Part:BBa_K1438025

jbfs_mil_Ferritin Expression Device

An optimized human ferritin protein

Constructing an optimized human ferritin BioBrick

The basic parts (human ferritin light and heavy chain) were provided by Chris Wintersinger from the iGEM Calgary Team 2013. Thanks again for sending us your part BBa_K1189018.

In general ferritins are found in all kingdoms of life and in many different cells of multicellular organisms. They fulfill manifold tasks synthesize iron concentrates required for cells to make cofactors of iron proteins (heme, FeS, mono and diiron) as well as caged ferritin Fe2O3*H2O is acting as an antioxidant crucial for bio metabolism of proteins [1]. Human Ferritin (huferritin) consits of two different domains, light and heavy chain. (See source part BBa_K1189018 from iGEM Calgary 2013). The presented plasmid DNA construct JBFS_Mil_Ferritin is an improvement of the existing part BBa_K1438022 from our iGEM Team Berlin 2014 is a standardized pQE80L plasmid backbone containing human ferritin. We created this construct according to a paper published 2010 in Journal of Biological Chemistry (JBC) [3], they presented different compositions of Ferritin made of light (L) and heavy (H) chain, linked with an Glycine-Serine - construct (GS-Linker,*). “We found that the L*H chimera exhibits significantly enhanced iron-loading ability [...] compared to wild-type ferritin [3].“ Therefore it was necessary to combine different methods used in standard molecular biology, polymerase chain reaction (PCR) for amplification of single light and heavy chain. Assembly PCR for combination of amplified light and heavy chain containing GS-Linker sequence and furthermore ligation and transformation [2,3]. Currently our Team is characterizing the optimzed ferritin construct against different terms and conditions e.g. supplementation of iron in media, to proof a possible magnetization of Escherichia coli.

References: [1] Ferritins for Chemistry and for Life; Coord Chem Rev. 2013 Jan 15;257(2):579-586. Epub 2012 May 18., Theil EC1, Behera RK, Tosha T. [2] Improved Coexpression and Multiassembly Properties of Recombinant Human Ferritin Subunits in Escherichia coli; J. Microbiol. Biotechnol. (2008), 18(5), 926–932; Lee, Jung-Lim, Robert E. Levin, and Hae-Yeong Kim [3] Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate; J Biol Inorg Chem. 2010 Aug;15(6):957-65. doi: 10.1007/s00775-010-0657-7. Epub 2010 Apr 17.; Iordanova B1, Robison CS, Ahrens ET.

SDS PAGE

We succesfully expressed jbfs_mil_ferritin in E. coli DH10b using our BFR expression Device BBa_K1438025

After induction with 1mM IPTG jbfs_mil_ferritin was expressed in DH10b over night. jbfs_mil_ferritin subunits were expected at a molecular weight of about 40 kDa

Quantification of jbfs_mil_ferritin mediated iron capacity increase

Expression of jbfs_mil_ferritin increased the cells capacity to store iron. Also note here that more the iron storage capacity of the normalized cell pellet is higher then the not optimized Hu-Ferritin

More iron can be stored inside of a cell that is overexpressing jbfs_mil_ferritin.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 88
  Illegal XbaI site found at 2291
  Illegal XbaI site found at 3581
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 88
  Illegal NheI site found at 1435
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 88
  Illegal BamHI site found at 145
  Illegal XhoI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 88
  Illegal XbaI site found at 2291
  Illegal XbaI site found at 3581
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 88
  Illegal XbaI site found at 2291
  Illegal XbaI site found at 3581
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 541
  Illegal BsaI.rc site found at 4954
  Illegal SapI site found at 3871