Part:BBa_K4669027

pET22b(+)_WT-Ferritin
Function	Plasmid
Use in	Bacterial cells
RFC standard	RFC 21
Submitted by	Hamburg 2023

pET22b(+)_WT-Ferritin

Introducing it to iGEM, we would like to nominate our plasmid pET22b(+)_WT-Ferritin. This plasmid integrates a ready-to-use construct for the expression of the wildtype human heavy chain ferritin in Escherichia coli.

Figure 1: Plasmid map for our composite part BBa_K4669027. WT-Ferritin, introduced in a pET22b(+) backbone, which we mutated to delete SapI and BsaI resitriction recognition sites. The backbone carries a ampicillin resistance and T7 promoters, inducible by adding IPTG.

Deletion of restriction recognition sites (RRS)

For contributing the full plasmid to iGEM, we have performed mutagenesis to delete restriction recognition sites for the enzymes BsaI and SapI. Thus, we established a plasmid, compatible with the iGEM regulations and are able to provide the plasmid to future iGEM teams for directly diving into cloning and expression experiments!

With simple PCR mutagenesis, we first deleted one of the RRS and then the other. To confirm the successful deletion of the RRSs, we performed a double digest with both enzymes and run the samples through a 1% agarose gel. As shown in figure 2, the samples with only one deleted RRSs remain higher in the gel due to being linearized by the digest and therefore run slower through the gel than the circular plasmid. The samples with both deletions remains circular and shows a band approximately at the same height as the positive control.

Figure 2: Agarose gel with samples after the double digest. The plasmid without added restriction enzymes serves as positive control (+), with enzymes as negative control (-). SapI Mut1 samples have only one RRS deleted and are digested, SapI (BsaI) Mut1 samples have both deleted and remain undigested.

Protein Expression

To express our protein, we transformed E. coli strain BL21(DE) star with our plasmid. Protein production then was induced with 0.25 mM IPTG and incubated the cells at 18 °C for 48 h at 180 rpm. By seperating the proteins in a 15% polyacrylamidgel, we could show that we successfully induced the ferritin expression (Figure 3).

Figure 3: Analysis of the expression. SDS-PAGE (15 % SDS gel) of whole cell samples of WT-Ferritin (WT-Ftn). The height where the ferritin protein construct is expected is marked by a red box.

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.

Our workflow for the purification consisted of heat and ammoniumsulfate precipitation, ion exchange chromatography (IEC), and two size exclusion chromatography (SEC) runs.

To verify if we purified our ferritin, we ran a SDS-PAGE. As you can see in figure 4, the gel only shows one band at the expected height of 21 kDa.

Figure 4: 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.

Imaging of ferritin

We were able to show that our expression was fruitful and we were capable to completely purify the ferritin in an IEC, followed by two rounds of SEC. With our purified ferritin container we performed negative staining using Transmission Electron Microscopy (TEM). The pictures of the ferritin protein can be seen in figure 5.

Figure 5: Negative stain image of WTFerritin, 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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 5338
  Illegal SpeI site found at 33
  Illegal PstI site found at 1318
  Illegal PstI site found at 5596
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 33
  Illegal PstI site found at 1318
  Illegal PstI site found at 5596
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5272
  Illegal XhoI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 5338
  Illegal SpeI site found at 33
  Illegal PstI site found at 1318
  Illegal PstI site found at 5596
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 5338
  Illegal SpeI site found at 33
  Illegal PstI site found at 1318
  Illegal PstI site found at 5596
  Illegal NgoMIV site found at 316
  Illegal NgoMIV site found at 3492
  Illegal NgoMIV site found at 3652
  Illegal NgoMIV site found at 5240
  
• 1000
  COMPATIBLE WITH RFC[1000]