli* DH5α cells incubated with eGFP (negative control) and TAT2.eGFP for 1 hour.**
 *E. coli* DH5α cells were cultured until OD600 0.7-0.8 and washed with 1X PBS. In 500 µL PBS, cells at a density of approximately 3 · 108 cells/mL were incubated with 10 µg of fusion protein - R9-eGFP, R12-eGFP, TAT2-eGFP, or eGFP without CPP as negative control - for 1 hour. After incubation, cells were washed with 1X PBS, fixed with 4 % formalin and mounted with Mowiol mounting medium. Leica Dmi8 Wide Field Microscope. DAPI channel (eGFP): Display range 0-10,000. DAPI channel (TAT2-eGFP): Display range 0-7,000. GFP channel (eGFP and TAT2-eGFP): Display range 0-3,000.

**Table 1: Fluorescence measurements of *E. coli* DH5α cells incubated with eGFP (negative control) and TAT2-eGFP for 1 hour.**
 Area of measurement ("Area"), average fluorescence ("Mean"), minimum values ("Min") and maximum values ("Max"). Measurements: Background, threshold 1 with MinThr=40 for differentiation of cells from background in negative control, threshold 2 (for TAT2-eGFP sample only) with MinThr=250 for more accurate detection of cells and exclusion of GFP signals from outside the focal plane.

|  |  | Area | Mean | Min | Max | MinThr | MaxThr |
| eGFP | Background | 1122.650 | 23.833 | 8 | 36 | 0 | 65535 |
|  | Threshold 1  (cells, autofluorescence) | 275.081 | **67.218** | 40 | 280 | 40 | 65535 |
| TAT2-eGFP | Background | 1054.966 | 7.466 | 0 | 24 | 0 | 65535 |
|  | Threshold 1 | 334.852 | 237.112 | 40 | 3635 | 40 | 65535 |
|  | Threshold 2 | 92.244 | **627.443** | 250 | 3635 | 250 | 65535 |

R9 and R12 showed different results in the penetration tests. R12-eGFP incubated cells show high GFP signals
indicating that R12 has bound or penetrated the plasma membrane. R9-eGFP incubated cells did not show any fluorescence intensity higher than autofluorescence.
The fluorescence intensities of R9-eGFP incubated cells and eGFP-incubated cells were measured by a threshold with a minimum value of 140.
The fluorescence intensity of with R9-eGFP incubated cells of approximately 145 is not higher than the fluorescence of cells in the negative
control (table 2).
R12-eGFP incubated cells show both visually (fig. 32) and by the measured fluorescence intensity of approximately 1098 (table 3) that R12 binds or penetrates the plasma
membrane. As with TAT2-eGFP (fig. 31), all cell groups stained with DAPI are
labeled with R12-eGFP (fig. 32) indicating a binding coverage of approx. 100 %.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/egfp-r9-r12.svg)

**Fig. 32: *E. coli* DH5α cells incubated with eGFP (negative control), with R9-eGFP, and with R12-eGFP.**
 Leica Dmi8 Wide Field Microscope. DAPI channel: Display range: 0-3000. GFP channel: Display range: 0-3000.

**Table 2: Fluorescence measurements of *E. coli* DH5α cells incubated with R9-eGFP, R12-eGFP, and eGFP (negative control) for 1 hour.**
Area of measurement ("Area"'), average fluorescence ("Mean"), minimum values ("Min") and maximum values ("Max"). Measurements: Background, threshold 1 with MinThr= 140 for differentiation of cells from background, threshold 2 (for R12-eGFP sample only) with MinThr=400 for more accurate detection of cells and exclusion of GFP signals from outside the focal plane.

|  |  | Area | Mean | Min | Max | MinThr | MaxThr |
| eGFP | Background | 1635.836 | 106.377 | 80 | 139 | 0 | 65535 |
|  | Threshold 1 | 0.211 | **164.020** | 140 | 235 | 140 | 65535 |
| R9-eGFP | Background | 935.111 | 106.325 | 66 | 129 | 0 | 65535 |
|  | Threshold 1 | 0.946 | **145.826** | 140 | 176 | 140 | 65535 |
| R12-eGFP | Background | 1954.789 | 106.471 | 79 | 150 | 0 | 65535 |
|  | Threshold 1 | 881.204 | 674.414 | 140 | 4416 | 140 | 65535 |
|  | Threshold 2 | 463.816 | **1098.096** | 400 | 4416 | 400 | 65535 |

In order to evaluate the capability of the CPP-eGFP fusion proteins to penetrate the plasma membrane, it is necessary to differentiate
whether the GFP signal is located at the plasma membrane or in the cytoplasm. For this, the penetration experiments
were repeated and examined on the SP8 Leica Confocal Microscope with Lightning Deconvolution.
Cells incubated with TAT2-eGFP and with R12-eGFP again showed increased fluorescence compared to the negative control (fig. 33).
In Z-stack images of single cell groups (fig. 34 to 36), GFP signals are low, possibly indicating a high level of photobleaching.
For each Z-stack, a single focal plane of high DAPI signal - to detect a cytoplasm-rich interface - and a projection of maximum intensity
are shown. Most Z-stacks do not show distinct fluorescence confirming CPP penetration. Z-stacks 2 and 4 from R12-eGFP incubation (fig. 34)
show GFP signal both within a cytoplasm-rich focal plane and in the projection of maximum intensities. It can be assumed that R12 penetrated the
plasma membrane. Further imaging is required. TAT2 shows minimal GFP signals, which is only visible in the projections of the maximum intensities
(fig. 35). Thus, differentiation between plasma membrane and cytoplasmic localization is not possible for TAT2-eGFP.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/egfp-tat2-r12-confocal.svg)

**Fig. 33: *E. coli* DH5α cells incubated with eGFP (negative control), with TAT2-eGFP, and with R12-eGFP in confocal microscopy.**
 Leica SP8 Confocal Microscope. DAPI channel: Display range (8-bit): 0-155. GFP channe: Display range (8-bit): 0-155.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/z-stacks-r12-egfp-1-5.png)

**Fig. 34: Z-stacks of *E. coli* DH5α cells incubated with R12-eGFP for 1.5 hours.**
 SP8 Leica Confocal Microscope with Lightning Deconvolution. Z-stacks of different cell groups. For each Z-stack: Maximum projection of the transmission. Focal plane with high DAPI-signals indicating cytoplasmatic localisation. GFP channel of the same focal plane. Maximum projection of DAPI and GFP signals. DAPI channel display range: Z-stack 1: 0-500. Z-stack 2: 0-2,000. Z-stack 3: 0-5,000. Z-stack 4: 0-300. GFP channel display range in all Z-stacks: 0-200.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/z-stacks-tat2-egfp-1-5.png)

**Fig. 35: Z-stacks of *coli* DH5α cells incubated with TAT2-eGFP for 1.5 hours.**
 Leica Confocal Microscope with Lightning Deconvolution. Z-stacks of two different cell groups. For each Z-stack: Maximum projection of the transmission. Focal plane with high DAPI-signals indicating cytoplasmatic localisation. GFP channel of the same focal plane. Maximum projection of DAPI and GFP signals. Display range of GFP channel: Z-stack 1: 1-1,000. Z-stack 2: 1-2,000. Display range of GFP channel for both Z-stacks: 0-200.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/z-stack-control.png)

**Fig. 36: Z-stack of *E. coli* DH5α cells incubated with eGFP (negative control) for 1.5 hours.**
 SP8 Leica Confocal Microscope with Lightning Deconvolution. Maximum projection of the transmission image, DAPI channel, and GFP channel. DAPI channel: Display range 0-1,000. GFP channel: Display range 0-200.

Further penetration experiments were performed with calcium-competent *E. coli* DH5α cells. The images obtained do not show increased GFP signals compared to the negative
control. Lower binding efficiency of GFP-CPP fusion proteins in 50 mM CaCl2
treated cells can be assumed. The results are shown in figure 37. To validate this assumption, the experiment will be repeated.
A possible explanation could be the the binding of Ca2+ to the negatively charged plasma membrane, inhibiting the binding of
the cationic CPPs used. DAPI signals are also of low intensities suggesting that fluorescence microscopy may have produced insufficient results.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/cacl2-egfp.png)

**Fig. 37: Penetration of CaCl2-chemically competent *E. coli* DH5α cells with eGFP, R9-eGFP, R12-eGFP, and TAT2-eGFP.**
 Leica SP8 Confocal Microscope with Lightning Deconvolution. Fusion proteins: eGFP (neg. control), R9-eGFP, R12-eGFP, and TAT2-eGFP. DAPI channel (8-bit): Display range 0-20. GFP channel (8-bit): Display range 0-20.

To determine the localization of CPPs, additional controls besides the negative
control - eGFP without fused CPP - will be implemented in the future. Additionally, a positive control for cytoplasmic
fluorescence could be used, for instance an *E.coli* DH5α mutant expressing an abundant cytosolic protein in fusion with GFP.
A suitable expression level resulting in GFP signal intensities similar to those expected upon successful CPP penetration could
be tested by different promoters. Another negative control that exhibits fluorescence at the plasma membrane but not in the cytoplasm
could be used. Additionally, a longer incubation period of three hours will be tested. The use of another microscope like the Nikon Spinning
Disc Confocal Microscope will be considered.

## Agar Diffusion Test

With a agar diffusion test with simple *E.coli* on an agar plate, we wanted to test the efficiency of antibacterial
plant substances in various concentrations. We plated out 100 µL *E.coli* BL21 (DE3) star bacteria on an regular agar plate and placed filter
paper soaked with flavone, quercetin, rutin and ethylparaben individually or in combination in following concentrations
in different parts of the plate as shown in figure 38: 1, 10, 25, 50, 75, and 100 µg/m. As controls, we used 70 % ethanol.
We then incubated the plates for 20-30 h at 37 °C. While we did this disk diffusion test several times, we only observed
significant and reproducable inhibition of cell growth for 70 % ethanol. The other compounds inhibited bacterial growth not significantly.

![Failed to load image](https://static.igem.wiki/teams/4669/wiki/pages/results/quercetin-plate.jpg)

**Fig. 38: LB agar plate with BL21 star *E. coli.* applied disks were soaked in 1, 10 or 25 µg/ml of a quercitin, flavone, ethylparaben and rutin mixture.**
A disk with water was applied as negative control (-), and one with 70 % ethanol as positive control (+).

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